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    • 专利摘要:
    The present invention discloses an efficient method for screening double-flowered mutant in Gypsophila paniculata, the method comprising: dividing G. paniculata seeds into groups, and performing ethyl methane sulfonate (EMS) mutagenesis on each group of seeds for different 5 times to obtain EMS-mutagenized seeds of G. paniculata; sowing the EMS-mutagenized seeds of G. paniculata on a substrate of peat and perlite mixture for germination and growing into seedlings of G. paniculata; growing the seedlings of G. paniculata into mutant plants; and performing statistical data analysis of the mutant plants, and isolating the double-flowered mutant of G. paniculata. The present invention provides an efficient method for screening 10 double-flowered mutant in Gypsophila paniculata by using a forward genetic approach, and identifies three double-flowered mutants with different variations in flower type and flower color, as well as many other phenotypes. The results of the EMS mutagenesis of the present invention are consistent with the expectation of obtaining several mutants, indicating that the method of the present invention successfully mutagenizes the seeds of G. paniculata.
    公开号:NL2028043A
    申请号:NL2028043
    申请日:2021-04-22
    公开日:2021-07-30
    发明作者:Wang Jihua；Cheng Ying；Li Fan；Ruan Jiwei；Yang Chunmei；Mo Xijun；Wu Lifang
    申请人:Floriculture Res Institute Yunnan Academy Of Agricultural Sciences；
    IPC主号:
    专利说明:

    [0001] [0001] The present invention relates to the technical field of biological breeding, and in particular to an efficient method for screening and identifying double-flowered mutant in Gypsophila paniculata.BACKGROUND
    [0002] [0002] Gypsophila paniculata, a flowering plant of the genus Gypsophila, is the only species used as cut flowers in the genus (Li et al, 2019). Due to its ornamental value, G. paniculata is one of the most important cut flowers in global commercial floriculture (Zvi et al., 2008). However, it is a major impediment that using traditional breeding methods to generate the genetic variabilities for specific traits of flower type (Wang et al., 2013). As a consequence, very few variations exist among the commercial cultivars in the world floricultural market (Zvi et al., 2008). Therefore, there is massive demand for new varieties with novel characteristics in ornamental traits over the past decade.
    [0003] [0003] The generation of diverse mutants with altered phenotypes provides critical tools to identify and characterize the biological functions of related genes. Various approaches and methodologies for mutagenesis have been developed, such as T-DNA insertion, irradiation, and chemical methods (Alonso et al, 2003; Belfield et al., 2012; Jansen et al., 1997). Each has advantages and disadvantages for creating mutation, but chemical mutagenesis is readily accessible and generates more subtle changes compared with other methods. For instance, ethyl methane sulfonate (EMS) induces chemical modification of nucleotides, resulting mispairing and base-pairs changes. EMS induced alkylation of guanine (G) residues results in pairing with thymine (T) but not with cytosine (C). Subsequently the original G/C pair is replaced with an A/T pair through DNA repair (Greene et al., 2003). EMS mutagenesis produces mutations evenly distributed through the genome in Arabidopsis, predominantly induces C-to-T changes resulting in C/G to T/A substitutions, and fewer G/C to C/G or G/C to T/A transversions or A/T to G/C transitions (Kim et al., 2006). Because mostly single- nucleotide polymorphisms or substitutions occur, loss- or gain-of-function mutants may be isolated. Together with forward genetics approach, the alterations in a large set of randomly mutagenized individuals could be monitored and detected (Alonso and Ecker, 2006).
    [0004] [0004] Flower type is a key ornamental trait and the top interest in G. paniculata breeding. Several commercial varieties of G. paniculata with double flowers have been released by breeders, such as ‘Million Stars’, ‘Bristol Fairy’, ‘Snowball’ and ‘Huixing 1’ {Li et al., 2020; Shibuya et al. 2017). Considering the challenges in genetic modification of related special ornamental traits, EMS mutagenesis is an ideal method to effectively induce mutations that may lead to improved traits. Moreover, several particular mutant phenotypes have been identified through EMS mutagenesis, such as the double-flower mutant of Portulaca grandiflora (Lokeshwar and Bhalla, 1982), dwarf and variegated plants of ornamental ginger (Sakhanokho et al, 2012), and valuable ornamental mutants in four Silene species (Jiang and Dunn, 2017). Therefore, the induction of gene mutations by EMS provides a simple method for the breeding of ornamental plants by creating beneficial mutations.SUMMARY
    [0005] [0005] For this reason, the present invention provides an efficient method for screening and identifying double-flowered mutant in Gypsophila paniculata.
