Effect On Male Sterility,ATPase Activity And ATP Content In Orf25 Transgenic Tobacco

Orf25 gene is one of the subunit gene in tobacco ATPase. Preliminary study confirmed that it has a difference in DNA sequence of orf25 gene between cytoplasmic male sterility lines and their corresponding maintainer lines. To uncover the relationship between the function of orf25 gene and the male sterility of tobacco, using Ca MV35 S and Pchs A as the promoters, we had already constructed the expression carrier with orf25 gene and the empty carrier without orf25 gene, and we had got the transgenic positive tobacco through transforming the vectors into the(CMS)Gexin3 by agrobacterium tumefaciens mediated genetic transformation method. This research used(CMS)Gexin3 and Gexin3,the expression carrier with orf25 gene(Z-35S-orf25) and the empty carrier without orf25gene(Z-35S) of Ca MV35 S promoter, the expression carrier with orf25 gene(Z-Pchs A-orf25)and the empty carrier without orf25 gene(Z-Pchs A) of Pchs A promoter, common tissue culture seedlings(CK) as experimental materials, discussing the influence of the orf25 gene to the cytoplasmic male sterility lines, ATPase and the content of ATP in the leaves, flowers,stems and roots of the experimental materials. The main results are as follows:1. The(CMS)Gexin 3 and Gexin 3 which were in reproductive growth stage were used as experimental materials. The real-time quantitative PCR(q RT-PCR) was used to analyze the expression patterns of the orf25 gene, and the activity of ATPase was also researched. The results show that the expression patterns of the orf25 gene in Gexin 3 were relatively higher than(CMS)Gexin 3, furthermore, the expression of the orf25 gene in flowers were the highest in the same materials, the leaves took the second place, the expression of orf25 gene in roots were relatively low. The activity of the ATPase in maintainer line was relatively higher than its male sterile line, the activity of the ATPase in the flowers was also the highest in the same materials, There was no obvious significant difference of ATPase in leaves and in roots. The correlation analysis indicated that the relative expression of orf25 gene had extremely significant positive correlation with the activity of ATPase in the same organ of tobacco. It shows that orf25 gene may be one of the causes of male sterility in tobacco.2. At the stage of early flowering period, no matter in which parts of the transgenic tobacco(leaves, flowers, stems or roots), the relative expression of orf25 gene of the Z-35S-orf25 and Z-Pchs A-orf25 were higher than the CK, Z-35 S and Z-Pchs A. The relative expression of the leaves, stems and roots in Z-35S-orf25 were higher than Z-Pchs A-orf25 except the flowers. In addition, the flowers relative expression was the highest than the other parts of the five experimental materials.This shows that the orf25 gene was successfully introduced into the transgenic tobacco, and its expression was the highest in reproductive growth stage.3. At the stage of early flowering period, no matter in which parts of the transgenic tobacco(leaves, flowers, stems or roots), the activity of ATPase of the Z-35S-orf25 and Z-Pchs A-orf25 were higher than the CK, Z-35 S and Z-Pchs A. The activity of ATPase in the leaves, stems and roots of the Z-35S-orf25 were higher than Z-Pchs A-orf25 except the flowers. In addition, the activity of ATPase in flowers was the highest than the other parts of the five experimental materials. This shows that the orf25 gene increased the activity of ATP in a certain extent, and the activity of ATPase was the highest in flower.4. At the stage of early flowering period, we used the methods of luciferasefluorescein to determin the content of ATP in the leaves, flowers and stems of the five experimental materials. The results show that content of ATP in the leaves of the Z-35S-orf25 and Z-Pchs A-orf25 were higher than the CK, Z-35 S and Z-Pchs A, but the flowers and stems were lower than the CK, Z-35 S and Z-Pchs A. The content of ATP in leaves, flowers and stems of the Z-35S-orf25 were higher than Z-Pchs A-orf25. In addition,the content of ATP in flowers was the highest than the other parts of the five experimental materials in general. It shows that the content of ATP has a different between the orf25 gene expression and ATPase activity.5. The relative expression and activity of ATPase of the leaves, stems and roots in Z-35S-orf25 were higher than Z-Pchs A-orf25 except the flowers. But the content of ATP in leaves, flowers and stems of the Z-35S-orf25 were higher than Z-Pchs A-orf25. It was proved that Ca MV35 S as a constitutive promoter could increase the orf25 gene expression,ATPase activity and ATP content in transgenic tobacco plants, while Pchs A as a flower specific promoter was only acted on flower.6. This research determined the number of leaves and height of the CK, Z-35S-orf25,Z-35 S, Z-Pchs A-orf25 and Z-Pchs A. We found the number of leaves and height in Z-35S-orf25 and Z-Pchs A-orf25 were lower than CK, Z-35 S and Z-Pchs A, but the number of leaves and height in Z-Pchs A-orf25 were higher than Z-35S-orf25. The results show that the over expression of Ca MV35 S resulted in the reduction of plant height and the number of leaves, while the flower specific promoter Pchs A had no effect on plant height and leaf number.