The Primary Research On The Effect Of Exogenous Inorganic Pyrophosphatase Gene (PPa) In Transformants Of Rice Variety’Hui236’

Pyrophosphatase is a hydrolytic enzyme with pyrophosphate as substrate. In order to evaluate the effect of PPa gene expressed in the mesophll cells of rice and to breed lines with value in use, we imported the PPa gene which was driven by the mesophyll cell specific promoter of cyFBPase, into rice variey,’Hui236’. After callus culture and Agrobacterium-mediated co-transformation, we obtained30transgenic plants, and there were16transformants after detection by PCR based on the inserted T-DNA sequence, the positive rate was53.3%.Two independent transformants obtained at first were planted in2009and we have got their homozygous transgenic lines in T2generation in the next year. For analyzing the genetic and expression characteristics of PPa in rice,6homozygous transgenic lines with3lines each transformant were selected to conduct following research. Since the summer of2010, we have investigated the agronomic traits and yield characteristics of the lines, arid detected mRNA expression of PPa gene, pyrophosphatase activity, soluble and total carbohydrate content and C/N and compared above results with that of the receptor parent’Hui236’(CK). The results were as follow:1、RT-PCR analysis was given to6homozygous transgenic lines during the third-leaf growing stage to detect the expression level of PPa gene in different rice organs. The results showed that there is no PPa gene expression in CK’s leaf, leaf sheath and root, while, in transgenic plants, the highest expression of PPa gene were found in leaves, a small amount of expression in leaf sheath, and no expression in root. The experimental results demonstrated that the PPa gene have transcribed successfully under the mesophyll cell-specific promoter.2、In the heading stage we determined PPase activity using leaves from this6homozygous transgenic lines. The analysis showed that PPase activities of all transgenic leaf were higher than that in CK; the activities of2transformants descendants were, respectively,2.2、3.5、3.1and2.6、3.4、4.2times as high as the CK. The results suggested that the target gene in transgenic plants was successfully expressed and its protein had high activity.3、Yield effect analysis showed that the main traits such as the tiller number, plant height and whole growth stage in transgenic lines had no significant change compared with the CK, which meant the genetic transformation did not have a negative impact on transgenic plants. The yield levels of most transgenic lines were much higher than the CK. The range of their yield increase was from about8.2%to15.4%. The increase was primarily due to the improvement of grain number per panicle and1000-filled grain weight.4、We determined carbohydrate in leaves and leaf sheath. Results showed, during the period of meddle and late growth stages, there was a significantly increased of total soluble sugar and sucrose in transgenic leaf sheath, but no significant difference in leaves between the transgenic plants and CK. The results indicated that the photosynthetic products of PPa transgenic line did not retard in the leaves but put out more to sheaths, which would promote photosynthesis in leaf through feedback regulation system.5、Compared with the CK, we found the nitrogen content and C/N ratio of transgenic leaves was no significant difference. But nitrogen content in the leaf sheath is generally lower than the CK, while the C/N ratio is generally higher. These results reflected a general relative increase for C in the leaf sheath of transgenic plants, and it would be better to maintain a good somatoplasm of plant under a higher-fertility level condition.In conclusion, we tried a new strategy to improve photo-production and increase rice yield through promoting photosynthesis and changing photoproduct transportation and distribution by means of over-expression of the PPa gene in mesophyll cells. The results suggested that the strategy seems feasible. However, the mechanism of molecular regulation for photosynthesis is quite complex and more research was required to continue to explore the function of PPa.