    [0006] [0006] In order to achieve the above objective, the present invention provides the following technical solutions:
    [0007] [0007] The present invention provides an efficient method for screening and identifying double-flowered mutant in Gypsophila paniculata, the method comprising:
    [0008] [0008] dividing G. paniculata seeds into groups, and performing ethyl methane sulfonate (EMS) mutagenesis on each group of seeds for different times to obtain EMS-mutagenized seeds of G. paniculata,
    [0009] [0009] sowing the EMS-mutagenized seeds of G. paniculata on a substrate of peat and perlite mixture for germination and growing into seedlings of G. paniculata;
    [0010] [0010] growing the seedlings of G. paniculata into mutant plants; and
    [0011] [0011] performing statistical data analysis of the mutant plants, and isolating the double- flowered mutant of G. paniculata.
    [0012] [0012] Preferably, the concentration of EMS is 150 mM.
    [0013] [0013] Preferably, the number of the G. paniculata seeds in reach group is 1,000.
    [0014] [0014] Preferably, the weight of the G. paniculata seeds in reach group is 0.641+0.02 g.
    [0015] [0015] Preferably, the EMS mutagenesis is performed by:
    [0016] [0016] putting dry G. paniculata seeds in a centrifuge tube, and pre-treating with a 100 mM potassium phosphate buffer solution at 4°C overnight;
    [0017] [0017] removing the potassium phosphate buffer solution, adding a 10 mL of 100 mM potassium phosphate buffer solution to the centrifuge tube, and then adding EMS to the potassium phosphate buffer solution to a final concentration of 150 mM/L; and
    [0018] [0018] incubating the G. paniculata seeds in the potassium phosphate buffer solution for 6 hours, then washing the G. paniculata seeds 10 times in 30 min successively with 100 mM sodium thiosulphate and distilled water, and then drying the G. paniculata seeds.
    [0019] [0019] Preferably, the different times are O hour, 4 hours, 6 hours, 8 hours and 12 hours.
    [0020] [0020] Preferably, the EMS mutagenesis lasts for 6h.
    [0021] [0021] Preferably, the EMS-mutagenized seeds of G. paniculata are sown within one week.
    [0022] [0022] The double-flowered mutant of G. paniculata produced by the above-mentioned method also belongs to the protection scope of the present invention.
    [0023] [0023] The present invention has the following advantages:
    [0024] [0024] The present invention provides an efficient method for screening double-flowered mutant in Gypsophila paniculata by using a forward genetic approach, and identifies three double-flowered mutants with different variations in flower type and flower color, as well as many other phenotypes. The results of the EMS mutagenesis of the present invention are consistent with the expectation of obtaining several mutants, indicating that the method of the present invention successfully mutagenizes the wild-type seeds of G. paniculata. In the absence of genomic information and effective genetic modification of G. paniculata, the present invention provides a simple method for producing multiple mutants of G. paniculata, and provides basic materials for genetic modification and molecular breeding of G. paniculata.BRIEF DESCRIPTION OF DRAWINGS
    [0025] [0025] In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following will briefly describe the drawings needed in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary. For those of ordinary skill in the art, other implementation drawings can be obtained without creative work in the light of the drawings.
    [0026] [0026] FIG. 1 shows the phenotype of the wild-type plant and flower of G. paniculata, provided in an embodiment of the present invention. The scale bar is 10 cm.
    [0027] [0027] FIG. 2 shows the germination rate of M1 seeds of G. paniculata treated with 150 mM EMS for different times, provided in an embodiment of the present invention. The average germination rate is derived from three replicates. One-way ANOVA is used to assess statistical significance, and p values are calculated with Tukey's HSD test (a=0.05).
    [0028] [0028] FIG. 3 shows the phenotype of EMS mutants with different types of plant and leaf, provided in an embodiment of the present invention. A: The various leaf types in the M1 population. Each leaf is collected from different mutants. B: The typical plant phenotype of mutants in the M1 population. The scale bar is 1 cm.
