Effect On Male Sterility In Orf25-RNAi And Atp9-RNAi Transgenic Tobacco

Tobacco is the vital economic crop of our country and even the world,which is also the commonly used model plant of plant genetic engineering at the same time.ATP synthase is a kind of complex enzyme in mitochondrion,it is mainly composed of two parts as F0 and F1.A single or multiple gene products abnormal coded of ATP synthase are associated with male sterility.orf25 gene is a subunit gene of F0,its abnormal expression may lead to tobacco male sterility.The study served atp9 as control gene,by analyzing the differences in morphology,ATPase activity,ATP content and pollen viability in comparison with the control plants between and RNAi-orf25 and RNAi-atp9 tobacco to explore the relationship between orf25 gene and tobacco male sterility.The main results were as follows:1.12 strains of RNAi-orf25 transgenic tobacco plants were obtained,one of which was died at seeding stage,and a strain of tobacco without blossomed.14 strains of RNAi-atp9 transgenic tobacco plants were obtained,two of which were less and smaller,and even without blossom.Phenotype characteristic of the transgenic castor were follows: plant height was dwarfed,poor growth and increased branching,leaves were less and smaller,plants were susceptible.2.In the non-transgenic plants,the relative transcript level of orf25 gene in maintainer line zy90 was significantly higher than male sterile line MSzy90 in these four organs of leaf,flower,steam and root.What’s more,the relative transcript level of orf25 gene in flower always kept in the highest level.The relative expression level of atp9 gene in maintainer line K326 was significantly higher than male sterile line MSK326 in these four organs,and that the relative expression level of atp9 gene in flower was always the highest.In the transgenic plants,RT-PCR analysis results indicated that the relative transcript level of orf25 and atp9 gene were significantly lower than the control plants in these four organs of leaf,flower,steam and root of transgenic tobacco plants,and that lowest in flower.At the same time,it is in accordance with the RNAi-orf25 tobacco that the relative transcript level of atp9 gene was lowest in flower,which were significantly lower than the control plants in these four organs.3.In the non-transgenic plants,the ATPase activity in leaf,flower,steam and root of MSzy90,zy90 and MSK326,K326 were compared in this study,which showed that ATPase activity in maintainer line were significantly higher than male sterile line,besides,the ATPase activity in floral organ was the biggest all in two lines.In the transgenic plants,the ATPase activity and ATP content were significantly lower than the control,and that in flowers were significantly higher than the other three organs.The above results indicated the two RNAi expression vectors all possessed interference effects.4.Pollen viability of transgenic tobacco plants decreased obviously compared with the control.One of RNAi-atp9 tobacco had a change in fertility,and formed a strain of male sterility tobacco.5.Interfering the normal expression of orf25 and atp9 gene can abate the expression of target gene specifically,it can also decrease ATPase activity,ATP content and pollen vitality of transgenic tobacco.These results suggest that the two subunit genes of ATP synthetase may have similar functions.Although aberrant mitochondrial gene orf25 can’t directly cause tobacco male sterility,but whether it regulates tobacco male sterility together with other tobacco CMS related gene awaits further research.