Evaluation Of Pod Shattering Resistant Mutants And Establishment Of ABE Base Editing Tool In Brassica Napus L

In the world,oilseed rape is one of the three main oil crops,and also the main source of edible vegetable oil in our country.At present,it is one of the main limiting factors of mechanized rapeseed harvest because the siliques are easy to dehisce during harvest,which usually leads to a great loss of yield.Therefore,it is of great significance to cultivate rapeseed varieties with suitable pod-shattering resistance to promote mechanized production of rapeseed and guarantee high and stable yield of rapeseed.Our laboratory has previously created the mutants of BnaIND,BnaSHP1,BnaSHP2,BnaCEL6 and BnaMAN7dehiscent-related genes in Brassica napus using CRISPR/Cas9 gene editing technology.On this basis,field agronomic characters and pod-shattering resistance of these mutants were investigated in this study,and their practical application value was evaluated comprehensively.At the same time,the mechanism of their participation in the regulation of silique dehiscence in Brassica napus L.In addition,this study established an effective single base editing system for ABE in Brassica napus for the first time.The main research results of this paper are as follows:1.Bioinformatics showed that the two copies of BnaIND were highly homologous with the protein encoded by Arabidopsis AtIND,both of which had the b HLH conserved domain.BnaSHP gene family proteins all contain MADS-box and K-box conserved domains.These two conserved domains are the important basis for regulating downstream genes as transcription factors.Two genes,BnaCEL6 and BnaMAN7,encode cellulase and hemicellulase,which are functionally conservative proteins with high amino acid sequence homology.2.The agronomic traits of the homozygous mutants of the pod-shattering resistant candidate genes were investigated and the pod-shattering resistant coefficient was measured.The results showed that the single copy A03 mutants and double copy mutants of BnaIND showed significant pod-shattering resistance,and there were no significant changes in other agronomic characters,such as plant height,thousand seeds weight,effective branch number,first branch height and inflorescence length,except for the silique length and the number of seeds per silique.The five-copy homozygous mutant of BnaSHP1 also showed significant pod-shattering resistant ability,but the pod-shattering resistant ability did not change significantly after BnaSHP2 mutation.There was no significant change in their main agronomic characters.The pod-shattering resistance of BnaCEL6 and BnaMAN7 mutants did not change significantly.3.The sequence alignment of BnaIND promoters showed that only the promoters of BnaA03.IND copy had 400 bp conserved domain similar to AtIND.Promoter truncation and double luciferase reporting experiments confirmed that this conserved domain plays an important role in up-regulating the expression of this gene.4.In order to create ABE single base editing system in Brassica napus,we modified 4ABE skeletons,Eighteen sgRNAs were designed to test the efficiency of different ABE systems for nine genes,namely BnaPDS,BnaALS,BnaCLV3,BnaCLV2,BnaTT12,BnaCRTISO,BnaDET2,BnaBRC1 and BnaZEP,respectively.The results showed that pBGE17 had the highest editing efficiency,with an average editing efficiency of 3.51%.The edit window is between A5 and A8.On this basis,the ABEmax-n Cas9 NG system is developed to recognize NGPAM.The test results show that ABE editing can be realized in Brassica napus,and the efficiency is up to 1.22%.In conclusion,in this study,excellent resources with pod-shattering resistance were selected by evaluating the pod-shattering resistance ability and comprehensive agronomic traits of BnaIND,BnaSHP1,BnaSHP2,BnaCEL6 and BnaMAN7 gene mutants in Brassica napus L.At the same time,the important promoter regulatory elements of BnaIND were preliminarily analyzed,which laid the foundation for the functional differentiation of BnaIND gene.In addition,this study created an effective ABE single base editing system for the first time in Brassica napus,providing an accurate editing tool for germplasm resource innovation.