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    • 专利摘要:
    The present invention belongs to the field of genetic engineering, and particularly relates to a malus domestica 4-coumaric acid: coenzyme A ligase gene Md4CL4 and an encoded protein and application thereof. The gene is the 4CL gene Md4CL4 cloned from malus domestica. The present invention is prepared by cloning from malus domestica for the first time, providing gene resources for plant molecular breeding. Vlfith the transgenic technology, the over-expression vector of the Md4CL4 gene is transferred into nicotiana tabacum to obtain transgenic nicotiana tabacum. In the present invention, high response of the Md4CL4 gene to salt stress is discovered in malus domestica for the first time, a DNA fragment of a complete coding section of a gene that is isolated and cloned from malus domestica and promotes tolerance to salt stress is obtained, and the functions of the gene are verified and used to finally find that the salt tolerance of the transgenic material after over-expression of the Md4CL4 gene in nicotiana tabacum is obviously improved. The use of the gene is conducive to breeding of new varieties of plants with high salt tolerance.
    公开号:NL2025428A
    申请号:NL2025428
    申请日:2020-04-26
    公开日:2020-11-23
    发明作者:Wang Haibo；Chang Yuansheng；Wang Sen；Wang Chuanzeng；He Xiaowen；Xu Li；Li Linguang；He Ping
    申请人:Shandong Inst Pomology；
    IPC主号:
    专利说明:

    Malus Domestica 4-Coumaric Acid: Coenzyme A Ligase 4 Gene and Encoded Protein and Application Thereof Technical Field The present invention belongs to the field of genetic engineering, and particularly relates to a malus domestica 4-coumaric acid: coenzyme A ligase gene Md4CL4 and an encoded protein and application thereof.
    Background Due to severe drought caused by global climate change and unreasonable farming, the problem of secondary salinization of soil has become increasingly prominent.
    Saline soil accounts for 7.6% of the world's land area, while the area of China's saline soil is 27 million hectares, accounting for 28.1% of the total area under cultivation.
    Soil salinization has become an important limiting factor restricting agricultural development.
    Malus domestica is one of the most economically important horticultural crops in the world and one of the most widely cultivated fruit tree species.
    Due to the different geographic origins of malus plants, the variety types are variant and abundant.
    At present, about 14 types, including cultivated apples, have been identified.
    The rich germplasm diversity also makes apples have different phenotypes, for example, M. xiaojinensis Chen et Jian., M. mandshurica XM, seboldii Rehd. and M. mandshurica (Maxim.) Kom. have strong salt tolerance, while M. prunifolia (Willd) Borkh., M. sieversii {Ledeb.) Roem. and M. micromalus Hemsl. have strong drought tolerance.
    In apple cultivars, the resistance of different varieties to stress also shows significant difference, on which many researches have been carried out.
    For example, the apple variety 'Golden Delicious’ which is primarily cultivated worldwide and the 'Qinguan’ which is primarily cultivated in the arid region of Northwest China have outstanding drought resistance, high utilization efficiency of limited water resources in the soil, and strong resistance to saline soil.
    The full excavation and application of excellent stress resistance genes contained in these specific varieties are of great significance to improve the resistance of malus domestica, widen the cultivation area and promote the product development.
    Salt stress is one of the main limiting factors affecting plant growth and development.
    In order to adapt to salt stress, plants have evolved to form a corresponding mechanism at the molecular level, i.e., regulating the physiological and biochemical activities of plants to resist stress through the expression of stress-induced genes.
    In higher plants, 4-coumaric acid: coenzyme A ligase (4CL) is an important class of enzyme, which catalyzes the last step of the phenylpropane metabolic pathway by converting 4-coumaric acid and other cinnamate derivatives into the corresponding coenzyme A thiol esters and is the key rate-limiting enzyme in the phenylpropane biosynthetic pathway.
    The synthesis of secondary compounds such as lignin and flavonoids through the phenylpropane metabolic pathway plays an extremely important role in plant physiology, ecology, growth and development, etc.
    Among them, lignin is one of the main components of the secondary walls of plant cells and is widely present in the ligneous tissues of vascular plants. It tightly adheres to cellulose and hemicellulose to form an interwoven net to harden cell walls, which plays an important role in plant growth and development. Flavonoids are involved in physiological processes such as protection against plant diseases and insect pests, UV protection, auxin transportation, root development and gravitropism, seed and pollen germination, symbiotic microorganism signaling and allelopathy among the plants. In the prior art, the 4CL coding gene has not been applied in the regulation of salt tolerance. Making full use of specific genes to carry out genetic engineering to improve plant resistance and improve the efficiency of plant variety breeding has received increasing attention, and the mining and functional study of key genes are the premise. Summary To solve the technical problem of genetic improvement of plant stress resistance, the present invention provides a malus domestica 4-coumaric acid: coenzyme A ligase gene Md4CL4.
    The present invention also provides an encoded protein and application of the malus domestica 4-coumaric acid: coenzyme A ligase gene Md4CL4 to achieve the purpose of providing gene resources for plant molecular breeding.
    To achieve the above purpose, the present invention adopts the following technical solution: The present invention provides a malus domestica 4-coumaric acid: coenzyme A ligase gene which is the 4CL gene Md4CL4 cloned from malus domestica, and the cDNA sequence is SEQ ID NO.1.
    The molecular weight of the encoded protein of the Md4CL4 gene is 65.59 kDa and the isoelectric point is 5.5.
    The present invention also provides an encoded protein of the malus domestica 4-coumaric acid: coenzyme A ligase gene, and the amino acid sequence of the encoded protein is shown as SEQ ID NO.2.
    In the present invention, the application of the malus domestica 4-coumaric acid: coenzyme A ligase gene Md4CL4 in improving plant salt tolerance is discovered for the first time. Further, the application specifically comprises the following steps: (1) analysis of Md4CL in response to salt stress: extracting the total RNA of ‘Golden Delicious’ leaves treated with salt stress by the "Trizol method", and using the extracted total RNA as the template for RT-PCR reverse transcription to synthesize the first strand of cDNA; and designing four pairs of specific primer sequences of Md4CL1, Md4CL2, Md4CL3 and Md4CL4: gPCRMd4CL1-F and qPCR4CL1-R, qPCRMd4CL2-F and qPCR4CL2-R, qPCRMd4CL3-F and gPCR4CL3-R, and gPCRMd4CL4-F and gPCR4CL4-R, analyzing the relative expression levels of Md4CL1, Md4CL2, Md4CL3 and Md4CL4 under salt stress, and screening the Md4CL4 gene significantly induced by salt stress;
    (2) cloning of malus domestica Md4CL4 gene: designing a pair of primer sequences Md4CL4-F and Md4CL4-R, and using the reverse-transcribed first strand of cDNA as the template for PCR amplification to obtain the full-length cDNA sequence of Md4CL4; (3) application of malus domestica Md4CL4 gene in improving plant salt tolerance: designing a pair of primers pBl121-Md4CL4-F and pBI121-Md4CL4-R containing BamHI and Sall restriction enzyme cutting sites, amplifying the coding region of the target gene Md4CL4 through PCR, constructing an expression vector pBl121-Md4CL4, transferring agrobacterium tumefacien EHA105, and transforming nicotiana tabacum ‘NC89' by the leaf disk transformation method to obtain positive transgenic nicotiana tabacum descendants.
