CN116042642A - Lotus bean No. 12 GmDFB1 gene related to soybean flower development, mutant and application thereof - Google Patents
Lotus bean No. 12 GmDFB1 gene related to soybean flower development, mutant and application thereof Download PDFInfo
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Abstract
The invention discloses a lotus bean No. 12 GmDFB1 gene related to soybean flower development, and the nucleotide sequence of the gene is shown as SEQ ID No. 1. The invention also discloses two mutant genes of the GmDFB1 genes, and the nucleotide sequences of the mutant genes are shown as SEQ ID NO.2 and SEQ ID NO. 5. The invention also discloses application of the GmDFB1 gene in regulating and controlling soybean flower development mechanism research and improving soybean flower type to realize soybean heterosis utilization, and application of the mutant gene of the GmDFB1 gene in cultivating improved soybean varieties. The invention provides more theoretical basis for exploring genes related to soybean flower development, can provide a new thought for research of soybean flower development mechanism, and provides a new means for improving soybean flower type so as to rapidly realize soybean heterosis utilization or cultivating improved soybean varieties.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a lotus bean No. 12 GmDFB1 gene related to soybean flower development, a mutant thereof and application thereof.
Background
Soybean (Glycine max (l.) Merr) is a world important grain and oil crop, and is also the most important source of vegetable proteins for humans. The soybeans cultivated and eaten in China have thousands of years history, but are limited by factors such as planting area, unit yield level and the like, and the total yield of the soybeans is low at present. According to the latest statistics data, the Chinese soybean yield only accounts for about 5% of the total world yield, and more than 80% of Chinese soybeans are imported in a relying way to meet domestic market demands, so that the improvement of the soybean yield has important significance for meeting internal demands of China and stabilizing economy. However, china has limited cultivated land area, and the cost of increasing soybean yield and relieving import pressure is too high and the possibility is low by enlarging the planting area, so that the effort of increasing the soybean yield per unit is also needed. The research on soybean heterosis utilization is developed only in the 80 s of the 20 th century, and the first soybean hybrid 'hybrid soybean No. 1' in the world is successfully cultivated through years of exploration and effort, sun in 2002 and the like. Compared with the control variety, the variety can increase the yield by more than 20%, and the breeding of the variety marks the breakthrough progress of soybean heterosis utilization in China. At present, a large number of hybrid soybean varieties with excellent properties, which can be applied to commercial production, are cultivated in China.
The reproduction process is the most important stage in the life history of higher plants, and is also the key period for obtaining products and improving yield in agricultural production, so that the development of flower organs closely related to plant reproduction is always one of the important research points of plant scientists. The characteristic that the gene related to the development of flowers is specifically expressed in different flower organs is utilized to apply the gene to genetic operations of different plants so as to modify the gardening characters of the plants, such as changing the flower type, the flower color, the flower period and the like of ornamental flowers, and the gene can also shorten the generation, reduce the cultivation time and improve the yield for economic crops. Therefore, the research on the gene related to the flower development plays an important role in researching the formation and development of flower organs, and has quite great theoretical significance and practical application prospect in agricultural production.
The soybean flowers are more, the flower device is smaller, pollen is sticky, when flowers are open, petals and keel petals tightly cover stamens and stigmas, flower structural characteristics such as anthers and stigmas are not exposed, so that soybean cross pollination is very difficult, soybean heterosis is difficult to use, if the flower organ structure of the soybean can be directionally changed, the soybean cross pollination is more favorable for attracting pollinating insects or for hand wind medium pollination, the cost of sterile line propagation or cross seed production is greatly reduced, and the soybean cross seed is favorable for large-area production. Feng et al realized petal modification in the lotus hundred roots (lotus japonica) of leguminous species by genetic manipulation of LjCYC2, and it is expected that with identification and cloning of more regulated floral development genes, it is entirely possible to directionally modify floral organ morphology in crops such as soybean. For such a strictly self-pollinated crop as soybean, genetic manipulation-based molecular design is performed or a change in the pollination pattern thereof is allowed.
At present, although some reports about soybean sterile mutants exist, researches about the development of flowers thereof are relatively lagging. Therefore, the soybean flower development mutant is isolated and positioned, and has important significance for soybean variety improvement. The applicant obtains two allelic mutants of flower dysplasia through EMS induction, locates that a target gene is a lotus bean No. 12 GmDFB1 gene through map cloning and BSA sequencing technology, searches to find that an arabidopsis thaliana homologous gene AtRST1 (AT 3G 27670) of the gene is reported to be related to lipid synthesis, but regarding a molecular mechanism of regulating soybean flower development of the lotus bean No. 12 GmDFB1 gene, and utilizes the gene to improve soybean flower types, so that the application in soybean heterosis utilization is not reported yet.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a lotus bean No. 12 GmDFB1 gene related to soybean flower development, a mutant and application thereof.