    [0029] [0029] FIG. 4 shows the phenotype of double-flowered mutants with different flower type and color. The same longitudinal line of flowers was derived from one mutant plant. The scale bar is 1 cm.
    [0030] [0030] FIG. 5 shows the alignment of amino acid sequence of AG in double-flowered mutants with different flower types, provided in an embodiment of the present invention. Identical amino acids are indicated with a black background, while sequence differences are highlighted with white.DETAILED DESCRIPTION
    [0031] [0031] The following specific examples are used to illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described examples are part of the examples of the present invention, rather than all of the examples. Based on the examples of the present invention, those of ordinary skill in the art
    [0032] [0032] Materials and Methods. Plant material. The wild type of G. paniculata was selected for this study, which has single pink petal flowers, as shown in FIG. 1. The seeds of the G. paniculata were obtained from Yuxi Yunxing Biological Technology Co., Ltd. (Yuxi, China). The thousand kernel weight (TKW) of the G. paniculata seed is around 0.641 g (n=10). Example 1
    [0033] [0033] This example provides an efficient method for screening double-flowered mutant in Gypsophila paniculata, comprising:
    [0034] [0034] In order to determine the optimal treatment time of EMS mutagenesis, a pre- treatment was conducted for different times (Oh, 4h, 6h, 8h, and 12h, three repetitions and 1000 seeds for each repetition) using 150 mM EMS before mutagenizing a large number of seeds. The best treatment was selected based on the criteria of visible traits, such as 50% seed germination rate, albinism and trichome phenotypes in seedlings (Li, 2018). The data of seeds germination rate for each treatment is shown in Table 1, which shows the seed germination rate of different mutagenesis time using 150 mM EMS in G. paniculata. One-way ANOVA was used to assess statistical significance, and p values were calculated with Tukey’s HSD test (a = 0.05). Sig. = significance. Table 1 Mutagenesis Repetition Mean SD Sig. time 1 2 3 Oh 77.20% 70.20% 66.00% 71.13% 5.66% a 4h 59.71% 52.50% 48.76% 53.65% 5.57% b 6h 38.60% 40.24% 36.56% 38.47% 1.84% cc 8h 15.60% 15.60% 12.60% 14.60% 1.73% d
    [0035] [0035] To avoid exposure of operators to the toxic and carcinogenic EMS, the process of mutagenesis was operated in a Plexiglas glove box in the fume hood. Approximately 30,000 well-dried seeds (19.23 g) were put in a 10 ml centrifuge tube and pre-treated with a 100 mM potassium phosphate buffer solution at 4 °C overnight. After removing the potassium phosphate buffer solution, a 10 mL of fresh 100 mM potassium phosphate buffer solution was added and then 154.4 ul EMS was added to a final concentration of 150 mM. The seeds were incubated in the solution for 6 h at room temperature with gentle shake. After incubation, the seeds were thoroughly washed 10 times in 30 min successively with 100 mM sodium thiosulphate and distilled water. The seeds were then carefully transferred to filter paper for totally dry overnight. The mutagenized seeds need to sow as soon as possible, since the EMS mutagenized M1 seeds (the first generation which was treated by mutagens) were very weak and couldn't be stored for a long time (even after 1 week, the seeds would all dead).
    [0036] [0036] After the EMS mutagenesis of the seeds of G. paniculata, all of the seeds were sown to a substrate of peat and perlite mixture for germination in the breeding bed. The mutagenized seedlings were then grown in the solar greenhouse under natural photoperiod at the experimental farm of Yunnan Academy of Agricultural Sciences. The dominant mutants showing any variation compared to wild type were investigated. The randomly planted plants in five fields (one square meter of each) were observed and investigated, including the number of total plants and dominant mutants.
