Transgenic Verification Of Candidate Genes In Flowering Time Of Brassica Napus.L

Brassica napus L.is one of the most essential oil crops in the world,which has important scientific research and practical value.Raising rapeseed yield has always been the primary goal of rapeseed research,and flowering time is an important factor affecting yield,the flowering of B.napus at an appropriate time can not only ensure its yield and quality,but also expand its planting area,therefore,the research on the flowering period is of great significance for rapeseed breeding.In this study,using the flowering candidate genes in the previous whole-genome association analysis（GWAS）as the verification object,18 flowering candidate genes were screened,named ZH1-ZH18,and bioinformatics analysis of ZH1-ZH18 showed that ZH7（BnaSVP）and ZH15（BnaSEP1）were members of the MIKC MADS-box family,and most of the genes with known functions in the family were related to flowering.CRISPR/Cas9gene editing and overexpression techniques were used to verify the function of genes,constructed 18 knockout vectors and transformed into Brassica napus Westar.6 genes were screened from 18 candidate genes to construct overexpression vectors,which were transformed into Arabidopsis and Westar,the flowering phenotype of transgenic offspring was investigated.The major results are as follows:1.The analysis of MIKC MADS-box gene family showed that the members of MIKC MADS-box family were related to flower development or flowering regulation.172 MIKC MADS-box genes were identified in the Westar genome,these 172 genes and 39 Arabidopsis MIKC MADS-box genes were used to construct a phylogenetic tree.211 genes were divided into 6 groups（A,B,C,D,E,F,H）according to the flower development model,the homologous genes of Arabidopsis and Brassica napus can be clustered on the same branch.Chromosome mapping of 172 Westar genes showed that 83 genes were distributed in A subgenome,86 genes were distributed in C subgenome,and 3 genes were distributed in random chromosomes,It can be seen that MIKC MADS-box genes were evenly distributed in the Westar genome.Motif,domain and gene structure analysis showed that six motif of motif 1,motif 2,motif 3,motif 4,motif 5 and motif 7 were highly conserved;K-box domain and MADS domain of MIKC MADS-box family existed simultaneously in most genes;and the gene structure of the same group or subfamily was relatively conservative,and most genes contained6-7 exon,5-6 introns.SEP1 in E class gene and SVP in H class gene were candidate genes for flowering period obtained from the GWAS study of our group.2.The positive detection of CRISPR/Cas9 knockout transgenic plants and overexpression transgenic plants showed that the average positive rate of CRISPR/Cas9 knockout transgenic plants with 17 genes was 93.11%;The average positive rate of T₀overexpression transgenic plants of BnaA02.FY,BnaA04.SPL9,BnaA05.COL9,BnaC09.COL4 and BnaA10.CO was 98.59%.High-throughput Tracking Of Mutations（Hi-TOM）sequencing technology was used to detect the editing efficiency of CRISPR/Cas9 knockout lines in T₀generation.The average editing efficiency of 10 genes:BnaFY,BnaLD,BnaSPL9,BnaCOL9,BnaCOL4,BnaSVP,BnaCSTF64,BnaCO,BnaSPL8,BnaSEP1 was 72.32%,and the editing efficiency of target 1 and target 2 was different in most genes.According to the statistics of editing types,it is found that the average efficiency of insertion,deletion,replacement and complex mutation is 41.67%,22.78%,2.89%and 32.66%,respectively,Most of editing types are insertion and complex mutation,and the probability of mutation on A/T base pair is higher than that on C/G base pair.3.Quantitative analysis of the overexpression transgenic lines of A.thaliana and Westar indicated that the expression levels of the transgenic lines in A.thaliana and Westar were significantly greater than that of wild type,and the plants with significantly higher expression levels were selected to investigate the phenotype:BnaA02.LD was genetically transformed into A.thaliana to obtain the early flowering phenotype,and the flowering period was advanced by 3 days;BnaA05.COL9 and BnaC05.COL9 were genetically transformed into Arabidopsis to obtain the late flowering phenotype,and the flowering was delayed by 5 days and 3 days,respectively.BnaA05.COL9 genetic transformation Westar got late flowering phenotype,flowering delay 12-17 days,BnaA10.CO genetic transformation Westar got early flowering phenotype,flowering advance 11-18 days.4.There were four homologous copies of BnaSVP in B.napus（BnaA09.SVP,BnaC08.SVP,BnaA04.SVP,BnaC04.SVP）.After knockout,the svp mutant showed early flowering in both indoor and outdoor environments.The flowering statistics showed that the flowering difference between the svp mutant and the wild type was8-31 days in the greenhouse environment.Using HI-TOM high-throughput sequencing,it was found that homozygous mutation of all homologous copy double targets had the greatest impact on flowering difference（the maximum flowering difference was 31days）;all homologous copies had mutations in the double targets,but the difference in flowering time was weakened when single target had heterozygous mutations（the maximum flowering difference was 21 days）;When double targets of multiple homologous copies appear heterozygous mutation will weaken the flowering difference（maximum flowering difference is 9 days）;When only two homologous copies were edited,the flowering time was not different from that of wild type.Fluorescence quantitative analysis of four homologous copies and pathway-related genes（FT,SOC1,GA20OX2,TFS1）of svp mutant showed that the expression levels of four copies were significantly lower than that of wild type,which weakened the effect of BnaSVP on the inhibition of flowering;The expression of FT and SOC1 were significantly greater than that of wild type.For example,the expression levels of FT and SOC1 in ZH7-7-2 plant were increased by 60 times and 6 times,respectively,and the flowering time was advanced by 29 days.In summary,BnaLD and BnaCO can promote flowering in Brassica napus,BnaCOL9 and BnaSVP can inhibit flowering in Brassica napus,other genes did not observe the change of flowering phenotype in Brassica napus,and their functions need to be further studied.