    Further, in the step (1), the primer sequences qPCRMd4CL1-F and qPCR4CL1-R, gPCRMd4CL2-F and qPCR4CL2-R, qPCRMd4CL3-F and qPCR4CL3-R, and qPCRMd4CL4-F and gPCR4CL4-R are shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQID NO.10.
    Further, in the step (2), the primer sequences Md4CL4-F and Md4CL4-R are shown as SEQ ID NO.11 and SEQID NO.12.
    Further, in the step (3), the primer sequences pBI121-Md4CL4-F and pBI121-Md4CL4-R are shown as SEQID NO.13 and SEQ ID NO.14.
    Further, in the step (4), the specific method of transferring agrobacterium tumefacien EHA105 into the expression vector pBI121- Md4CL4 is as follows: inoculating the agrobacterium tumefacien EHA105 into 10 mL of LB liquid selection medium containing rifampicin (50 mg L-1), streptomycin (30 mg L-1) and kanamycin (30 mg L-1) for shake culture for 24 h (28°C); then continuing to transfer 0.4 mL of culture into 10 mL of LB liquid medium to continue culturing to make ODB00 reach 0.6 to 0.8; after that, placing the culture into an ice bath for 30 min, and centrifuging at 5,000 rpm at 4°C for 5 min; after removing the supernatant, adding 5 mL of pre- cooled CaCl2 (0.05 mol L-1) suspension bacteria solution, and continuing centrifugation at 5,000 rpm at 4°C for 5 min; and after removing the supernatant, adding 0.4 mL of pre-cooled CaCl2 (0.02 mol L-1) suspension bacteria solution, continuing to add 1/3 volume of glycerin, quick freezing with liquid nitrogen, and storing in a refrigerator of -80°C.
    Further, in the step (4), the specific method of transforming nicotiana tabacum 'NC89' by the leaf disk transformation method is as follows: germinating and sowing nicotiana tabacum 'NC89' seeds, and conducting management by routine to 2- or 3-leaf stage; disinfecting nicotiana tabacum leaves with 70% ethyl alcohol for 10 s, then disinfecting with 0.1% HgCI2 for 8 min, finally cutting the leaves into small leaf blocks of 0.5 cm x 0.5 cm after rinsing with sterile water for several times, and placing on an MS differential medium for pre-culturing at 28°C for 2 d with the illumination time of 16h/d and the illumination intensity of 2,000 Lx; immersing the pre-cultured nicotiana tabacum leaves into the prepared EHA105 bacteria solution for 10 min, then blotting up the excess bacteria solution with sterile absorbent paper, and placing on the MS basic medium for co-culturing at 28°C in weak light for 2 d; rinsing the co-cultured explants first with sterile water
    (containing 250 mg/L carbenicillin) for three times and then with MS basic culture solution (containing 250 mg/L carbenicillin) for one time, blotting up the liquid with sterile absorbent paper, and placing the explants on the MS differential medium (containing 100 mg/L kanamycin and 250 mg/L carbenicillin) for selective culture at 28°C; when buds grow to about 1 cm, cutting off the buds and moving into a rooting medium (containing 50 mg/l kanamycin and 200 mg/l cephalosporin) to promote rooting; after the root system is well developed, moving the root system into a flowerpot with sterile soil, moisturizing with a plastic film for 2 d, and placing in a greenhouse for routine management; and continuing propagation and cultivation to obtain T3 transgenic nicotiana tabacum strains.
    After scanning, identification and salt tolerance analysis of the domain of the malus domestica genome 4CL, a malus domestica Md4CL4 gene improving plant salt tolerance is obtained, which is the 4CL gene cloned from malus domestica. The total length of cDNA is 1833 bp and the cDNA sequence is SEQ ID NO.1. The amino acid sequence of the encoded protein is SEQ ID NO.2, and the nucleotide sequence contains one AMP-binding domain, one AMP-binding domain_C and two conserved domains: Box | and Box Il, which belong to Class II member of angiosperm 4CL gene family.
    For the encoded protein of the Md4CL4 gene provided by the present invention, the molecular weight is 65.59 kDa, the isoelectric point is 5.5, and the encoded protein contains 610 amino acids. The Md4CL4 gene is significantly induced by salt stress and can increase the growth of transgenic nicotiana tabacum under salt stress.
    Identification of the malus domestica 4CL gene of the present invention: HMMER software is used to scan the gene containing conserved domains: AMP-binding domain (PF00501) and AMP- binding domain_C (PF13193) from the malus domestica genome, and the malus domestica 4CL (Md4CL) genes: Md4CL1, Md4CL2, Md4CL3 and Md4CL4 are identified by the methods such as conserved domain search, phylogenetic analysis and gene function annotation; The identification method is as follows: downloading the malus domestica reference genome (v1.0) sequence from the GDR database; downloading HMM (Hidden Markov Model) files PFO0501 (AMP-binding domain) and PF13193 (AMP-binding domain_C) from the Pfam database (31.0), and searching the malus domestica genome (the default E value is 0.05) with the hmmsearch function of the HMMER software (version 3.1b2); obtaining the protein sequences of four 4CL (At4CL) and nine 4CL-like (At4CL-like) of arabidopsis thaliana from the TAIR database; conducting phylogenetic analysis on the screened protein sequences and the protein sequences of four At4CL and nine At4CL-like by MEGA 7.0; analyzing the presumptive malus domestica 4CL (Md4CL) protein according to the clustering result, further confirming the reliability of the protein sequence of the candidate Md4CL through NCBI conserved domain search and the Pfam database to ensure that each candidate protein contains conserved domains: AMP-binding domain and AMP-binding domain_C; further annotating the presumptive protein with blastp and the NCBI nr protein database; and further using four At4CL and nine At4CL-like proteins as inquiry sequences with the blastp method to search predicted malus domestica proteins from the GDR database so as to verify the above results, and obtaining four final Md4CL candidate protein sequences which are named Md4CL1to Md4CL4 and twenty-five final Md4CL-like candidate protein sequences which are named Md4CL-like1 to Md4CL-like25. 5 The present invention has the following advantageous effects that: (1) The malus domestica 4-coumaric acid: coenzyme A ligase 4 gene provided by the present invention is cloned from malus domestica for the first time, which provides gene resources for plant molecular breeding. In the present invention, high response of the Md4CL4 gene to salt stress is discovered in malus domestica for the first time, a DNA fragment of a complete coding section of a gene that is isolated and cloned from malus domestica and promotes tolerance to salt stress is obtained, and the functions of the gene are verified and used to finally find that the salt tolerance of the transgenic material after over-expression of the Md4CL4 gene in nicotiana tabacum is obviously improved. The use of the gene is conducive to breeding of new varieties of plants with high salt tolerance.
    (2) The present invention can be used to improve plant salt tolerance with the genetic engineering technology, laying a foundation for breeding new varieties of stress-resistant plants by genetic engineering, which has important theoretical significance and practical application value.