    [0037] [0037] Inthe pre-treatment of EMS mutagenesis, the seed germination rate of the control {0 h} was 71.13% (n=3, FIG. 2}, while the germination rate of the seeds significantly decreased with the increase of mutagenesis time (a = 0.05). After a 6 hours treatment, the seed germination rate reduced to 38.47% (n=3), which was approximately half of that of the
    [0038] [0038] It was estimated that 5,000 EMS-mutagenized M1 populations are enough to produce a mutation in any given gene in Arabidopsis, since EMS causes multiple point mutations in each individual (Qu and Qin, 2014). There are more than 56000 genes in the genome of G. paniculata, which is over two-fold than that of Arabidopsis (27655 genes) (Initiative, 2000). Therefore, at least 10 000 M1 plants are required for EMS mutagenesis of G. paniculata to produce at least one mutation in any given gene. Thus, we mutagenized 30,000 seeds with 150 mM EMS for 6 hours in order to reach the needed minimum M1 population in the example. These seeds were sown in the breeding bed in the green house for further mutant screen. FIG. 2 shows the germination rate of M1 seeds of G. paniculata treated with 150 mM EMS for different time. The average germination rate was derived from three replicates. One-way ANOVA was used to assess statistical significance, and p values were calculated with Tukey's HSD test (a = 0.05).
    [0039] [0039] After the seed germination, the plants with different mutant phenotypes were observed and investigated. In order to determine the effect of the EMS treatment, we did the sample survey in five fields in the example. The dominant mutants were counted and the rate of dominant mutations was around 54.63% (n = 5, Table 2, the dominant mutations rate in the EMS mutagenized population). Therefore, our EMS mutagenesis generated sufficient mutations with variety phenotypes in G. paniculata.
    [0040] [0040] For example, several kinds of mutants with different type of plant and leaf were isolated, as shown in FIG. 3, such as dwarf plants, leaf shape and color variations. The results indicated that EMS mutagenesis can cause leaf texture, shape, and color variation in G.
    [0041] [0041] In all, since there is no efficient approach of molecular genetic engineering in G. paniculata, like the T-DNA insertion mutant library or gene edition system in Arabidopsis, the protocol of the example provides an easy and efficient way to generate useful mutations in G. paniculata.
    [0042] [0042] In order to identify double-flowered mutants, we performed a mutant screen for the flowers type in the flowering M1 plants. As a consequence, three different types of double-flowered mutants have been isolated, as shown in FIG. 4. Comparing with the flower of wild type, the phenotype of these three double-flowered mutants displayed unequal variation in flower type and color, including one white colored and two pink colored double- flowers. More specifically, mutagenesis caused stamens weaker or even disappeared, but the pistils seem didn’t changed. Further, no seeds were generated in all mutants. It is speculated that the extra petals were likely transformed from stamens, resulting sterile of the mutants. Using the mutants with less pollen or no pollen as females in breeding will facilitate the hybridization of G. paniculate, since this species has a high self-pollination rate based on our observation.
    [0043] [0043] The discovery of double-flowered mutants with difference in flower type and color indicated that multiple base pairs or genes were targeted during the EMS mutagenesis. Since the M1 generation only harbored the dominant mutants, the targeted gene(s) regulating double flower is/are dominant. Furthermore, the results were consistent with the theoretical
    [0044] [0044] To further investigate the regulatory gene of double flower in G. Paniculata, we cloned the AGAMOUS (AG) genes of the three double-flowered mutants. Through gene sequencing, we identified several SNP switch or deletion and synonymous substitutions in AG genes from these mutants. Although the flower types of the mutants were similar, the detected mutation sites are different from each other in AGa and AGb genes. For instance, we detected a single base pair deletion (T/-) at the position 540 in AGa gene from M1, resulting in an amino acid change from leucine to serine (L181S), as well as four amino acids substitution and a premature stop codon (1185>STOP). Moreover, we also identified the same 6 bp base pair deletion in AGb from M1 and M3, causing two non-synonymous amino acids deletion compared with the reference genome sequence. This suggested that the loss function of AG genes could cause semi-double or double flower type. Table 3 shows the mutation information of AG genes detected from double-flowered mutants of G. Paniculata. FIG. 5 shows the alignment of amino acid sequence of AG in double-flowered mutants with different flower types. Identical amino acids are indicated with a black background, while sequence differences are highlighted with while.