The lotus bean No. 12 GmDFB1 gene related to soybean flower development is characterized in that: the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
The amino acid coded by the lotus bean No. 12 GmDFB1 gene related to the soybean flower development is characterized in that: the amino acid sequence is shown as SEQ ID No. 3.
The invention provides a mutant gene of a lotus bean No. 12 GmDFB1 gene related to soybean flower development, which is characterized in that: the nucleotide sequence of the gene is shown as SEQ ID NO. 2; the mutation shows that the 3886 base of the ninth exon of the nucleotide sequence of the GmDFB1 gene shown in SEQ ID NO.1 is changed from wild-type G to A.
The amino acid encoded by the allelic mutant gene of the lotus bean No. 12 GmDFB1 gene related to the soybean flower development is characterized in that: the amino acid sequence is shown as SEQ ID No.4, and is truncated as shown as SEQ ID No. 3; the plant character shows sterility and no petals, and the mutant is named Gmdfb1-1.
The invention also provides a mutant gene of the lotus bean No. 12 GmDFB1 gene related to the soybean flower development, which is characterized in that: the nucleotide sequence of the gene is shown as SEQ ID NO. 5; the mutation shows that the 4804 base of the ninth exon of the nucleotide sequence of the GmDFB1 gene shown in SEQ ID NO.1 is changed from wild type G to A.
The amino acid encoded by the allelic mutant gene of the lotus bean No. 12 GmDFB1 gene related to the soybean flower development is characterized in that: the amino acid sequence is shown as SEQ ID No.6, and is truncated as shown as SEQ ID No. 3; the plant character shows sterility and no petals, and the mutant is named Gmdfb1-2.
F produced by the applicant by crossing Gmdfb1-1 (homoline fertile plants) with williams82 2 The generation performs map cloning and BSA sequencing, the GmDFB1 gene is obtained, and the accuracy of the gene is determined through allelic system verification. Experiments prove that: the GmDFB1 gene has the function of regulating the development of soybean flowers, which is a new function of the GmDFB1 gene discovered for the first time. Meanwhile, compared with the wild lotus bean No. 12, the mutant and allelic mutant of the GmDFB1 gene have the defects of no petals, malformation of male and female stamen, sterility and the like, and the phenomenon has important significance for researching the plant flower development mechanism.
The application of the lotus bean No. 12 GmDFB1 gene related to flower development in research of plant flower development mechanism, wherein the plant is preferably soybean.
The invention relates to application of lotus bean No. 12 GmDFB1 gene related to flower development in improving soybean flower type to realize soybean heterosis utilization.
The invention relates to application of a mutant gene of a lotus bean No. 12 GmDFB1 gene related to soybean flower development in cultivation of improved soybean varieties.
Compared with the prior art, the invention has the beneficial effects that: the stable genetic mutants Gmdfb1-1 or Gmdfb1-2 are obtained through EMS mutagenesis, and the mutation of the GmdFB1 gene of the lotus bean No. 12 related to flower development is found to cause the abnormal development of the lotus bean No. 12 flowers. The invention provides more theoretical basis for exploring genes related to soybean flower development, can provide a new thought for research of soybean flower development mechanism, and provides a new means for improving soybean flower type so as to rapidly realize soybean heterosis utilization or cultivating improved soybean varieties.
Drawings
Fig. 1: flower morphology and flower anatomy photographs of wild type (left) and Gmdfb1-1 mutant (right).
Wherein: A. lotus bean 12 flower morphology structure (left), gmdfb1-1 flower morphology structure (right); B. lotus bean 12 flower anatomy; gmdfb1-1 flower anatomy.
Fig. 2: the Gmdfb1-1 mutant map clones the coarse localization interval.
Fig. 3: BSA sequencing ED correlated Manhattan plots.
Fig. 4: schematic representation of the mutation site of Gmdfb1-1 mutant.
Fig. 5: flower morphology and flower anatomy photographs of wild type (left) and Gmdfb1-2 allelic lines (right).
Wherein: A. lotus bean 12 flower morphology structure (left), gmdfb1-2 flower morphology structure (right); B. lotus bean 12 flower anatomy; gmdfb1-2 flower anatomy.