    Description of Drawings To more clearly describe the technical solution in the embodiments of the present invention or in the prior art, the drawings required to be used in the description of the embodiments or the prior art will be simply presented below.
    Fig. 1 is a schematic diagram showing multi-species evolution analysis of a malus domestica Md4CL gene disclosed in the embodiment of the present invention and 4CL genes of other species; Fig. 2 is a schematic diagram showing comparison of the amino acid sequence of the encoded Md4CL protein of a malus domestica Md4CL gene disclosed in the embodiment of the present invention with the amino acid sequences of the 4CL proteins of arabidopsis thaliana, poplar and oryza sativa ; Fig. 3 is a schematic diagram showing the expression of Md4CL1, Md4CL2, Md4CL3 and Md4CL4 genes under stress of 200 mmol NaCl disclosed in the embodiment of the present invention; Fig. 4 is an electrophoretic diagram of a malus domestica Md4CL4 gene disclosed in the embodiment of the present invention; Fig. 5 is an electrophoretic diagram showing PCR detection of over-expressed nicotiana tabacum strains 'OE-2', 'OE-3' and 'OE-4' of a malus domestica Md4CL4 gene disclosed in the embodiment of the present invention and the target gene Md4CL4 of the wild-type strain "WT",
    Fig. 6 is a schematic diagram showing qRT-PCR detection of expression of over-expressed nicotiana tabacum strains 'OE-2', 'OE-3' and 'OE-4' of a malus domestica Md4CL4 gene disclosed in the embodiment of the present invention and the target gene Md4CL4 of the wild-type strain
    WT Fig. 7 is a schematic diagram showing growth situations of over-expressed nicotiana tabacum strains 'OE-2', 'OE-3' and 'OE-4' of a malus domestica Md4CL4 gene disclosed in the embodiment of the present invention and the wild-type strain "WT" in the reference MS medium and the MS media respectively added with 200 mmol NaCl and 300 mmol NaCl; Fig. 8 is a schematic diagram showing root lengths and fresh weights of plants of over- expressed nicotiana tabacum strains 'OE-2', 'OE-3' and 'OE-4' of a malus domestica Md4CL4 gene disclosed in the embodiment of the present invention and the wild-type strain "WT" in the reference MS medium and the MS media respectively added with 200 mmol NaCl and 300 mmol NaCl; Fig. 9 is a schematic diagram showing content of malondialdehyde (MDA) in leaves of plants of over-expressed nicotiana tabacum strains 'OE-2', 'OE-3' and 'OE-4' of a malus domestica Md4CL4 gene disclosed in the embodiment of the present invention and the wild-type strain "WT in the reference MS medium and the MS media respectively added with 200 mmol NaCl and 300 mmol NaCl; Fig. 10 is a schematic diagram showing relative electrolytic leakage of leaves of plants of over-expressed nicotiana tabacum strains 'OE-2', 'OE-3' and 'OE-4' of a malus domestica Md4CL4 gene disclosed in the embodiment of the present invention and the wild-type strain 'WT" in the reference MS medium and the MS media respectively added with 200 mmol NaCl and 300 mmol NaCl; Fig. 11 is a schematic diagram showing content of free proline in leaves of plants of over- expressed nicotiana tabacum strains 'OE-2', 'OE-3' and 'OE-4' of a malus domestica Md4CL4 gene disclosed in the embodiment of the present invention and the wild-type strain 'WT" in the reference MS medium and the MS media respectively added with 200 mmol NaCl and 300 mmol NaCl. Detailed Description The technical solution in the embodiments of the present invention will be clearly and fully described below in combination with the drawings in the embodiments of the present invention. The present invention provides a malus domestica 4-coumaric acid coenzyme A ligase (Md4CL4) gene and an encoded protein and application thereof, and the specific embodiment is as follows: A malus domestica 4-coumaric acid coenzyme A ligase (Md4CL4) gene and an encoded protein and application thereof, comprise the following steps:
    (1) Identification of malus domestica 4CL gene: using malus domestica reference genome v1.0 sequence (downloaded from GDR database, http://www.rosaceae.org/). Downloading HMM (Hidden Markov Model) files PF00501 (AMP-binding domain) and PF13193 (AMP-binding domain_C) from the Pfam database (http://pfam.xfam.org; version 31.0}, and searching the malus domestica genome (the default E value is 0.05) with the hmmsearch function of the HMMER software (version 3.1b2). Obtaining the protein sequences of four 4CL (At4CL) and nine 4CL-like (At4CL-like) of arabidopsis thaliana from the TAIR database (http://www.arabidopsis.org/). Conducting phylogenetic analysis on the remaining protein sequences and the protein sequences of four At4CL and nine At4CL-like by MEGA 7.0. Analyzing the presumptive malus domestica 4CL (Md4CL) protein according to the clustering result. To confirm the reliability of the protein sequence of the candidate Md4CL, ensuring that each candidate protein contains conserved domains: AMP-binding domain and AMP-binding domain_C. For this purpose, using NCBI conserved domain search (CDD: http://www.ncbi.nlm.nih. gov/Structure/cdd/wrpsb.cgi} and the Pfam database (Pfam31.0:http://pfam.xfam.org/}. Further annotating the presumptive protein with blastp and the NCBI nr protein database (https://www.ncbi.nlm.nih.gov) to further verify the above results, using four At4CL and nine At4CL-like proteins again as inquiry sequences with the blastp method to search predicted malus domestica proteins from the GDR database (http://www.rosaceae.org/tools/ncbi_blast), and obtaining four final Md4CL candidate protein sequences which are named Md4CL1to Md4CL4 and twenty-five final Md4CL-like candidate protein sequences which are named Md4CL-like1 to Md4CL-like25.