    [0045] [0045] Although the present invention has been described in detail with general descriptions and specific examples above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, all these modifications or improvements made without departing from the spirit of the present invention belong to the scope of the present invention.REFERENCES
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    GBCYO86-NL-SEQUENCE LISTING ST25
    SEQUENCE LISTING <110> Floriculture Research Institute, Yunnan Academy of Agricultural Sciences <120> Efficient method for screening and identifying Double-flowered Mutant in Gypsophila paniculata <130> GBCY986-NL <150> CN 202110361816.7 <151> 2021-04-02 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 1 Met Leu Thr Thr Asn Ser Gln Gly Val Glu Glu Glu Arg Pro Lys Met 1 5 10 15 Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Thr Thr Ser Arg Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala Tyr 40 45 Glu Leu Ser Val Leu Cys Asp Ala Gln Leu Ala Leu Ile Val Phe Ser 50 55 60 Ser Arg Gly Arg Val Tyr Glu Tyr Ser Asn His Asn Ile Arg Ser Ile 65 70 75 80 Ile Glu Arg Tyr Lys Lys Ser Thr Asp Gly Ser Asn Ser Ser Ser Val 85 90 95 Thr Glu Ile Asn Ala Gln Tyr Tyr Gln Gln Glu Ser Thr Lys Leu Arg Pagina 1
    GBCYO86-NL-SEQUENCE LISTING ST25 100 105 110 His Gln Ile Gln Val Met Gln Asn Ser Asn Lys Asn Leu Met Gly Glu 115 120 125 Cys Leu Glu Asn Leu Thr Val Lys Glu Met Lys Gln Val Glu Asn Arg 130 135 140 Leu Glu Arg Gly Ile Ser Arg Ile Arg Ser Lys Lys His Glu Leu Leu 145 150 155 160 Leu Ala Asp Ile Glu Phe Leu Gln Lys Arg Glu Thr Asp Leu Glu His 165 170 175 Glu Asn Ala Phe Leu Arg Ser Lys Ile Ala Glu Val Glu Asn Leu Gln 180 185 190 Gln Leu Asn Pro Asn Glu Asn Leu Ser Val Tyr His Gly Phe Val Pro 195 200 205 Arg Asp His Ile His Ile Pro Gln Asn Ser Cys Pro Phe Ala Ile Ser 210 215 220 Thr Asp Lys Lys Phe Leu His 225 230 <210> 2 <211> 184 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 2 Met Leu Thr Thr Asn Ser Gln Gly Val Glu Glu Glu Arg Pro Lys Met 1 5 10 15 Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Thr Thr Ser Arg
    Pagina 2
    GBCYO86-NL-SEQUENCE LISTING ST25 Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala Tyr 40 45 Glu Leu Pro Val Leu Cys Asp Ala Gln Leu Ala Leu Ile Val Phe Ser 50 55 60 Ser Arg Gly Arg Val Tyr Glu Tyr Ser Asn His Asn Ile Arg Ser Ile 65 70 75 80 Ile Glu Arg Tyr Lys Lys Ser Thr Asp Gly Ser Asn Ser Ser Ser Val 85 90 95 Thr Glu Thr Asn Ala Gln Tyr Tyr Gln Gln Glu Ser Thr Lys Leu Arg 100 105 110 His Gln Ile Gln Val Met Gln Asn Ser Asn Lys Asn Leu Met Gly Glu 115 120 125 Cys Leu Glu Asn Leu Thr Val Lys Glu Met Lys Gln Val Glu Asn Arg 130 135 140 Leu Glu Arg Gly Ile Ser Arg Ile Arg Ser Lys Lys His Glu Leu Leu 145 150 155 160 Leu Ala Asp Ile Glu Phe Leu Gln Lys Arg Glu Thr Asp Leu Glu His 165 170 175 Glu Asn Ala Phe Ser Glu Ala Arg 180 <210> 3 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 3 Met Leu Thr Thr Asn Ser Gln Gly Val Glu Glu Glu Arg Pro Lys Met Pagina 3
    GBCYO86-NL-SEQUENCE LISTING ST25 1 5 10 15 Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Thr Thr Ser Arg
    Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala Tyr 40 45 Glu Leu Ser Val Leu Cys Asp Ala Gln Leu Ala Leu Ile Val Phe Ser 50 55 60 Ser Arg Gly Arg Val Tyr Glu Tyr Ser Asn His Asn Ile Arg Ser Ile 65 70 75 80 Ile Glu Arg Tyr Lys Lys Ser Thr Asp Gly Ser Asn Ser Ser Ser Val 85 90 95 Thr Glu Ile Asn Ala Gln Tyr Tyr Gln Gln Glu Ser Thr Lys Leu Arg 100 105 110 His Gln Ile Gln Val Met Gln Asn Ser Asn Lys Asn Leu Met Gly Glu 115 120 125 Cys Leu Glu Asn Leu Thr Val Lys Glu Met Lys Gln Val Glu Asn Arg 130 135 140 Leu Glu Arg Gly Ile Ser Arg Ile Arg Ser Lys Lys His Glu Leu Leu 145 150 155 160 Leu Ala Asp Ile Glu Phe Leu Gln Lys Arg Glu Thr Asp Leu Glu His 165 170 175 Glu Asn Ala Phe Leu Arg Ser Lys Ile Ala Glu Val Glu Asn Leu Gln 180 185 190 Gln Leu Asn Pro Asn Glu Asn Leu Ser Val Tyr His Gly Phe Val Pro 195 200 205 Arg Asp His Ile His Ile Pro Gln Asn Ser Cys Pro Phe Ala Ile Ser Pagina 4
    GBCYO86-NL-SEQUENCE LISTING ST25 210 215 220 Thr Asp Lys Lys Phe Leu His 225 230 <210> 4 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 4 Met Leu Thr Thr Asn Ser Gln Gly Val Glu Glu Glu Arg Pro Lys Met 1 5 10 15 Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn Thr Thr Ser Arg
    Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala Tyr 40 45 Glu Leu Ser Val Leu Cys Asp Ala Gln Leu Ala Leu Ile Val Phe Ser 50 55 60 Ser Arg Gly Arg Val Tyr Glu Tyr Ser Asn His Asn Ile Arg Ser Ile 65 70 75 80 Ile Glu Arg Tyr Lys Lys Ser Thr Asp Gly Ser Asn Ser Ser Ser Val 85 90 95 Thr Glu Thr Asn Ala Gln Tyr Tyr Gln Gln Glu Ser Thr Lys Leu Arg 100 105 110 His Gln Ile Gln Val Met Gln Asn Ser Asn Lys Asn Leu Met Gly Glu 115 120 125 Cys Leu Glu Asn Leu Thr Val Lys Glu Met Lys Gln Val Glu Asn Arg 130 135 140 Pagina 5
    GBCYO86-NL-SEQUENCE LISTING ST25 Leu Glu Arg Gly Ile Ser Arg Ile Arg Ser Lys Lys His Glu Leu Leu 145 150 155 160 Leu Ala Asp Ile Glu Phe Leu Gln Lys Arg Glu Thr Asp Leu Glu His 165 170 175 Glu Asn Ala Phe Leu Arg Ser Lys Ile Ala Glu Val Glu Asn Leu Gln 180 185 190 Gln Leu Asn Pro Asn Glu Asn Leu Ser Val Tyr His Gly Phe Val Pro 195 200 205 Arg Asp His Ile His Ile Pro Gln Asn Ser Cys Pro Phe Ala Ile Ser 210 215 220 Thr Asp Lys Lys Phe Leu His 225 230 <210> 5 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 5 Met Glu Phe Ser Ser Gln Ile Thr Arg Glu Glu Gly Ser Pro Ser Ser 1 5 10 15 Gln Arg Lys Leu Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn
    Thr Thr Asn Arg Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu 40 45 Lys Lys Ala Tyr Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Leu 50 55 60 Ile Val Phe Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ser Asn His Ser Pagina 6
    GBCYO86-NL-SEQUENCE LISTING ST25 65 70 75 80 Cys Ser Val Lys Gly Thr Ile Glu Lys Tyr Lys Lys Thr Cys Ser Asp 85 90 95 Ser Ser Ala Thr Ser Ala Ala Glu Ala Asn Ala Gln Tyr Tyr Gln Gln 100 105 110 Glu Ser Ala Lys Leu Arg Asn Gln Ile Arg Thr Met Thr Glu Asn Asn 115 120 125 Arg Ser Leu Ser Arg His Leu Met Gly Glu Gly Leu Ser Ala Leu Asn 130 135 140 Met Lys Glu Leu Lys Asn Leu Glu Gly Lys Leu Glu Arg Gly Ile Ser 145 150 155 160 Arg Ile Arg Ser Lys Lys Asn Glu Leu Leu Phe Ala Glu Ile Glu Phe 165 170 175 Met Gln Lys Arg Glu Val Asp Leu His Asn Asn Asn Gln Leu Leu Arg 180 185 190 Ala Lys Ile Ala Glu Asn Glu Arg Ala Gln Gln Ser Met Ser Leu Met 195 200 205 Pro Gly Gly Gly Asn Asp Tyr Glu Leu Ala Pro Pro Pro Gln Ser Phe 210 215 220 Asp Ser Arg Thr Tyr Phe Gln Val Asn Ala Leu Gln Pro Asn Asp Gln 225 230 235 240 Tyr Ser Arg Gln Asp Gln Thr Pro Leu Gln Leu Val 245 250 <210> 6 <211> 250 <212> PRT <213> Artificial Sequence Pagina 7
