Cloning Of Tef Related To Reproductive Development In Arabidopsis Thaliana

The food(seeds and fruits)that humans depend on is the product of plant sexual reproduction?In angiosperms,successful sexual reproduction requires a series of events,such as the normal development of pollen and embryo sacs,the growth of pollen tube correctly targeting ovule,and the fusion of sperm and egg cells.For a long time,one of the focuses of angiosperm sexual reproduction research is to isolate the key genes involved in these events.In recent years,with the development of Agrobacterium mediated genetic transformation methods,a large number of T-DNA insertion mutants have been obtained and used in the study of sexual reproduction in angiosperms.In this study,we isolated and cloned a gene tef involved in reproductive process.The main research contents and results are as follows:(1)By screening the transgenic seed bank of our laboratory,we found a transgenic plant with abnormal segregation ratio of resistance genes.The segregation ratio of the resistance gene of the transgenic plant in the offspring was 8.5:1,which clearly deviated from the normal segregation ratio(1:3).The mutant showed normal nutrition growth and flowering,but the number of seeds in siliques is less than that of wild-type Arabidopsis thaliana.We named this mutant tef.(2)Through TADEA-PCR,we cloned the insertion position of the T-DNA of tef in the Arabidopsis thaliana chromosome.The results showed that the T-DNA insertion replaced a segment of Arabidopsis thaliana chromosome 4 containing about 30,000 nucleotides.This fragment contains 8 coding genes: AT4G02890,AT4G02900,AT4G02910,AT4G02920,AT4G02930,AT4G02940,AT4G02950,AT4G02960.(3)Using the PCR method,we further tested the conductivity of the insertion site in selfed and hybrid progeny.In selfed offspring,the conductivity of the insertion site was 12.95%;In the hybrid test with wild-type plants as the female parent,tef as the male parent,and wild-type plants as the male parent and tef as the female parent,the conductivity rates were5.6% and 5.4% respectively.Moreover,in the offspring plants,we have not been able to obtain a homozygous mutant of tef,which implies that tef is a homozygous lethal gene.(4)In order to explore which gene deletion caused the abnormal conductivity of the tef mutant,we analyze the genotypes of the T-DNA mutants corresponding to AT4G02890,AT4G02900,AT4G02910,AT4G02920,AT4G02940,AT4G02960 in the offspring one by one.The results show that these T-DNA mutants can detect homozygous mutant plants in selfed progenies,suggesting that these gene mutations are not the cause of abnormal tef conductivity.(5)Because AT4G02950 lacks effective T-DNA mutants,in order to verify whether the AT4G02950 gene is the target gene that causes the abnormal transmission of tef,we produced a transgenic line Pro AT4G02950: AT4G02950 and introduced it into the tef plant through hybridization.In F2 plants,we isolated Pro AT4G02950:AT4G02950 homozygous plants containing tef background,and tested the conductivity of tef in F3 generation plants.The results showed that the conductivity of tef in the F3 generation was 8.30%,suggesting that the AT4G02950 gene was not the target gene that causes abnormal conduction of tef.(6)Because AT4G02930 lacks effective T-DNA mutants,in order to explore whether the AT4G02930 gene is the target gene that causes abnormal conduction of tef,we used CRISPR/Cas9 technology to knock out the AT4G02930 gene and obtained its missing mutant tuf.Hybrid experiments with wild-type plants show that the conductivity of tuf was4.3%(wild-type plants are female parents)and 4.5%(wild-type plants are male parents);at the same time,the conductivity of tuf in inbred progeny was 9.1%.The conductivity of tuf is almost equal to that of tef,suggesting that the absence of AT4G02930 gene may be the cause of abnormal conductivity of tef.We also produced Protuf: tuf-GFP transgenic plants and introduced them into tuf mutants.The experimental results showed that the introduction of Protuf: tuf-GFP increased the conductivity of tuf in F2 generation plants to52.50%.Based on the above experimental results,we preliminarily concluded that the abnormal transmission of tef plants is caused by the loss of AT4G02930 gene.