Study On The Creation Of New Male Sterile Lines Of Rice By Genome Editing Technology

There are two main ways to use hybrid vigor in rice,three-line method and two-line method.However,these two kinds of methods all have some limitations on practice.The main disadvantage of the three-line is its lower range of germplasm resources utilized as restorer line.Although the two-line method has an obvious advantage compared with the three-line,the male fertility fluctuation of photo-thermo-sensitive genic male sterile(PTGMS)lines under extreme changes in weather is an important problem troubling two-line system.But there are a lot of genic male sterile mutants with stable fertility have not been utilized because of unavailable strategies to maintain the male sterility.Currently,seed production technology(SPT)technology and the intelligent sterile technology were developed to solve these problems which opens up a new way for rice heterosis utilization.This study intends to site-direct mutate 3 male sterile genes and 1 anther-specific gene by CRISPR/cas9 system to create genic male sterile in MingHui 86,a elite rice restorer line.After that we construct a engineering vector and then transformed into the corresponding male sterile mutant to create a intelligent system to utilize recessive genic male sterility,creation of new materials,open up new ways to utilization of heterosis in rice.The main results are list as follows:1,Three male sterility genes CYP704B2,TIP2 and DPW were site-directed in elite rice variety Minghui 86 by CRISPR/Cas9 technology respectively.Through each edit vector transgenic plants screened for gene CYP704B2 we got a total of 13 kinds of mutations,which biallelic homozygous mutation j3-1 and biallelic heterozygous mutation j3-4,k2-2 representatives now at T0 pollen completely sterile;for DPW received a total of seven kinds of gene mutations,which in homozygous mutant 01-4 T0 representative of pollen abortion now,two allelic heterozygous mutation 1-1 isolated in Ti progenies ingredient plant pollen abortion completely,and the separation ratio of 3:1 is consistent with the Mendel separation law;for TIP2 gene obtained 14 kinds of mutations,which biallelic heterozygous or homozygous mutant M1-1,Ml-2,M2-7 and M22-9 representatives at To plant type is now completely normal and pollen sterility.2,Anther-specific OsIPK gene was mutated by CRISPR/Cas9 technology.Through the screening of transgenic plants,16 kinds of mutation types were obtained for 1 target sites,T0 and T1 of all mutant plants were not observed.The subcellular localization showed that OsIPK gene was expressed specifically in the nucleus.In addition,the over expression vector was constructed,and the gene function was still need to be further analyzed.3,In order to keep after site directed mutagenesis of cyp704b2 mutant male sterility,pollen lethal gene dam,wild-type cyp704b2 gene and herbicide resistance gene OsEPSP series was cloned into the plant expression vector pCAMBIA1300.The work of the vector into the cyp704b2 mutant is under way,it makes preparation for the maintenance of the cyp704b2 mutant material.