    (2) Analysis of Md4CL in response to salt stress: RNA extraction: treating three-month 'Golden Delicious’ apple seedlings with 200 mM NaCl and collecting the middle leaves of the seedling stem at 0, 3, 8, 12, 24 and 48 h of treatment. Extracting the total RNA of the ‘Golden Delicious' leaves of each treatment stage by the "Trizol method". The specific method is as follows: weighing 0.2 g of leaf tissues, grinding into powder in liquid nitrogen, adding 2 mL of Trizol into the mortar for homogeneous mixing to form homogenate, and placing at room temperature for 10 min; sucking the homogenate into a 1.5 mL centrifuge tube, adding 200 pL of chloroform, fastening the cover on the centrifuge tube, vigorously shaking the centrifuge tube, and placing at room temperature for 10 min; centrifuging at 12,000 r/min at 4°C for 15 min, and moving the upper liquid phase into another tube; adding 500 UL of isopropyl alcohol of equal volume, gently turning the centrifuge tube upside down to fully blend the liquid, and placing at room temperature for 10 min; centrifuging at 12,000 r/min at 4°C for 15 min, and carefully sucking out all the supernatant with a gun; rinsing with 1 mL of 75% ethyl alcohol twice, centrifuging at 8,000 r/min at 4°C for 10 min, carefully sucking out all the supernatant with a gun, and drying in a super clean bench for 5 min; and adding an appropriate amount of DEPC treating water, promoting dissolution at 85 °C for 10 to 15 min, and storing at -80°C for later use. cDNA synthesis: using the extracted total RNA as the template for RT-PCR reverse transcription to obtain the first strand of cDNA, wherein the reverse transcription reaction system contains 2 ug of total RNA, 10 HL of 2 x TS Reation Mix, 2 pmol of random primers and 1pL of TransScript RT/RI Enzyme Mix; adding ddH 2 O to 20 pL, and mixing well; and incubating at 42°C for 30 min, cooling on ice for 2 min, and storing at -20°C for later use. According to the sequence information of the reference genome, designing four pairs of candidate gene specific primer sequences: gPCRMd4CL1-F and qPCR4CL1-R, qPCRMd4CL2- F and gPCR4CL2-R, qPCRMd4CL3-F and qPCR4CL3-R, and qPCRMd4CL4-F and qPCR4CL4- R, wherein the sequences are respectively shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ IDNO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, and analyzing the relative expression levels of Md4CL1, Md4CL2, Md4CL3 and Md4CL4 under salt stress by real- time fluorescent quantitative PCR (qRT-PCR). qRT-PCR is conducted on BIO-RAD iQ5 equipment, and the reaction system is 20 yL, including 10 pL of SYBR ® Premix Ex Taq™ (TaKaRa), 1.0 JL of cDNA, 0.5 ‚LL of forward primer, 0.5 pL of reverse primer and 9 JL of ddH 2 O. The amplification procedure comprises: at 95°C for 3 min; at 95°C for 10 s, at 58°C for 30 s and at 72°C for 15 s which are cycled for 40 times; and then at 72°C for 3 min. Setting three repeated determinations for each sample. Using malus domestica MdMDH as the reference gene to calculate the ACt value. Calculating the relative expression level according to the 2 -AACt method, and determining the specific amplification based on the solubility curve.
    (3) Cloning of gene: designing a pair of primer sequences Md4CL4-F and Md4CL4-R, wherein the sequences are respectively shown as SEQ IDNO.11 and SEQ ID NO.12, and using the reverse-transcribed first strand of cDNA as the template for PCR amplification to obtain the full-length cDNA sequence of Md4CL4.
    The PCR amplification system is 50 pL. Respectively adding the following components into a 200 pL centrifugal tube: 1 pL of Md4CL4-F (10 uM),1 pL of Md4CL4-R (10 pM), 10 pL of 5x TransStart FastPfu Buffer, 5 uL of 2.5 mM dNTPs, 1 pL of TransStart FastPfu DNA polymerase and 2 pL of template, adding ddH 2 O to 50 uL, and mixing well.
    The PCR reaction conditions are as follows: pre-denaturing at 94°C for 5 min; denaturing at 94°C for 30 s, annealing at 56°C for 1 min, and extending at 72°C for 10 min which are cycled for 35 times; and finally extending at 72°C for 5 min.
    Finally, detecting the obtained PCR products by 1.0 agarose gel electrophoresis.
    Recovering the target fragments with an agarose gel DNA extraction kit: cutting off a single target DNA band from the agarose gel (removing the unwanted parts as much as possible), placing in a clean centrifugal tube, weighing the gel, and calculating the gel weight (recording the weight of the centrifugal tube in advance); adding one volume of Buffer PG into the gel block (if the gel weight is 100 mg, the volume can be regarded as 100 pL, and so on); and incubating in a 50°C water bath, during which the centrifuge tube is gently turned upside down every 2 to 3 min until the sol solution is yellow to ensure that the gel blocks are fully dissolved. If gel blocks that are not dissolved still exist, adding some more sol solution or continuing to place for a few minutes until the gel blocks are completely dissolved; column equilibration: adding 200 uL of Buffer PS into a spin column (DM) placed into a collection tube, centrifuging at 13,000 rpm for 1 min, discarding the waste solution in the collection tube, and putting the spin column back into the collection tube; adding the above solution into the spin column (DM) placed into the collection tube, placing at room temperature for 2 min, centrifuging at 13,000 rpm for 1 min, discarding the waste solution in the collection tube, and putting the spin column back into the collection tube; adding 450 pl of Buffer PW (checking whether absolute ethyl alcohol is added before use) into the spin column, centrifuging at 13,000 rpm for 1 min, discarding the waste solution in the collection tube, and putting the spin column back into the collection tube; centrifuging at 13,000 rpm for 1 min, and discarding the waste solution in the collection tube; and putting the spin column into a new 1.5 mL centrifugal tube (self-provided), adding 50 uL of Buffer EB in the air into the intermediate position of an adsorption film, and placing at room temperature for 2 min. Centrifuging at 13,000 rpm for 1 min, and collecting the DNA solution.
    Ligating the recovered products to the pEASY-Blunt vector. The ligation system is 5 pL. Gently mixing 4 pL of PCR products with 1 pL of pEASY-Blunt vector, and keeping reaction at 37°C for 5 min. Placing on ice after completion of the reaction. Then transforming enterobacteria DHS5a by heat shock: taking 100 HL of DH5a competent cells stored at -80°C, quickly adding 5 HL of ligation products after unfreezing on ice, gently mixing well, placing in an ice bath for 30 min, conducting heat shock in a 42°C water bath for 1 min, immediately placing on ice for 3 min, adding 1 mL of antibiotic-free LB liquid medium, conducting shake culture at 37°C for 1 h, centrifuging at 5,000 rpm for 10 min, sucking out the supernatant, suspending the precipitation in 100 JL of LB culture solution, evenly applying the bacteria solution to the LB solid medium containing antibiotic, and cultivating at 37°C overnight until single colonies grow. Screening positive clones, detecting the colonies by PCR, selecting five positive colonies and submitting to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.
    Finding the Open Reading Frame (ORF) of the gene by the online ORF Finder (http://www.ncbi.nim.nih.gov/gorf/gorf.ntml) provided by NCBI. Predicting the physiochemical properties including molecular weight and isoelectric point of the encoded protein by ProtParam (http://us.expasy.org/tools/ProtParam.html).
    (4) Over-expression of malus domestica Md4CL gene in nicotiana tabacum: constructing an expression vector pBI121-Md4CL4: designing a pair of primers pBI121-Md4CL4-F and pBI121- Md4CL4-R containing BamHI and Sall restriction enzyme cutting sites to amplify the coding region of the target gene Md4CL4, wherein the primer sequences are shown as SEQ ID NO.13 and SEQ ID NO.14. The PCR reaction conditions are as follows: pre-denaturing at 95°C for 10 min; denaturing at 95°C for 60 s, annealing at 80°C for 1 min, and extending at 72°C for 2 min which are cycled for 30 times; and extending at 72°C for 10 min. Then recovering and sequencing the PCR products with the same methods as gene cloning. Enzyme cutting the cDNA with Sall and BamHI, and ligating to Sall and BamHI sites of a binary vector pBl121 (Clontech) under the control of CaMV35S promoter. The 10 pL ligation system comprises 4 uL of target fragments. Recovering 1 pL of PBI vector and 5 pL of Ligation Solution.
