Genetic Transformation Of PmPT3 And PmPAP1 In Tobacco (Nicotiana Tabacum) And Their Functional Analysis

Plants adapted to low phosphorus stress through a series of physiological and biochemical changes,including the activation of insoluble phosphorus on soil,the effective absorption,transportation and effective utilization of the root to low concentration organic phosphorus,among which phosphorus transporter(PT)and purple acid phosphatase(PAP)are crucial in response to low phosphorus stress.Masson pine(Pinus massoniana)is an important pioneer tree in subtropical regions,especially for the low phosphorus environment in the south of China,which demonstrated a strong adaptation to the lack of phosphorus on soil.In the previous work,Pinus massoniana phosphorus transporter gene PmPT3 and purple acid phosphatase gene PmPAP1 were cloned and their expression in transcription level was analyzed under phosphorus stress.To further validate the function of the two genes in low phosphorus stress,PmPT3 and PmPAP1 were genetically transformed into tobacco(Nicotiana tabacum)�Xanthin' via Agrobacterium-mediated method,transgenic plants were treated with different concentration of phosphorous,then the growth,physiological and biochemical reaction were detected,aiming at providing new information for PmPT3 and PmPAP1,as well as laying the foundation for the creation of new tobacco germplasm with high tolerance to low phosphorus stress,the main results are as follows:1.Gene over-expression vector pSH737-35S-PmPT3 and pSH737-35S-PmPAP1 were constructed and genetically transformed into tobacco via Agrobacterium-mediated method,totally,30 transgenic lines of PmPT3 and 25 transgenic lines of PmPAP1 were obtained respectively.Results of RT-PCR and qPCR indicated that PmPT3 was highly expressed in 16 transgenic lines,PmPAP1 was highly expressed in 13 transgenic lines.2.The transgenic and wild-type tobacco were treated with different concentrations of phosphorus stress for 45 d,and then physiological and biochemical responses were detected.The results showed that the activity of protective enzyme on transgenic tobacco was significantly higher than that of wild type,while the content of MDA decreased significantly under low phosphorus stress.The contents of inorganic phosphorus and total phosphorus of transgenic plants were higher than that of wild type.The ratio of root to shoot increased significantly,demonstrated a strong resistance to low phosphorus stress.Which suggested that over-expression of PmPT3 and PmPAP1 could significantly improve the utilization of phosphorus in tobacco and enhance the resistance to low phosphorus under low phosphorus stress.3.The expression of exogenous genes in transgenic tobacco under different P treatments showed that the expression of PmPT3 in transgenic plants was significantly increased under P deficiency.The results indicated that the expression of PmPAP1 were up-regulated in leaves and roots of the transgenic PmPAP1 tobacco as subjected to phosphorus deficiency in comparison with the normal condition(control),and the expression of its was barely in roots and leaves with high phosphorus treatment.Under the condition of low phosphorus,the activities of acid phosphatase(APase)in roots and leaves of transgenic PmPAP1 tobacco were significantly higher than wild type,the up-regulation of PmPAP1 in transgenic plant root and leaf tissues under P deficiency may improve the activity of APase in plant roots and leaves indirectly to further improve the absorption capacity and efficiency of phosphorus reuse to respond to phosphorus deficiency.4.Effect of over-expression of PmPT3 on Pht1 family gene(NtPT1,NtPT2,NtPT3,NtPT4)in tobacco were detected,the results showed that expression of high affinity phosphate transporter genes(NtPT1,NtPT2,NtPT3)was significantly increased in the transgenic and wild-type tobacco with the phosphorus deficiency,and the expression of these 3 genes in transgenic tobacco were significantly higher than that of wild type,while there was no significant difference in the expression of NtPT4 in transgenic and wild-type plants.5.Effects of Pm PAP1 on the expression of endogenous purple acid phosphatase in tobacco showed that over-expression of Pm PAP1 enhanced the expression of NtPAP1 significantly.However,the transcription level of NtPAP4;12;19 in wild type and transgenic plants was almost the same under p-sufficient and P-deficient conditions.Therefore,we concluded that over-expression of PmPAP1 in tobacco had no significant influence on the 3 endogenous gene of tobacco.