| During the growth period of soybean,the reproductive growth is most critical for the formation of seed yield and quality.Flower is one of the important organs in reproductive growth,which has a direct impact on the final yield.Therefore,the research of the regulation network of reproductive growth has laid a foundation for soybean breeding.MYB transcription factor is one of the largest members of the transcription factor family,involved in plant development,primary and secondary metabolism,cell fate and identity and responses to biotic and abiotic stresses,which has an important regulatory effect on floral organs.Soybean seeds are rich in storage proteins,which are mainly divided into 11S and 7S globulins.However,the amino acid composition is unbalanced in soybean seeds,and the low content of sulfur amino acids limits the nutritional value of soybean.Increasing the ratio of 11S/7S by molecular breeding can enhance the content of sulfur amino acid and improve the quality of soybean without changing the total protein content.The CRISPR/Cas9 is the third generation of gene editing that can be used to accurately knock out genes.This study used the CRISPR/Cas9 multiply-target system modified by our laboratory to construct a vector pSC-M-MYB,targeting the MYB transcription factor gene GmMYB181 and its homologous gene GmMYB182,which were dominant in floral organ expression,and another vector pSC-M-7S,targeting the β-conglycinin a and α’ subunit genes Gm7Sa and Gm7Sα’.The targeting efficiency of the multiply-target system was preliminarily determined by the transformation system of soybean hairy roots.The site directed mutagenesis plants were created by the genetic transformation system of soybean cotyledonary node,which were used to study the biological functions of GmMYB181,GmMYB182,Gm7Sα and Gm7Sα’ in soybean.The main results of the study were as followed:1.Using the constructed pSC-M-MYB and pSC-M-7S multiply-target vector to transform Agrobacterium rhizogenes K599 to induce soybean hairy roots,and to measure the frequency of hairy root mutation:the single gene mutation rate caused by pSC-M-MYB was 18.75%and 20.83%respectively,and the simultaneous mutation rate of GmMYB181 and GmMYB182 was 18.75%;the single gene mutation rate caused by pSC-M-7S was 22.92%and 18.75%respectively,and the simultaneous mutation rate of Gm7Sα and Gm7Sα’ was 18.75%.These results showed that the vector can be used to simultaneously knock out multiple target genes with a targeting efficiency of about 20%,and it was speculated that multiple target sites on the vector are simultaneously targeted or simultaneously off-target.2.The pSC-M-MYB and pSC-M-7S multiply-target vector were transformed into Agrobacterium tumefaciens EHA105 to infect the soybean cotyledonary node,and detected 11 and 8 transgenic plants,respectively.The mutation efficiency of the transgenic plants was found to be 72.7%and 75.0%,which was higher than the hairy root system.3.The relative expression of GmMYB181 and GmMYB182 in the T1 and T2 generation of site directed mutagenesis lines was significantly decreased,indicating that the target genes were successfully knocked out.Directed mutagenesis of GmMYB181 and GmYB182 resulted in increased branching and delayed flowering,affecting soybean plant type and floral development.4.The relative expression of Gm7Sα and Gm7Sα’ in the T1 and T2 generation of site directed mutagenesis lines was significantly decreased,indicating that the target genes was successfully knocked out.The content of 7S globulin in homozygous mutagenesis plants was significantly lower than that of the control,and the content of 11S globulin was significantly higher than that of the control,indicating that the knockout of Gm7Sa and Gm7Sα’ increased the ratio of 11S/7S.Further detection showed that the proportion of Met and Cys in homozygous mutagenesis plants was higher than that of the control,and the content of sulfur amino acids was increased. |
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