    Transferring agrobacterium tumefacien EHA105 into the expression vector pBl121- Md4CL4.
    Inoculating the agrobacterium tumefacien EHA105 into 10 mL of LB liquid selection medium containing rifampicin (50 mg L-1), streptomycin (30 mg L-1) and kanamycin (30 mg L-1) for shake culture for 24 h (28°C); then continuing to transfer 0.4 mL of culture into 10 mL of LB liquid medium to continue culturing to make OD600 reach 0.6 to 0.8; after that, placing the culture into an ice bath for 30 min, and centrifuging at 5,000 rpm at 4°C for 5 min; after removing the supernatant, adding 5 mL of pre-cooled CaCl2 (0.05 mol L-1) suspension bacteria solution, and continuing centrifugation at 5,000 rpm at 4°C for 5 min; and after removing the supernatant, adding 0.4 mL of pre-cooled CaCl2 (0.02 mol L-1) suspension bacteria solution, continuing to add 1/3 volume of glycerin, quick freezing with liquid nitrogen, and storing in a refrigerator of -80°C.
    Transforming nicotiana tabacum 'NC89' by the leaf disk transformation method. Germinating and sowing nicotiana tabacum 'NC89' seeds and conducting management by routine to 2- or 3- leaf stage. Disinfecting nicotiana tabacum leaves with 70% ethyl alcohol for 10 s, then disinfecting with 0.1% HgCl2 for 8 min, finally cutting the leaves into small leaf blocks of 0.5 cm x 0.5 cm after rinsing with sterile water for several times, and placing on an MS differential medium for pre- culturing at 28°C for 2 d with the illumination time of 16h/d and the illumination intensity of 2,000 Lx. Immersing the pre-cultured nicotiana tabacum leaves into the prepared EHA105 bacteria solution for 10 min, then blotting up the excess bacteria solution with sterile absorbent paper, and placing on the MS basic medium for co-culturing at 28°C in weak light for 2 d. Rinsing the co- cultured explants first with sterile water (containing 250 mg/L carbenicillin) for three times and then with MS basic culture solution (containing 250 mg/L carbenicillin) for one time, blotting up the liquid with sterile absorbent paper, and putting the explants on the MS differential medium (containing 100 mg/L kanamycin and 250 mg/L carbenicillin} for selective culture at 28°C. When buds grow to about 1 cm, cutting off the buds and moving into the rooting medium (containing 50 mg/l kanamycin and 200 mg/l cephalosporin} to promote rooting. After the root system is well developed, moving the root system into a flowerpot with sterile soil, moisturizing with a plastic film for 2 d, and placing in a greenhouse for routine management. Continuing propagation and cultivation to obtain T3 transgenic nicotiana tabacum strains.
    (5) Evaluation of salt tolerance of transgenic nicotiana tabacum strain: respectively transplanting 5 d transgenic nicotiana tabacum strains (OE-2, OE-3 and OE-4) and wild-type (WT) seedlings to the reference MS medium, the MS medium added with 200 mM NaCl and the MS medium added with 300 mM NaCl to verify the effect of Md4CL4 in improving plant salt tolerance.
    After culturing for 14 d, determining the indexes such as fresh weight, root length, and malondialdehyde, electrolytic leakage and free proline of leaves of plants of nicotiana tabacum strains (OE-2, OE-3, OE-4 and WT) in different media.
    MDA determination: adding 0.1 g of leaves or tissues into 1.2 mL of pre-cooled 0.1% TCA (trichloroacetic acid) solution, grinding, centrifuging at 10,000 rpm at 4°C for 15 min, adding 0.5 mL of supernatant into 1.0 mL of 0.65% TBA (thiobarbituric acid), putting in a boiling water bath for 30 min after mixing well, immediately terminating the reaction with an ice bath, and centrifuging at 5,000 rpm at 4°C for 10 min. Determining the light absorption value of the sample solution at the wave lengths of 532 nm and 600 nm, and calculating the MDA content.
    Determination of relative electrolytic leakage: weighing 0.1 g of leaves, rinsing with distilled water for 3 to 5 times, immersing in 10 mL of deionized water, placing at room temperature for 12 h, and determining the initial conductivity (E1) with a Mettler Toledo Inlab 738 conductivity meter; then boiling the sample for 30 min, and cooling and placing at room temperature for 12 h; finally, determining the final conductivity (E2) of the sample electrolyte; and calculating the electrolytic leakage by the formula EL (%) =E1/E2*100.
    Determination of content of proline: weighing 0.2 g of leaves or tissues, placing in a test tube, adding 5 mL of 3% sulfosalicylic acid solution, sealing the mouth of the test tube, and placing into aboiling water bath for 10 min. Cooling at room temperature, taking 2.0 mL of supernatant, adding
    2.0 mL of glacial acetic acid and 2.0 mL of 2.5% acidic ninhydrin coloring solution, continuing to place into a boiling water bath for 40 min, adding 5.0 mL of toluene and shaking for mixing well after cooling, and extracting red substances. After standing and layering, determining the light absorption value of the toluene layer at the wavelength of 520 nm, and calculating the content of proline.
    The detection results are as follows: HMMER software is used to scan the gene containing conserved domains: AMP-binding domain (PF00501) and AMP-binding domain_C (PF13193) from the malus domestica genome, four malus domestica 4CL (Md4CL) genes: Md4CL1, Md4CL2, Md4CL3 and Md4CL4 are identified by the methods such as conserved domain search, phylogenetic analysis and gene function annotation, and the multi-species evolution analysis of four malus domestica Md4CL genes and other species is shown in Fig. 1. The result shows that Md4CL1, Md4CL2, Md4CL3 and Md4CL4 have a close evolution relationship with prunus persica, wherein Md4CL1 is close to PPE_003G09700, Md4CL2 is close to PPE_001G08080, and Md4CL3 and Md4CL4 are clustered with PPEO02. The clustering result of four Md4CL genes with arabidopsis thaliana 4CL (At4CL) and oryza sativa 4CL (Os4CL) shows that Md4CL1 and Md4CL2 belong to Class | member of angiosperm 4CL gene family and Md4CL3 and Md4CL4 belong to Class II member of angiosperm 4CL gene family.
    DNAMAN 8.0.3.99 software is used to compare and analyze four malus domestica 4CL protein sequences derived from the malus domestica v1.0 reference genome and four known arabidopsis thaliana 4CL protein (At4CL) sequences, five known poplar 4CL protein (Ptr4CL) sequences and four known oryza sativa ACL (Os4CL) protein sequences, as shown in Fig. 2. The result indicates that multi-sequence alignment shows that the 4CL protein sequences of the four plants all contain conserved Box | and Box Il in addition to AMP-binding domain and AMP-binding domain_C, wherein 1%, 19% 215 23" to 33™ and 37 amino acids in the Box | domain and 1%, 3d 5h to 8h 10! to 13 28 31%! to 34™ and 36™ to 37" amino acids in the Box II domain are highly conserved in the 4CL proteins of arabidopsis thaliana, poplar, oryza sativa and malus domestica, which verifies the correctness of malus domestica Md4CL screening.
