Creat A Brassica Napus Mutant Library Of MIKC~C Type MADS-box Gene By Using CRISPR/Cas9 Technique

Brassica napus is one of the most important oil crops in China,and the development of flower organs is closely related to yield.MADS-box genes that are very important for plant growth and development,and its regulatory effect runs through the process of plant,from vegetative growth to reproductive growth,until aging and death.MIKC~C type gene is a subfamily of MADS-box genes.Most genes in ABCDE model of flower organ development belong to MIKC~C subfamily and are very important for flower organ development.At present,the function of MIKC~C subfamily genes in Arabidopsis thaliana is basically clear,but there are few studies on the gene function of MIKC~C subfamily genes in Brassica napus.In this study,a mutant library of MIKC~C subfamily genes was established in Brassica napus and the functions of some MIKC~C subfamily genes were preliminarily studied.The main results obtained are as follows:We analyzed the nucleotide sequence of MIKC~C subfamily genes in Brassica napus,and designed 60 multi-targeted sg RNA for 74 MIKC~C subfamily genes.30 double sg RNA gene editing vectors were constructed.Through the genetic transformation of Brassica napus Westar,1428 transformed plants were obtained by mixing pool with 3-5 or 30agrobacterium strains,and the number of Cas9 positive plant was 961(67.30%).Specific primers were used to amplify sg RNA segments and high-throughput sequencing was performed.The detection rate of sg RNA was 25%,20 kinds of sg RNAs which from 20different vectors were detected in 355 transgenic plants,and 55 MIKC~C subfamily genes were targeted.Data comparison showed that the Cas9 positive rate,sg RNA detection rate and single plant multiple vector transfer rate of 30 strains were higher than those of 3-5strains in mixed pool.Phenotypic observation of the transformed population showed that 310 plants had phenotypic mutations,and 302 plants(97.42%)had phenotypic mutations related to the development of flower organs,mainly showing the variation in number,morphology or function of flower organs.These results indicated that the gene functions of 55 MIKC~Csubfamily genes covered by the transformed population were mostly related to flower organ development.In this study,genotypes of three phenotypes were identified.Phenotypic one:The phenotype of agl1(shp1)mutants(15 plants)was early termination of floral organ differentiation.Combined with gene expression,we speculated that AGL1 genes involved in the regulation of floral organ differentiation and gametes or embryos development,is the indispensable important genes reproduce Brassica napus were used,play a flower organ development ABCDE model class D function of gene;Phenotypic 2:The phenotype of agl2(sep1)mutants(1 plants)was five buds in one sepal("lowers inlay flower").Combined with gene expression,we speculated that AGL2 genes involved in pistil and petals of determining identity and position,and determine the pistil fertility,play a flower organ development ABCDE model class E function of gene;Phenotype 3:The phenotype of agl8(ful)mutants(3 plants)siliques became shorter and dehiscent prematurely.Phenotype separation of plants in T1 generation,and silique length of plants gradually shortened with the increase of the number of AGL8 homologous gene edited.Combined with the expression level of AGL8 gene,it is speculated that AGL8 has anti-pod splitting function,and several homologous genes of AGL8 gene have small cumulative effect.