    GBCYO86-NL-SEQUENCE LISTING ST25 <220> <223> The sequence is synthesized. <400> 6 Met Glu Phe Ser Ser Gln Ile Thr Arg Glu Glu Gly Ser Pro Ser Ser 1 5 10 15 Gln Arg Lys Leu Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn
    Thr Thr Asn Arg Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu 40 45 Lys Lys Ala Tyr Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Leu 50 55 60 Ile Val Phe Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ser Asn His Ser 65 70 75 80 Val Lys Gly Thr Ile Glu Lys Tyr Lys Lys Thr Cys Ser Asp Ser Ser 85 90 95 Ala Thr Ser Ala Ala Glu Ala Asn Ala Gln Tyr Tyr Gln Gln Glu Ser 100 105 110 Ala Lys Leu Arg Asn Gln Ile Arg Thr Met Thr Glu Asn Asn Arg Ser 115 120 125 Leu Ser Arg His Leu Met Gly Glu Gly Leu Ser Ala Leu Asn Met Lys 130 135 140 Glu Leu Lys Asp Leu Glu Gly Lys Leu Glu Arg Gly Ile Ser Arg Ile 145 150 155 160 Arg Ser Lys Lys Asn Glu Leu Leu Phe Ala Glu Ile Glu Phe Met Gln 165 170 175 Lys Arg Asp Phe Asp Leu His Asn Asn Asn Gln Leu Leu Arg Ala Lys 180 185 190 Pagina 8
    GBCYO86-NL-SEQUENCE LISTING ST25 Ile Ala Glu Asn Glu Arg Ala Gln Gln Ser Met Ser Leu Met Pro Gly 195 200 205 Gly Gly Asn Asp Tyr Glu Leu Ala Pro Pro Pro Gln Ser Phe Asp Ser 210 215 220 Arg Thr Tyr Phe Gln Val Asn Ala Leu Gln Pro Asn Asp Gln Tyr Ser 225 230 235 240 Arg Gln Asp Gln Thr Pro Leu Gln Leu Val 245 250 <210> 7 <211> 154 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 7 Met Glu Phe Ser Ser Gln Ile Thr Arg Glu Glu Gly Ser Pro Ser Ser 1 5 10 15 Gln Arg Lys Leu Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn
    Thr Thr Asn Arg Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu 40 45 Lys Lys Ala Tyr Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Leu 50 55 60 Ile Val Phe Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ser Asn His Ser 65 70 75 80 Cys Ser Val Lys Gly Thr Ile Glu Lys Tyr Lys Lys Thr Cys Ser Asp 85 90 95 Ser Ser Ala Thr Ser Ala Ala Glu Ala Asn Ala Gln Tyr Tyr Gln Gln Pagina 9
    GBCYO86-NL-SEQUENCE LISTING ST25 100 105 110 Glu Ser Ala Lys Leu Arg Asn Gln Ile Arg Thr Met Thr Glu Asn Asn 115 120 125 Arg Ser Leu Ser Arg His Leu Met Gly Glu Gly Pro Ser Ala Leu Asn 130 135 140 Met Lys Glu Leu Lys Asn Leu Glu Gly Lys 145 150 <210> 8 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> The sequence is synthesized. <400> 8 Met Glu Phe Ser Ser Gln Ile Thr Arg Glu Glu Gly Ser Pro Ser Ser 1 5 10 15 Gln Arg Lys Leu Gly Arg Gly Lys Ile Glu Ile Lys Arg Ile Glu Asn
    Thr Thr Asn Arg Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Leu 40 45 Lys Lys Ala Tyr Glu Leu Ser Val Leu Cys Asp Ala Glu Val Ala Leu 50 55 60 Ile Val Phe Ser Ser Arg Gly Arg Leu Tyr Glu Tyr Ser Asn His Ser 65 70 75 80 Val Lys Gly Thr Ile Glu Lys Tyr Lys Lys Thr Cys Ser Asp Ser Ser 85 90 95 Ala Thr Ser Ala Ala Glu Ala Asn Ala Gln Tyr Tyr Gln Gln Glu Ser 100 105 110 Pagina 10
    GBCYO86-NL-SEQUENCE LISTING ST25 Ala Lys Leu Arg Asn Gln Ile Arg Thr Met Thr Glu Asn Asn Arg His 115 120 125 Leu Met Gly Glu Gly Leu Ser Ala Leu Asn Met Lys Glu Leu Lys Asn 130 135 140 Leu Glu Gly Lys Leu Glu Arg Gly Ile Ser Arg Ile Arg Ser Lys Lys 145 150 155 160 Asn Glu Leu Leu Phe Ala Glu Ile Glu Phe Met Gln Lys Arg Glu Val 165 170 175 Asp Leu His Asn Asn Asn Gln Leu Leu Arg Ala Lys Ile Ala Glu Asn 180 185 190 Glu Arg Ala Gln Gln Ser Met Ser Leu Met Pro Gly Gly Gly Asn Asp 195 200 205 Tyr Glu Leu Ala Pro Pro Pro Gln Ser Phe Asp Ser Arg Thr Tyr Phe 210 215 220 Gln Val Asn Ala Leu Gln Pro Asn Asp Gln Tyr Ser Arg Gln Asp Gln 225 230 235 240 Thr Pro Leu Gln Leu 245 Pagina 11