    The relative expression levels of Md4CL1, Md4CL2, Md4CL3 and Md4CL4 under salt stress are analyzed to screen the Md4CL gene significantly induced by salt stress. The result shows that compared with the control, Md4CL1 and Md4CL2 genes have the expression significantly down- regulated at 2 h of salt treatment and have the expression always significantly suppressed as the salt stress continues, while Md4CL3 and Md4CL4 show the opposite gene expression trend, i.e., the expression is significantly up-regulated at 2 h of salt stress treatment and always significantly induced as the stress continues, wherein Md4CL3 reaches the induction peak at 24 h of stress, which is 5.5 times of control, while Md4CL4 reaches the induction peak at 6 h of stress, which is
    69.9 times of control, and the expression is significantly high as the stress continues (Fig. 3). The above results show that Md4CL3 and Md4CL4 are significantly induced by salt stress to express, and especially, the expression level of Md4CL4 is extremely high under salt stress, which indicates that the gene plays an important role in response to salt stress.
    The Md4CL4 gene is cloned from ‘Golden Delicious’ malus domestica, and the reverse- transcribed first strand of cDNA is used as the template for PCR amplification to obtain the full- length cDNA sequence of Md4CL4. The electrophoretogram is shown in Fig. 4. The total length of cDNA is 1833 bp, and the sequence is SEQ ID NO.1; and the encoded protein contains 610 amino acids, the encoded amino acid sequence is SEQ ID NO.2, and the nucleotide sequence contains one AMP-binding domain, one AMP-binding domain_C and two conserved domains: Box | and Box Il. The molecular weight of the encoded protein of the Md4CL4 gene is 65.59 kDa and the isoelectric point is 5.5.
    Application of malus domestica Md4CL4 gene in improving plant salt tolerance: a pair of primers containing Sall and BamHI restriction enzyme cutting sites are designed to amplify the coding region of the target gene Md4CL4 for genetic transformation, an expression vector pBI121- Md4CL4 is constructed, and agrobacterium tumefacien EHA105 is transferred. Nicotiana tabacum 'NC89' is transformed by the leaf disk transformation method to obtain positive transgenic nicotiana tabacum descendants. The PCR and qRT-PCR detection results of the over- expressed Md4CL nicotiana tabacum strains are respectively shown in Fig. 5 and Fig. 6. In Fig. 5, "+" is positive control (plasmid DNA of 35S::Md4CL pRI101AN vector), "-" is negative control (water), and "M" is DL2000 DNA marker. The salt stress treatment of transgenic nicotiana tabacum strains is evaluated to verify the effect of Md4CL4 in improving plant salt tolerance. The result shows that 5 d transgenic Md4CL nicotiana tabacum strains OE-2, OE-3 and OE-4 and wild-type 'WT' seedlings are transplanted to the MS solid medium and the MS solid media respectively added with 200 mM NaCl and 300 mM NaCl to evaluate the effect of the over-
    expressed Md4CL4 on salt stress tolerance of nicotiana tabacum.
    In the reference medium, nicotiana tabacum wild-type "WT' and transgenic strains show no significant difference in growth, but the addition of NaCl shows inhibition on the growth of nicotiana tabacum wild-type and transgenic strains, and the inhibition enhances as the salt concentration increases, and is stronger on the growth of wild-type "WT" than transgenic strains (Fig. 7). Among the strains under stress, the fresh weight and the root length of the wild-type plants are the lowest (Fig. 8). The result shows that the over-expression of Md4CL4 under salt stress can alleviate the adverse effect of this stress on the growth of nicotiana tabacum plants and improve the salt tolerance of nicotiana tabacum.
    The MDA, electrolytic leakage and content of free proline of leaves of various strains are further determined.
    The result shows that the MDA content and electrolytic leakage of leaves of three transgenic strains are lower than those of wild-type strains, and the content of proline is higher than that of the wild-type strains, but these indexes of the strains have no significant difference before treatment (Fig. 9 to Fig. 11), which physiologically explains that the over- expression of the Md4CL gene enhances the salt tolerance of nicotiana tabacum.
    The research can provide gene resources for plant salt-resistant molecular assistant breeding and lay a foundation for breeding new varieties of stress-resistant plants by genetic engineering, which has important theoretical significance and practical application value.SEQUENCE LISTING
    <110> Shandong Institute of Pomology <120> Malus Domestica 4-Coumaric Acid: Coenzyme A Ligase 4 Gene and
    Encoded Protein and Application Thereof <130> Apple CoEnzA ligase NL <150> CN201919371292.2 <151> 2019-85-06 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 1833 <212> DNA <213> Malus domestica <400> 1 atgaccattg cttccagttc cgtcgaaact caaaagccgg cagacatacc taccaatctc 60 atgccgtctg agattaattc tacctctcaa caaaatctaa cccaattgca acccgccgcc 120 tgcaccaaca atattattga ttccaccacc gccacctcca ccgccactgc caccactaac 180 catgtattca gatcaaaact accagacata ccyatcccca accacctccc tctccacact 240 tactgcttcc agaacctccc cgagttctcc gacagaccct gcttgatagt gggctcaacc 300 ggaaaatcat actctttctc cgagactcac ctcattgctc agaagaccgg cgcaggcctc 360 tccaacctcg gcatccaaaa aggtgaggtc atcatgattc tcctccaaaa ctgtgctgag 420 ttegtctteg ccttcatggg cgcttccatg atcggcgccg tcaccaccac cCgccaacccc 480 ttttacactg ccgccgaggt tttcaagcag gtcaaggccg ctaatgccaa actcatcatc 540 actcaatccc agtacgtcaa taagctccgc gaacatccct cctccgccga cggcacggac 600 caaaataact tcccgaaact cggcgaagac tttaagatcg tcacgatcga caatcctccg 660 gagaattgct tgcatttctc agtgctctcc gaggccaacg agaaggagct tccggacgtg 720 gtgatcgacg cagaggaccc ggtggccctc ccgttctctt cggggacgac cgggctcccc 780 aagggagtca ttcttacaca