Fig. 6: schematic representation of the mutation site of the Gmdfb1-2 allele.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are merely for explaining the present invention, and are not limiting in any way, and any simple modification, equivalent variation and modification of the embodiments according to the technical principles of the present invention are within the scope of the technical solutions of the present invention.
The present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
In the following examples, materials, reagents, strains, plasmids, enzymes, kits and the like were used, and were obtained commercially unless otherwise specified. Among them, EMS mutagens (ethyl methylsulfonate, ethyl methansulfonate), manufactured by Sigma Co., USA.
Example 1: screening of soybean flower dysplasia mutant Gmdfb1
The wild type seed No. 12 of the lotus seed is subjected to mutagenesis treatment by adopting an EMS mutagen with the concentration of 0.6 percent. The specific method comprises the following steps: selecting about 5000 wild type lotus seeds with consistent and full size and intact seed coats, soaking the wild type lotus seeds for about 4 hours at room temperature, pouring out water, soaking the wild type lotus seeds for about 8 hours by using 0.6% EMS mutagen, adding 5% sodium thiosulfate as a terminator and a antidote, flushing with running water for about 1 hour, airing the wild type lotus seeds for sowing, and harvesting the mature wild type lotus seeds by single plants to obtain M 1 Generation material 1231 individual plants. Next year, sow M 1 Seed generation, wherein 30 seeds are sown in each single plant, namely M 2 Substituted material, pair M 2 The generation material was subjected to field phenotype observation to determine that one of the soybean flower dysplasia mutant plants was named Gmdfb1-1 (see FIG. 1), which exhibited a petal-free, stamen-free, and eventually sterile phenotype. FIG. 1 is a flower morphology and flower anatomy of wild type and Gmdfb1-1 mutants. The left side of fig. 1A is a lotus bean 12 flower morphology structure, the right side of fig. 1A is a Gmdfb1-1 flower morphology structure, fig. 1B is a flower anatomy structure of H12, and fig. 1C is a flower anatomy structure of Gmdfb1-1.
Example 2: genetic analysis of Soybean sterile mutant Gmdfb1-1
Hybridization is carried out by taking a fertile plant in soybean flower dysplasia mutant Gmdfb1-1 plant homoline as a female parent and a sequenced variety Williams82 as a male parent to obtain F 1 Hybrid seeds of generation F 1 Obtaining F by substitution selfing 2 Colony, pair F 2 The generation population was phenotyped, at 161 strain F 2 Separating 37 flowers dysplasia mutants from the generation group, and comparing F 2 The generation population is subjected to a χ2 test, and the calculated χ2= 0.1343 is df=1, the significant level alpha=0.05, and the χ2= 0.1343 is obtained by looking up a table<3.84, the flower dysplasia mutant Gmdfb1-1 is proved to accord with the Mendelian genetic segregation ratio of 3:1, thus determining that the mutation trait is controlled by a single recessive nuclear gene.
Example 3: determination and cloning of soybean regulatory flower development gene
Localization was performed using the method of map-based cloning.
1) Plant DNA extraction
About 0.8g of soybean leaf material is taken and added into a 1.5mL centrifuge tube, magnetic beads and a buckle cover are added, the mixture is rapidly placed into liquid nitrogen for precooling, and a sample is ground into powder by a sample grinding machine; after the sample is crushed into powder, 1mL of 2% CTAB extracting solution preheated at 65 ℃ is rapidly added, and the mixture is gently and reversely mixed according to the 'infinity' shape, and water bath is carried out for 0.5-2h at 65 ℃ and reversely mixed every 5 min; centrifuge at 12000rpm for 10min, take 600. Mu.L of supernatant in a new 1.5mL centrifuge tube. Adding equal volume of phenol/chloroform/isoamyl alcohol, gently reversing and mixing according to the + -shaped, and centrifuging at 12000rpm for 10min; transferring the supernatant to a new 1.5mL centrifuge tube, adding equal volume chloroform/isoamyl alcohol, gently reversing and mixing according to the 'infinity' shape, and centrifuging at 12000rpm for 10min; taking 400 mu L of supernatant to a new clean centrifuge tube, adding 400 mu L of pre-cooled isopropanol with the temperature of-20 DEG, gently reversing and mixing uniformly according to the 'infinity' shape, and standing for more than 30 minutes at the temperature of-20 ℃; centrifuging at 12000rpm at 4deg.C for 10min, discarding supernatant, washing with 75% ethanol once, and blow-drying in an ultra-clean bench; 600. Mu.L of RNase A-containing double distilled water is dissolved and precipitated, digested for 15min at 37 ℃, and sequentially extracted once with equal volumes of phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol; taking 400 mu L of supernatant to a new 1.5mL centrifuge tube, adding 1/10 times of 3M NaAc (pH 5.2) and 2 times of precooled absolute ethyl alcohol, and standing at-20 ℃ for 30min; centrifuging at 12000rpm at 4deg.C for 10min, discarding supernatant, washing with 75% ethanol twice, and blow-drying in an ultra-clean bench; 40 mu L of double distilled water is dissolved and precipitated and is stored in a refrigerator at the temperature of minus 20 ℃ for standby.