    权利要求:
    Claims (8)
    [1]
    An efficient method for screening a double-flowered mutant in Gypsophila paniculata, the method comprising: dividing seeds of G. paniculata into groups, and performing ethyl methanesulfonate (EMS) mutagenesis on each group of seeds for different time periods to obtain EMS mutagenized seeds of G. paniculata; sowing the EMS mutagenized seeds of G. paniculata on a substrate of a mixture of peat and perlite for germination and growing into seedlings of G. paniculata; growing the seedlings of G. paniculata into mutant plants; and performing statistical data analysis of the mutant plants, and isolating the double flowered mutant of G. paniculata.
    [2]
    An efficient method for screening a double-flowered mutant in Gypsophila paniculata according to claim 1, wherein the EMS concentration is 150 mM.
    [3]
    The efficient method of screening a double-flowered mutant in Gypsophila paniculata according to claim 1, wherein the number of the seeds of G. paniculata in each group is 1,000.
    [4]
    An efficient method for screening a double flowered mutant in Gvpsophila paniculata according to claim 3, wherein the weight of the seeds of G. paniculata in each group is 0.641 + 0.02 g.
    [5]
    An efficient method for screening a double-flowered mutant in Gypsophila paniculata according to claim 1, wherein the EMS mutagenesis is performed by: placing dry seeds of G. paniculata in a centrifuge tube, and pretreating overnight with a 100 mM potassium phosphate buffer solution at 4°C; removing the potassium phosphate buffer solution, adding a 10 mL of
    15-100 mM potassium phosphate buffer solution to the centrifuge tube, then adding EMS to the potassium phosphate buffer solution to a final concentration of 150 mM/L; and incubating the seeds of G. paniculata in the potassium phosphate buffer solution for 6 hours, then washing the seeds of G. paniculata 10 times in 30 minutes sequentially with 100 mM sodium thiosulfate and distilled water, and then drying the seeds of G. paniculata.
    [6]
    An efficient method for screening a double flowered mutant in Gypsophila paniculata according to claim 1, wherein the different time periods are 0 hours, 4 hours, 6 hours, 8 hours and 12 hours.
    [7]
    An efficient method for screening a double-flowered mutant in Gypsophila paniculata according to claim 5, wherein the EMS mutagenesis lasts for 6 hours.
    [8]
    The efficient method of screening a double flowered mutant in Gypsophila paniculata according to claim 1, wherein the EMS mutagenized seeds of G. paniculata are sown within one week.
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    同族专利:
    公开号 | 公开日
    CN113142048A|2021-07-23|
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    CN202110361816.7A|CN113142048A|2021-04-02|2021-04-02|Effective method for screening double-petal mutants of cone stone flower|
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