caagagcttg gtcaccagcg tggcccaaca ggtggacgga 840 gagaatccaa acctctactt gaaggaggac gatgtcgtat tgtgcgtgct gccgttgttt 900 cacatattct cgttgaacag cgtgctgctg tgctcgctgc gagcaggggc gggagttctg 960 ctgatgcaca agtttgagat aggtacgctg ctggagctga ttcagcggta ccgagtgtcg 1020 gtggcggcgg tggtgccgcc gctggtgata gcactggcga agaacccaat ggtggcggag 1080 ttcgacctga gctctattag ggtggtgttg tctggagcgg cgccgctggg gaaggagctg 1140 gaggaggcgc tcaagagccg agtccctgag gcagtgttgg gtcagggtta tgggatgacg 1200 gaggcagggc cggtgttgtc aatgtgcatg gcatttgcaa aggaaccgat gccaaccaag 1260 tcagggtcgt gtgggacggt ggtccgaaat gcagagctca aggtccttga ccttgaaact 1320 ggtctctcac tcggctataa ccaatccggc gagatttgca tccgtggctc tcaaatcatg 1380 aaaggatatt tgaatgatgt tgcggctacg gcaaccaccg tagacacgga gggctggctt 14409 cacactggtg acgtgggtta tgtggatgat gacaatgaga ttttcatcgt tgatagagcc 1500 aaggagctca tcaaattcaa aggcttccaa gtgccaccag ctgagctgga gtccctactt 1560 ataagccatc catccattgc agatgcagcc gtcgttccgc aaaaagatga tgctgctggt 1620 gaggttcccg ttgcatttgt ggttcggtct aatggtctcg aacttactga agaggctgta 1680 aaagaattta tagcaaaaca ggtagtgttt tacaagagac tgcacaaggt gcacttcgtc 1740 catgcaattc caaagtctcc gtctggaaag atcttgagaa aagacctcag agccaagctt 1800 gcaaccgcaa ccccttctgc cctggctaat taa 1833 <210> 2 <211> 610 <212> PRT <213> Malus domesticus <400> 2 Met Thr Ile Ala Ser Ser Ser Val Glu Thr Gln Lys Pro Ala Asp Ile 1 5 10 15 Pro Thr Asn Leu Met Pro Ser Glu Ile Asn Ser Thr Ser Gln Gln Asn
    Leu Thr Gln Leu Gln Pro Ala Ala Cys Thr Asn Asn Ile Ile Asp Ser 40 45 Thr Thr Ala Thr Ser Thr Ala Thr Ala Thr Thr Asn His Val Phe Arg 50 55 60 Ser Lys Leu Pro Asp Ile Pro Ile Pro Asn His Leu Pro Leu His Thr 65 70 75 80
    Tyr Cys Phe Gln Asn Leu Pro Glu Phe Ser Asp Arg Pro Cys Leu Ile 85 90 95 Val Gly Ser Thr Gly Lys Ser Tyr Ser Phe Ser Glu Thr His Leu Ile 100 105 110 Ala Gln Lys Thr Gly Ala Gly Leu Ser Asn Leu Gly Ile Gln Lys Gly 115 120 125 Glu Val Ile Met Ile Leu Leu Gln Asn Cys Ala Glu Phe Val Phe Ala 130 135 140 Phe Met Gly Ala Ser Met Ile Gly Ala Val Thr Thr Thr Ala Asn Pro 145 150 155 160 Phe Tyr Thr Ala Ala Glu Val Phe Lys Gln Val Lys Ala Ala Asn Ala 165 170 175 Lys Leu Ile Ile Thr Gln Ser Gln Tyr Val Asn Lys Leu Arg Glu His 180 185 190 Pro Ser Ser Ala Asp Gly Thr Asp Gln Asn Asn Phe Pro Lys Leu Gly 195 200 205 Glu Asp Phe Lys Ile Val Thr Ile Asp Asn Pro Pro Glu Asn Cys Leu 210 215 220 His Phe Ser Val Leu Ser Glu Ala Asn Glu Lys Glu Leu Pro Asp Val 225 230 235 240 Val Ile Asp Ala Glu Asp Pro Val Ala Leu Pro Phe Ser Ser Gly Thr 245 250 255 Thr Gly Leu Pro Lys Gly Val Ile Leu Thr His Lys Ser Leu Val Thr 260 265 270 Ser Val Ala Gln Gln Val Asp Gly Glu Asn Pro Asn Leu Tyr Leu Lys 275 280 285 Glu Asp Asp Val Val Leu Cys Val Leu Pro Leu Phe His Ile Phe Ser 290 295 300
    Leu Asn Ser Val Leu Leu Cys Ser Leu Arg Ala Gly Ala Gly Val Leu 305 310 315 320 Leu Met His Lys Phe Glu Ile Gly Thr Leu Leu Glu Leu Ile Gln Arg 325 330 335 Tyr Arg Val Ser Val Ala Ala Val Val Pro Pro Leu Val Ile Ala Leu 340 345 350 Ala Lys Asn Pro Met Val Ala Glu Phe Asp Leu Ser Ser Ile Arg Val 355 360 365 Val Leu Ser Gly Ala Ala Pro Leu Gly Lys Glu Leu Glu Glu Ala Leu 370 375 380 Lys Ser Arg Val Pro Glu Ala Val Leu Gly Gln Gly Tyr Gly Met Thr 385 390 395 400 Glu Ala Gly Pro Val Leu Ser Met Cys Met Ala Phe Ala Lys Glu Pro 405 410 415 Met Pro Thr Lys Ser Gly Ser Cys Gly Thr Val Val Arg Asn Ala Glu 420 425 430 Leu Lys Val Leu Asp Leu Glu Thr Gly Leu Ser Leu Gly Tyr Asn Gln 435 440 445 Ser Gly Glu Ile Cys Ile Arg Gly Ser Gln Ile Met Lys Gly Tyr Leu 450 455 460 Asn Asp Val Ala Ala Thr Ala Thr Thr Val Asp Thr Glu Gly Trp Leu 465 470 475 480 His Thr Gly Asp Val Gly Tyr Val Asp Asp Asp Asn Glu Ile Phe Ile 485 490 495 Val Asp Arg Ala Lys Glu Leu Ile Lys Phe Lys Gly Phe Gln Val Pro 500 505 510 Pro Ala Glu Leu Glu Ser Leu Leu Ile Ser His Pro Ser Ile Ala Asp 515 520 525
    Ala Ala Val Val Pro Gln Lys Asp Asp Ala Ala Gly Glu Val Pro Val 530 535 540 Ala Phe Val Val Arg Ser Asn Gly Leu Glu Leu Thr Glu Glu Ala Val 545 550 555 560 Lys Glu Phe Ile Ala Lys Gln Val Val Phe Tyr Lys Arg Leu His Lys 565 570 575 Val His Phe Val His Ala Ile Pro Lys Ser Pro Ser Gly Lys Ile Leu 580 585 590 Arg Lys Asp Leu Arg Ala Lys Leu Ala Thr Ala Thr Pro Ser Ala Leu 595 600 605 Ala Asn 610 <2105 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 3 gaagccattc cgaagtcacc 20 <2105 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 4 ttccttcaaa atggcagggc 20 <216> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer
    <400> 5 ggctccttct ggcaaaatct 20 <210> 6
    <211> 18
    <212> DNA
    <213> Artificial Sequence
    <220>
    <223> Primer
    <400> 6 cgcccacacg ctgatatg 18 <210> 7
    <211> 20
    <212> DNA
    <213> Artificial Sequence
    <220>
    <223> Primer
    <400> 7 ttcatttctg ctagcctgct 20 <210> 8
    <211> 20
    <212> DNA
    <213> Artificial Sequence
    <220>
    <223> Primer
    <400> 8 cggaactgga agcaatggag 20 <210> 9
    <211> 20
    <212> DNA
    <213> Artificial Sequence
    <220>
    <223> Primer
    <400> 9 cgatcgatgc gtgttgactt 20 <210> 10
    <211> 20
    <212> DNA
    <213> Artificial Sequence
    <220>
    <223> Primer
    <400> 10 gatccccgac ttcagaagct 20 <210> 11
    <211> 23
    <212> DNA
    <213> Artificial Sequence
    <220>
    <223> Primer
    <400> 11 atgaccattg cttccagttc cgt 23 <210> 12
    <211> 23
    <212> DNA
    <213> Artificial Sequence
    <220>
    <223> Primer
    <400> 12 ttaattagcc agggcagaag ggg 23 <210> 13
    <211> 25
    <212> DNA
    <213> artificial sequence
    <220>
    <223> Primer
    <400> 13 cgggatccat gaccattgct tccag 25 <210> 14
    <211> 25
    <212> DNA
    <213> artificial sequence
    <220>
    <223> Primer
    <400> 14 gcgtcgactt aattagccag ggcag 25
    权利要求:
    Claims (10)
    [1]
    A 4-coumarinic acid-coenzyme ligase A gene from Malus domestica, characterized in that the gene is the 4CL gene Md4CL4 cloned from Malus domestica and the cDNA sequence is that of SEQ ID NO.
