Target Editing Of A Fertility-controlling Gene By A CRISPR/Cas9 Syestem In Rice

Rice(Oryza sativa L.)is one of three major crops in our country.Nearly half of the population in the world,including almost the entire population of east and southeast Asia,put rice as the staple food,so the quality and quantity of the rice contact the focus of present food security problem intimately.Rice sterility plays a decisive role on rice yield.Arabidopsis,as a model plant,in the study of the molecular mechanism of male gametophyte development,is more mature than rice.ACOS5(ACYL-COA SYNTHETASE 5),as specifically expressed gene in Arabidopsis anther,is the key to the control of Arabidopsis pollen sterility genes,and also is one of the gene encoding the fatty acyl-CoA synthetase,which is a conservative protein with special functions.Recently,the CRISPR/Cas system is becoming the most popular gene editing technology,using of RNA to guide Cas9 nucleases to cut and modify targeted sites.Compared with the ZFN?TALEN,CRISPR/Cas system is clearly easier to operate,obviously higher efficiency of mutations,easier to obtain homozygous in the first generation,and may distinctly import multiple mutations simultaneously at different sites.CRISPR/Cas9,which belongs to Type?,is a relatively simple system component,and widely used in a variety of species.In this research,firstly,we find the rice gene LOC_Os04g24530,that is similar to arabidopsis ACOS5 reached 61.8%for cognate degree,by amino acid homology sequence alignment,.Then,we use the CRISPR/Cas9,system to study the function of LOC_Os04g24530.The way we used is the genetic transformation system by agrobacterium mediated,with the vector of the CRISPR/Cas9 pCAS9-Os04g24530-sgRNA to Nipponbare,and then obtains transgenic knockout plants,using sequence alignment and phenotypic validation.Specific conditions as follows:1.Through amino acid homologous sequence alignment,we find,the identity of ACOS5 in Arabidopsis and LOC_Os04g24530 in rice is 61.8%,and both of them are in the same evolutionary tree branches;2.In order to knock out the LOC_Os04g24530,a target spot for CRISPR/Cas9 in the first exon of that gene was designed,followed with the PAM sequence,choosing high GC content is better,according to the sequence from the gene of Gramene website;3.The CRISPR/Cas9 vector containing the target oligos was designed into transformation system of rice callus receptor by agrobacterium mediated.Through hygromycin selection,differentiation,and rooting,obtained 143 transgenic plants;4.By PCR amplification for Hygromycin gene fragment of the transgenic plants,we comfirmed 29 Hygromycin positive transgenic plants,and the enetically modified probability are 20.28%.Using PCR amplification to amplificat gene fragments of the target in these positive transgenic plants,then sub-clone amplification products to pEasy-Blunt carrier,in the end,obtained 5 TO generation knockout plants,and knockout rate is 17.24%;5.Sequencing results show that there are three types of mutations in these 5 mutant plants,including a A of insertion,a G of insertion,and a G of deletion.Among them,Plant4-1 is a A of insertion,and a G of insertion,Plant4-2 a G of deletion and a A of insertion,Plant4-3 a A of insertion,and Plant4-4 heterozygous mutant a G of insertion,Plant4-5 heterozygous mutant a A of insertion.The protein levels of LOC_Os04g24530 that has been successfully knockout show that encoding amino acids appear abnormal N prime;6.Through the observation in the growth period of these 5 mutants,found that seed by selffertilization for Plant4-4andPlant4-5,and the phenotypes of Plant4-1,Plant4-2andPlant4-3 are similar,inclunding anther dysplasia,failure of pollen iodine dyeing,no seeds by selffertilization.The results show that knockout successfully lead to male sterility in rice.we can conclude the gene is the gene that can be related of male gametophyte development.