Using CRISPR/Cas9 Gene Editing System To Obtain Mutants Of Genes Related To Stress Tolerance And Disease Resistance In Cabbage

Cabbage(Brassica oleracea var.Capitate L.)is an important leafy vegetable of Brassicaceae.Due to global climate change and intensive production,the frequency of abiotic stress such as drought,cold as well as biological stress such as bacteria,fungi have greatly increased in the process of cabbage cultivation,which has a huge impact on the yield and quality of cabbage production.The response of crops to stress and pathogen invasion is achieved mainly by a series of physiological and biochemical response produced by the associated genes.A few earlier studies had prove that the AtOCP3 and AtESK1 genes are involved in drought and cold stress response in Arabidopsis thaliana.Down-regulated gene expression or mutations can improve their drought or cold resistance.The AtBIK1 and AtLACS2 genes are involved in the resistance of Plasmodiophora brassicae and the resistance of fungal diseases of B.cinerea and Sclerotinia.And the resistance level of the corresponding disease is greatly improved in the mutants of the corresponding genes.Therefore,mutation of the homologous genes in cabbage is expected to obtain resistant germplasm.CRISPR/Cas9 gene editing system is the most commonly used tool for site-directed gene mutation.It is widely used to obtain mutants of various crops and achieve improvement of variety traits and multi-gene stacking.After the target gene mutation was achieved by CRISPR/Cas9 gene editing system,the Cas9-free mutant could be isolated from the self-pollination progeny population.The common method is to screen transgenic and non-transgenic individuals by means of molecular detection,which greatly increases the workload and cost.Therefore,it is of great value to explore a rapid and simple transgenic individual identification system to apply the CRISPR/Cas9 gene editing system to the creation of high-throughput crop mutants.By introducing the visible selection marker gene into the CRISPR/Cas9 gene editing vector,the visual screening of transgenic individuals and non-transgenic individuals can be performed at the seed or seedling stage,which will undoubtedly greatly improve the screening efficiency of non-transgenic mutants.Therefore,the study analyzed the expression of BoOCP3 gene after drought stress,BoESK1 gene after cold stress,BoBIK1 gene after Plasmodiophora brassicae inoculation,BoLACS2 gene after Sclerotinia inoculation,which confirmed whether they are involved in the biotic stress or abiotic stress.In addition,two visual selection marker genes were introduced into the CRISPR/Cas9 gene editing vector to construct gene knockout vectors for the above genes.One of the two visible selective genes is BoMYB,a key regulatory gene derived from the anthocyanin synthesis in purple cauliflower,which was fused to herbicide resistance gene BAR to identify genetically modified individuals at all stages of the transgenic individuals.Another visible selective marker gene is the seed-specific fluorescent protein mCherry gene,which can be used to screen transgenic individuals at the seed stage.Agrobacterium-mediated genetic transformation method was used to introduce the targeted knockout vector of the above genes into the cabbage inbred line F416,and the target genes were successfully knocked out and the corresponding mutants were obtained.The main results are as follows:1.The expression of stress-related gene in B.oleracea.After the drought stress on F416,the expression of BoOCP3 was significantly down-regulated 2 days after stress,up-regulated 4 days later,and the expression rose to the pre-stress level after rehydration.In the range of 1-4 days after cold stress treatment in a 4℃ incubator,BoESK1 gene expression level increased steadily.The results showed that BoOCP3 was involved in the regulation of drought stress response of cabbage,while BoESK1 gene was involved in cold stress response of cabbage.2.The expression of resistance-related gene in B.oleracea.One day after inoculation of sclerotinia on F416,the expression level of BoLACS2 gene increased significantly,and the expression level reached the highest within 2 days,and obvious symptoms of plant victimization after inoculation were observed.The expression of BoBIK1 gene in leaves increased steadily in a period of 4-14 days after inoculation of Plasmodiophora brassicae.The above results showed that BoLACS2 and BoBIK1 are involved in the process of infecting and reproducing of the pathogen ion leaves and roots3.Application of visual marker-assisted selection of genes.Two visible selective marker genes,MYB and mCherry,were introduced into the CRISPR/Cas9 gene editing vector and transformed into cabbage.And significant accumulation of red anthocyanins was observed in the PPT resistant callus,buds and plants.At the same time,mCherry gene was detected in the red callus or plants without exception.The results showed that the introduction of the visual selection marker gene into the CRISPR/Cas9 gene editing vector did not affect the transformation,screening efficiency,growth and development of transgenic plants.4.Obtain ocp3/esk1 mutant background cabbage material.A double knockout targeted vector PCAMC-tBoOCP3/tBoESK1 was constructed to target the abiotic stress related genes BoOCP3 and BoESK1.It was introduced into cabbage self-incompatible line F416 by agrobacterium-mediated method.Twenty transgenic plants were generated,and BoOCP3 gene mutation was detected in 19 plants,all of them were detected BoESK1 gene mutation,and double mutation efficiency is 95%.Five double-knockout plants were selected randomly for monoclonal sequencing.The BoOCP3 gene mutation was detected in 5 transgenic cabbage at position D,while only 1 plant was detected at position B.The mutation of BoESK1 gene was detected in A,B and C sites.5.Obtain bik1/lacs2 mutant background cabbage material.Double knockout targeted vector PCAMC-tBoBIK1/tBoLACS2 of abiotic stress related genes BoBIK1 and BoLACS2 was constructed to introduced into cabbage self-incompatible line F416.Five red transgenic calli and two transgenic plants were obtained,and the mutation detection results of the targeted genes showed that the mutation frequency of BoBIK1 gene was 71.43%,and that of BoLACS2 gene was 28.57%.Among them,BoBIK1 and BoLACS2 genes were double knocked out in two calli.Based on monoclonal sequencing analysis,two of the calli were BoOCP3 heterozygous mutations,and no mutation of the BoLACS2 gene was detected.