Cloning Fertility Restorer Genes And Constructing The Overexpression Plants In Tobacco

Male sterility of tobacco is widely cultivated in practice and plays an important role in cross breeding.It belongs to cytoplasmic male sterility,male sterility is caused by mitochondrial genes,PPR protein(pentatricopeptide repeat)which is product in nuclear fertility genes that regulates fertility restoration.The research group conducted the study on the hybrid breeding of the cultivated tobacco male sterile line Nicotiana tabacum L.cv.(gla.)S K326(Nta(gla.)S K326)and Nicotiana alata in the earlier period.The hybrids were obtained and divided into two groups,a kind of hybrid can recover the fertility,while another hybrid?s stamen are able to development but the pollen aborted.It is speculated that restorer of fertility exists in the nucleus of male parent,acting on the gene products of maternal sterile and regulating the fertility restoration of the hybrids.In this study,Nta(gla.)S K326,N.alata and their hybrid offsprings were used to cloning candidate fertility restorer genes in N.alata,combining with bioinformatics analysis and expression characteristics of genes,we first screened fertility restorer genes and transformed candidate genes into male sterile female parent,to exploring the mechanism of fertility restoration about hybrids,laying a theoretical foundation for the extensive application of cytoplasmic male sterile lines and wild species in the production process about hybrid seeds.The main findings are as follows:(1)The homologous cloning technique was used to obtaining the 27 homologous ppr genes in the anthers of paternal N.alata,according to subcellular locations,the structure of PPR protein and PPR motif characteristics,18 candidate fertility restorer genes were further screened out,10 possible sequences of fertility restorer genes were finally obtained by comparing the similarity of these sequences;(2)q RT-PCR was used to examinating the transcriptional level of the above 10 genes,to detecting the expression of anthers in different parents and different hybrids,the expression pattern accorded with the predicting results that fertility restorer genes in anthers of different fertility tobacco,it showed these 10 genes were probably the fertility restorer genes in this experiment;(3)The above candidate ppr genes were respectively ligated to the p BI 121,which was the plant expression vector,to constructing the recombinant plasmids,and using the agrobacterium-mediated transformed into male sterile lines of sterile seedlings, detectig the target fragments which obtained from some transgenic plants.The results showed that the amplified bands in the transgenic plants were consistent with plasmids,and the negative control didn?t have any bands.The experimental results initially showed that the exogenous genes has been integrated into the genome of tobacco,it can be used for phenotypic identification and functional validation in the later period.Among them,the conversion rate of ppr582-2 is 30 %,the conversion rate of ppr582-9 is 20 %,the conversion rate of ppr581 is 40 %,the conversion rate of ppr320 is 62.5 %.In this study,a series of PPR sequences were cloned from N.alata,combining with bioinformatics analysis and expression characteristics,10 candidate fertility restorer genes were screened out,and overexpression vectors of 10 candidate genes were successfully constructed and transformed into male sterile female parent,the fertility restoration function of these sequences will be further verified by phenotypic identification.This study laid the foundation for exploring the mechanism of hybrid fertility,and provided a thought for cloning and identificating fertility restorer genes in tobacco.