Study On The Function Of BoEMS1 And BoDAD Genes To Control Male Sterility In Brassica Oleracea

Cabbage(Brassica oleracea L.var.capitata L.)is an important biennial Cruciferous vegetable.Cabbage is a typical allogamy plant with self-incompatibility,the reproduction depend on self-cross pollination at bud stage,it increase the cost of seed production and it is difficult to achieve 100% F1 seed purity.At present,the emphasis of cabbage hybrids have shifted to the breeding and utilization of male sterile lines.Cytoplasmic male sterility(CMS)is the most common use method.But the cytoplasmic male sterility have some problems as follow,such as fertility restoration source lacking,negative effect of cytoplasm,and the simplification of the cytoplasm.There are several problems in cabbage breeding based on male sterility approach:(1)limited genetic resources for male sterility;(2)simplex Ogura CMS source from radish;(3)potential risk of single cytoplasmic use;(4)lack of self-compatibility lines that restrict the development and application of male sterile hybrid breeding in cabbage;and so on.This study intends to create a cooperative mutant of nuclear sterile and self-compatibility in the cabbage,to expand the male sterility source in cabbage,avoid the risk of single cytoplasm using,promote the development of cabbage male sterile hybrid breeding.Based on the understangding of male sterility and self-incompatibility regulation in cabbage,reverse genetics technology-genome editing,can be used to carry out specific gene mutation,which provide possibilities for beeding compatible cabbage male sterile lines with its maintainer.In this experiment,the BoEMS1,BoSRK and BoDAD gene of cabbage were regulated and edited by RNAi and CRISPR/Cas9 technologies,the aim is to obtain a stable male sterile with comprehensive characters and solve the compatibility problem between maintainer line and sterile line.The mainly results are as follows: 1.The binary expression vector PCA13BarPAtEMS1-EMS1 was constructed with Bar gene as a screening marker,and the mutant of Arabidopsis thaliana ems1/+ was transformed by inflorescence staining.1 positive transgenic plant with bar gene and ems1/ems1 background was genotyped.Stereo microscope observation showed that the anther has obvious pollen,and with full seeds after self-pollinated.The results showed that BoEMS1 gene of Brassica oleracea L.could restore the sterility phenotype of ems1/ems1 mutant of Arabidopsis thaliana,demonstrated the BoEMS1 gene was related to male sterility of cabbage.2.The interference binary expression vector PCA1300PEMS-iEMS1 and PCA1300PEMS-iEMS1/iSRK were constructed with hygromycin(Hyg)as the screening marker.They were transformed into the cabbage self-incompatibility line 'F416'(belongs to SRK3 haplotype)by agrobacterium tumefaciens mediated method.7 transgenic plants(PCA1300PEMS-iEMS1)from 2 calluses and 25 transgenic plants(PCA1300PEMS-iEMS1/iSRK)from 7 calluses were obtained,respectively.The flowering phenotype showed that 1 of the 7 PCA1300PEMS-iEMS1 interference transgenic plants of was sterile,2 of them were seriously degraded,the other 4 were normal.And only 6 of the 25 PCA1300PEMS-iEMS1/iSRK transgenic plants showed significant stamen degeneration.The test of pollen vitality identification showed that transgenic plants pollen viability is far lower than that of wild type plants pollen viability.Pollens from 'F416' in situ germination experiment on the PCA1300PEMS-iEMS stigma showed that pollen grains adsorbed on the surface of stigma and germination,but did not form the pollen tube through the style of PCA1300PEMS-iEMS1 transgenic plants.However,PCA1300PEMS-iEMS1/iSRK transgenic plants showed difference,we found the pollen grain absorbed on the stigma surface and part of the pollen grain formed the pollen tubes through the stigma into style.Based on the RNA interference experimentation,it preliminarily indicated that the fertility and self-incompatibility of transgenic plants were affected.At the same time,an gene knocked expression vector PCA13BarCasEMSABC/tSRK was transformed into the self-incompatibility line 'F416' by agrobacterium mediated method,total 60 transgenic positive individual plants were produced,target loci in BoEMS1 gene are edited in 7 individual plants,the editing efficiency is 11.7%.9 plants(including 7 plants that BoEMS1 gene had edited and two randomly selected individuals)were found BoSRK gene mutation at the target sites,the editing efficiency is 100%.3.An interfering binary expression vector PCA13BariDAD/iSRK and a gene knock-out vector PCA13BarCasDADAB/tSRK were constructed with bar gene as a screening marker,respectively.60 PCA13BarCasDADAB/tSRK positive transgenic plants were produced,1 plant was detected DAD gene mutations,the editing efficiency is poor 1.7%,but the BoSRK gene editing efficiency is 100%.There were 26 PCA13BariDAD/iSRK transgenic plants were obtained.Randomly seleted 4 individual plants and a wild type were performed real-time quantitative PCR analysis for the expression of DAD gene as well as downstream LOX,AOS,AOC and OPR3 genes of Jasmonic acid biosynthesis pathway.The results showed the accumulation of mRNA in transgenic plants was lower significantly than that of the wild type 'F416' before soft rot bacteria inoculating,the accumulation of target genes mRNA in both transgenic plants and wild type increased after soft rot bacteria inoculating,however,the accumulation were significantly lower than that of wild type accumulation.In addition,Comparing the development of anther and observing the powder of stamen on the day of flowering,we found that anthers in some transgenic plants were severely degenerated,and anther was delayed powder on the day of flowering and had less powder;Pollen in situ germination results showed that pollen grains adsorbed on the surface of stigma and germination,and part of them formed the pollen tubes through the stigma into style.The results showed that the fertility and self-incompatibility are affected.