Respectively extracting the fields according to the stepsGrown wild type lotus bean No. 12, BC1F 2 Wild type plants isolated from the generation, flower dysplasia mutant soybean leaf DNA.
2) PCR amplification
PCR amplification reaction (20. Mu.L system) was performed with ordinary easy Taq enzyme:
the amplification conditions were as follows:
2) Initial localization of soybean regulatory flower development genes
In the period of growing soybean to V6 (full development of 6 th three-leaf complex leaf), wild lotus seed No. 12, F 1 Hybrid plants of the generation, sequencing varieties Williams82 and F 2 Extracting DNA from Gmdfb1-1 plant separated from the generation group, wherein the lotus bean genotype 12 is a band type, the Williams82 genotype is b band type, and F 1 The genotype of the hybrid is h band type, 128 Indel molecular markers with polymorphism uniformly distributed on lotus bean No. 12 20 chromosomes are used as PCR amplification primers, the PCR amplification primers are used for amplifying, and then acrylamide gel is used for electrophoresis separation of PCR products, so that the PCR products are determined to belong to a band type b band type or h band type.
According to the formula recombination rate= (h+2b)/(a+b+h) ×2, searching for a chromosome band with the lowest recombination rate, and carrying out statistical analysis on the amplified product band type, the result shows that the number of exchange individuals corresponding to the 6 pairs of molecular markers on chromosome 19 is gradually decreased from right to left (see fig. 2). The number of single plants was minimal at the Gm19002 marker and the Gm19002 marker was tightly linked to the phenotype of Gmdfb1-1, thus initially mapping the mutation site to chromosome 19. Then, the development of a new Indel marker was continued at the right end of the Gm19002 marker, and the minimum number of exchange lines was detected at the Gm190012 marker, indicating that the mutation candidate gene was located between the Gm 19002-Gm 190012 markers, with a physical distance between intervals of about 9.82Mb.
3) Fine localization of soybean regulatory flower development genes
On the basis of initial positioning, the segregation population is further expanded, new Indel molecular markers are continuously developed, and the mutation sites are further positioned in a 124kb interval between dCAPS-Gm19-23 to Gm19-SSR7 markers on chromosome 19 by using 588 mutation individuals obtained through segregation.
By looking up the gene sequences contained within the candidate interval provided on the https:// phytozome. Jgi. Doe. Gov website, it was found that this interval contained 10 candidate genes. At the same time utilize F 2 BSA sequencing is carried out on the generation population, ED correlation analysis is adopted on the sequencing result, a remarkable peak is found at chromosome 19 at 388033 (see figure 3), the mutation site is cloned, the site is found to be located in a gene9 sequence of a map-based cloning region, and a base at 3886 position downstream of ATG is mutated from G to A, so that protein coding is terminated in advance, and therefore gene9 (GmDFB 1) is presumed to be a candidate gene for regulating and controlling soybean flower development.
Sequencing and determining: the nucleotide sequence of the lotus bean No. 12 GmDFB1 gene related to the soybean flower development is shown as SEQ ID No. 1.
The amino acid coded by the lotus bean No. 12 GmDFB1 gene related to the soybean flower development has an amino acid sequence shown in SEQ ID No. 3.
The nucleotide sequence of the mutant gene of the lotus bean No. 12 GmDFB1 gene related to the soybean flower development is shown as SEQ ID No. 2; the mutation shows that the 3886 base of the ninth exon of the nucleotide sequence of the GmDFB1 gene shown in SEQ ID NO.1 is changed from wild-type G to A.