    [2]
    The 4-coumarinic acid-coenzyme ligase A gene from Malus domestica, according to claim 1, characterized in that the molecular weight of the protein encoded by the Md4CL4 gene is 65.59 kDa and its isoelectric point is 5.5.
    [3]
    A protein encoded by the 4-coumarinic acid-coenzyme ligase A gene from Malus domestica, according to claim 1 or 2, characterized in that the amino acid sequence of the encoded protein is as shown in SEQ ID 2.
    [4]
    4. An application of the 4-coumarinic acid-coenzyme ligase A gene from Malus domestica Md4CL4 in improving salt tolerance in plants.
    [5]
    The use of claim 4, characterized in that it comprises the following steps: (1) analysis of Md4CL in response to salt stress: - extracting total RNA from leaves of 'Golden Delicious' subjected to salt stress according to the “TRIzol method ”, - applying the extracted total RNA as template for RT-PCR reverse transcription to synthesize a first strand of cDNA, - designing four pairs of specific primer sequences from Md4CL1, Md4CL2, Md4CL3 and Md4CL4: o qPCRMd4CL1-F and qPCR4CL1- R, o qPCRMd4CL2-F and qPCR4CL2-R, o qPCRMd4CL3-F and qPCRA4CL3-R, and o qPCRMd4CL4-F and qPCRA4CLA4-R, - analyzing the relative expression values of Md4CL1, Md4CL2, Md4CL3, and Md4CL3, and Md4 under screen of the Md4CL4 gene significantly induced by the salt stress; (2) cloning the Malus domestica Md4CL4 gene: - design a pair of primer sequences Md4CL4-F and Md4CL4-R, - use the reverse transcribed first strand cDNA axis template for PCR to generate the full length cDNA sequence of Md4CL4. to gain,
    (3) Using the Malus domestica Md4CL4 gene in improving salt tolerance: - designing a pair of primers pBl121-Md4CL4-F and pBI121-Md4CL4-R that cleave sites for the restriction enzymes Bam HI and Sal | - Using PCT amplify the coding region of the target gene Md4CL4, - Construct an expression vector pBI121-Md4CL4, (4) transfer the expression vector pBI121-Md4CL4 into Agrobacterium tumefaciens EHA105 and transform Nicotiniana tabacum 'NC89 with using the leaf disc transformation method to obtain positive transgenic Nicotiniana tabacum progeny.
    [6]
    The use according to claim 5, characterized in that in step (1) the primer sequences of gPCRMd4CL1-F and gPCR4CL1-R, qPCRMd4CL2-F and qPCR4CL2-R, qPCRMd4CL3-F and qPCR4CL3-R, and gqPCRMd4CL4-F and gPCR4CL4-R are as shown in SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and SEQ ID No. 10.
    [7]
    The use according to claim 5, characterized in that in step (2) the primer sequences Md4CL4-F and Md4CLA4-R are as shown in SEQ ID 11 and SEQ ID 12, respectively.
    [8]
    The use according to claim 5, characterized in that in step (3) the primer sequences pBI121-Md4CL4-F and pBI121-Md4CL4-R are as shown in SEQ ID 13 and SEQ ID 14, respectively.
    [9]
    The use according to any one of claims 5 to 8, characterized in that in step (4) the method for transferring the expression vector pBI121-Md4CL4 into Agrobacterium tumefaciens EHA105 is as follows: - inoculating the Agrobacterium tumefaciens EHA105 into 10 ml liquid LB selection medium containing 50 mg / l rifampicin, 30 mg / l streptomycin and 30 mg / l kanamycin, - shaking the culture for 24 hours at 28 ° C, - transferring 0.4 ml of the culture to 10 ml liquid LB medium, - Soak until an ODs of 0.6 - 0.8 is reached, - Place the culture in an ice bath for 30 minutes, - Centrifuge for 5 minutes at 4 ° C at 5000 rpm, - Remove the supernatant,
    - add 0.4 ml of a pre-cooled solution of 0.02 mol / l CaCl to the scraped material to obtain a bacterial suspension, - add 1/3 ° volume of glycerine, - freeze quickly with liquid nitrogen, and - Store in a freezer at -80 ° C until use.
    [10]
    The use according to any one of claims 5 to 9, characterized in that in the step (4) the process for transforming Nicotiniana tabacum 'NC89 by the leaf disc transformation process proceeds as follows: - seeding and leaving germination of seeds of Nicotiniana tabacum 'NC89', - routine care to the stage where the germ has 2 or three leaves, - sanitizing the leaves of Nicotiniana tabacum for 10 seconds with 70% ethanol, - then disinfecting for 8 minutes with 0, 1% HgCls, - rinsing the leaves several times with sterile water, - cutting the leaves into small leaf fragments of 0.5 cm x 0.5 cm, - placing the leaf fragments on an MS differential medium, - pre-cultivation of the leaf fragments during 2 days at 28 ° C with an exposure time of 16 hours per day at a light intensity of 2000 Lux, - immersion of the leaf fragments of Nicotiniana tabacum in the prepared suspension of EHA for 10 minutes 105 bacteria, - taking up the excess bacterial suspension with absorbent paper, - placing the leaf fragments on MS base medium for co-culturing for 2 days in dim light; - rinse the co-cultured explants three times with sterile water containing 250 mg / ml carbenicillin, - rinse once with MS base culture solution containing 250 mg / ml carbenicillin, - absorb the liquid with absorbent paper, - place the explants on the MS differential medium containing 100 mg / l kanamycin and 250 ml / l carbenicillin for selective cultivation at 28 ° C, - cutting of callus buds when they have grown to about 1 cm, - placing the cut callus buds in a rooting medium containing 50 mg / l kanamycin and 200 ml / cephalosporin to promote root formation, - after proper development of the root system, place the root system in a flower pot containing sterile soil, - moisten for two days using a plastic film, - place in a greenhouse for routine care,
    - continue propagation and growth to obtain T3 transgenic varieties of Nicotiniana tabacum. from - until use store in a freezer at -80 ° C.
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