Effect Of Knocking Out Chalk5 Using CRISPR/Cas9 Gene Editing On Chalkiness In Rice

Rice is the largest food crop in China,and with the improvement of people’s living standards,the requirements for rice quality are getting higher and higher,and the selection and breeding of high-quality and high-yielding rice varieties is an important goal for breeders.The current CRISPR/Cas9 technology is widely used for rice variety improvement with its advantages of high efficiency,short cycle time and high purposefulness.In this study,a CRISPR/Cas9 vector controlling the chalk gene Chalk5was constructed,and seven T₂generation mutant lines were obtained by transforming rice variety 9311.These transgenic lines were sown in stages and tested with different nitrogen fertilizer gradients to verify the changes of rice chalk under different environmental conditions after gene editing,and also mated with sterile lines to study their application potential as two-line recovery lines.The results were as follows:1.The pBWA（V）H＿cas9i2-chalk5 vector was successfully constructed and the target fragment was transferred into the rice recipient parent 9311,and the gene editing was proven successful by sequencing.The vector skeleton assay was performed on T₁generation transgenic plants,and two empty vector lines TC1 and TC6 were screened,among which the target gene of TC1 was edited successfully,i.e.,one knockout individual without vector skeleton was obtained（traceless edited plants）,and TC6 had trait variation but the target gene was not edited successfully.In addition,TC2,TC3,TC4,TC5 and TC7 transgenic strains with better agronomic traits were screened,and a total of seven mutant strains with research and utilization value were obtained.2.The agronomic traits of these seven strains were analyzed,in which the transgenic strains TC1-TC7 all had a significantly longer reproductive period than the control.The relative chlorophyll content（SPAD）of TC1-TC7 was significantly lower than that of the control at about two weeks after tasseling,and the blade length of TC3,TC5,TC6 and TC7 was significantly longer than that of the control.The rice quality analysis of these seven strains showed that the chalkiness of TC1-TC5 was 0%-0.6%and chalkiness was 0%-0.2%,while the chalkiness of TC6 and TC7 was 1.1%and 0.9%,and chalkiness was 0.7%and 0.6%,respectively,all of which were significantly lower than the chalkiness（5.6%）and chalkiness（2.1%）of the control variety 9311.The seed aspect ratio and thousand grain weight of these seven strains were not significantly different from the control.3.Under four different sowing conditions,the chalkiness of the successful Chalk5-edited lines ranged from 0%to 0.2%,the chalkiness of the wild type ranged from 0.8%to 8.5%,and the chalkiness of the unedited lines ranged from 0.3%to 1.1%,indicating that the chalkiness could be significantly improved by Chalk5 gene editing under different sowing conditions.Under three different nitrogen fertilizer gradients,the chalkiness of the edited lines ranged from 0%-0.1%,the control from 0.4%-1.1%,and the unedited lines from 0.1%-0.6%,indicating that knocking out the Chalk5 gene also significantly improved chalkiness under different nitrogen fertilizer conditions.4.By examining the F₁of the crosses formulated with the transgenic strain and the two sterile lines,it was found that the progeny of the crosses produced by the successful Chalk5 gene editing strain and the sterile lines had significantly lower chalkiness and chalkiness compared with the progeny of the crosses between the wild type and the sterile lines,while the differences in other quality traits were not significant.This indicates that the editing technology of Chalk5 gene can be applied in the genetic improvement of chalkiness traits in rice.In conclusion,a number of transgenic strains with low chalkiness,excellent overall traits and potential use were created and screened by editing the Chalk5 gene,which provided new breeding materials for genetic improvement of chalkiness traits in rice.