Example 4: acquisition and Gene determination of allelic mutant Gmdfb1
The next year in the laboratory, seeds of wild type lotus seed No. 12 were mutagenized with EMS mutagen at a concentration of 0.6%. The specific method comprises the following steps: selecting about 5000 wild type lotus seeds with consistent and full size and intact seed coats, soaking for about 4 hours at room temperature, pouring out water, soaking for about 8 hours with 0.6% EMS mutagen, adding 5% sodium thiosulfate as terminator and antidote, washing with running water for about 1 hour, and air dryingSowing, separating single plant after maturation, collecting seeds to obtain M 1 Generation material 1231 individual plants. Next year, sow M 1 Seed generation, wherein 30 seeds are sown in each single plant, namely M 2 Substituted material, pair M 2 The generation materials are subjected to field phenotype observation, and the generation materials are in M 2 A mutant completely consistent with the phenotype of Gmdfb1-1 is found in the generation mutant (see figure 5), and the soybean flower dysplasia mutant plant is named as Gmdfb1-2 (see figure 5), and the mutant shows the phenotype of no petals, malformation of male and female stamen and final sterility. FIG. 5 is flower morphology and flower anatomy of wild type and Gmdfb1-2 allelic lines. The left side of fig. 5A shows the lotus bean 12 flower morphology structure, the right side of fig. 5A shows the Gmdfb1-2 flower morphology structure, fig. 5B shows the flower anatomy structure of H12, and fig. 5C shows the flower anatomy structure of Gmdfb1-2.
Through sectional cloning and sequencing of the GmDFB1 gene of the plant, the base at 4804 downstream of ATG of the GmDFB1 gene of the plant is mutated from G to A, and the mutation of the site leads to early termination of protein coding, so that the GmDFB1 can be determined to be an important gene for regulating soybean flower development.
The nucleotide sequence of the mutant gene of the lotus bean No. 12 GmDFB1 gene related to the soybean flower development is shown as SEQ ID No. 5; the mutation shows that the 4804 base of the ninth exon of the nucleotide sequence of the GmDFB1 gene shown in SEQ ID NO.1 is changed from wild type G to A.
Hybridizing a fertile heterozygous single plant of the Gmdfb1-1 hybrid female parent with a fertile heterozygous single plant of the Gmdfb1-2 hybrid female parent, F 1 The phenotype of the mutant in the generation is completely consistent with the phenotype of Gmdfb1-1 and Gmdfb1-2, and the mutant is not provided with petals, and the male and female stamen are abnormal in development, so that sterility is finally caused, and the accuracy of the lotus bean No. 12 Gmdfb1 gene is verified.
Claims (10)
1. A lotus bean number 12 GmDFB1 gene related to soybean flower development, characterized in that: the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
2. The amino acid encoded by the soybean flower development-related GmDFB1 gene of lotus bean No. 12 of claim 1, wherein: the amino acid sequence is shown as SEQ ID No. 3.
3. A mutant gene of lotus bean No. 12 GmDFB1 gene related to soybean flower development, which is characterized in that: the nucleotide sequence of the gene is shown as SEQ ID NO. 2; the mutation shows that the 3886 base of the ninth exon of the nucleotide sequence of the GmDFB1 gene shown in SEQ ID NO.1 is changed from wild-type G to A.
4. The amino acid encoded by an allelic mutation of the GmDFB1 gene of lotus bean No. 12 associated with soybean flower development according to claim 3, wherein: the amino acid sequence is shown as SEQ ID No.4, and is truncated as shown as SEQ ID No. 3; the plant character shows sterility and no petals, and the mutant is named Gmdfb1-1.
5. A mutant gene of lotus bean No. 12 GmDFB1 gene related to soybean flower development, which is characterized in that: the nucleotide sequence of the gene is shown as SEQ ID NO. 5; the mutation shows that the 4804 base of the ninth exon of the nucleotide sequence of the GmDFB1 gene shown in SEQ ID NO.1 is changed from wild type G to A.
6. The amino acid encoded by an allelic mutation of the GmDFB1 gene of lotus bean No. 12 associated with soybean flower development according to claim 5, wherein: the amino acid sequence is shown as SEQ ID No.6, and is truncated as shown as SEQ ID No. 3; the plant character shows sterility and no petals, and the mutant is named Gmdfb1-2.
7. The use of the lotus bean number 12 GmDFB1 gene related to flower development as claimed in claim 1 in research of regulating soybean flower development mechanism.
8. The use of the lotus bean number 12 GmDFB1 gene related to floral development of claim 1 for improving soybean floral type to realize soybean heterosis utilization.
9. Use of the mutant gene of GmDFB1 gene of lotus bean No. 12 related to soybean flower development as claimed in claim 3 for breeding improved soybean varieties.
10. The use of the mutant gene of the GmDFB1 gene of lotus bean No. 12 related to soybean flower development according to claim 5 for breeding improved soybean varieties.
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| CN114875038A (en) * | 2022-03-25 | 2022-08-09 | 山东大学 | GmILPA1 gene mutant causing soybean dwarfing and application thereof |
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