Mutant Creation Of The Bsr-k1 Gene In Cotton And Arabidopsis Thaliana

Long-term cotton production practice shows that mining resistance genes can effectively control cotton Verticillium wilt,mining disease resistance-related genes and analyzing their disease resistance mechanisms,which are of great significance to molecular breeding of cotton disease resistance.In the early stage,it was preliminarily proved that the cotton GhBsr-kl gene negatively regulated the broad-spectrum disease resistance of cotton by VIGS technology.In order to finally clarify the disease resistance function of Bsr-kl gene in plants,further in-depth research is needed on the basis of stable Bsr-kl gene mutant materials.Therefore,this study used CRISPR/Cas9 gene editing technology and cotton leaf shrink virus CLCrV-mediated VIGE technology to create Arabidopsis and cotton mutant materials,in order to clarify the disease resistance function of Bsr-kl gene and evaluate it in cotton disease resistance breeding Its application value provides key genetic material.The main results of this study are as follows:(1)GhBsr-kl and AtBsr-k1/AtSKI3 genes were cloned.Both GhBsr-kl and AtBsr-kl/AtSKI3 proteins have TPR domains and Bsr-kl proteins are highly conserved in different species;at the same time,bioinformatics showed that they have many similarities may have similar biological functions.Expression pattern analysis showed that both GhBsr-kl and AtBsr-kl/AtSK13 genes responded to cotton Verticillium wilt infection.(2)Based on the cotton GhBsr-kl gene,6 gene editing vectors were successfully constructed,of which 2 gene editing vectors achieved targeted editing of the cotton GhBsr-kl gene,and two effective sgRNAs were successfully screened.The carrying capacity of the CLCrV vector was explored,the results show that the CLCrV vector carrying the complete CRISPR/Cas9 gene editing system components is difficult to achieve efficient gene editing in cotton.(3)Using traditional plant tissue culture techniques,the pRGEB32-GhU6.7 gene editing vector was transformed into cotton by infecting the hypocotyl with Agrobacterium,and cotton mutants with GhBsr-kl gene editing were successfully obtained.Based on the CLCrV-mediated VIGE technology established in cotton by the research group,the gene editing efficiency and heritability of the florigen FT gene fused to the 3’ end of GhBsr-k1-sgRNA were investigated for the cotton GhBsr-k1 gene.The results showed that The gene editing efficiency fused to the 3’ end of the sgRNA is higher than that of the gene fused to the 5’end.The heritability of gene editing in offspring is a work in progress.(4)The AtBsr-k1/AtSKI3-pHEE401 gene editing vector was successfully constructed,and the Arabidopsis thaliana was transformed by the flower dip method,and the T1 generation positive plants were obtained.Through direct sequencing and high-throughput sequencing of PCR products,Arabidopsis plants with AtBsr-k1/AtSKI3 gene editing were successfully screened,which provided a material basis for the subsequent study of the value of Bsr-k1 gene in broad-spectrum disease resistance and breeding.In this study,three GhBsr-k1 gene edited cotton mutants and ten AtBsr-k1/AtSKI3 gene edited Arabidopsis mutant plants were obtained,providing a material basis for clarifying the disease resistance function of the Bsr-k1 gene and its value in cotton breeding.