Genetic Transformation And Functional Analysis Of PmMYB169 From Pinus Massoniana

Phosphorus plays an important role in the whole life of plants,however,the concentration of available phosphorus is low and the element is easily fixed in the soil,which causes the plant to be deficient in phosphorus and seriously affects the growth and development of plants.Therefore,the discovery and utilization of low-phosphorus-related genes are beneficial for improving plant tolerance to low phosphorus.It was justified that MYB transcription factors played an important role in plant response to low phosphorus stress.Masson pine is the main forestation species in the subtropical area of China.However,many soils are deficient in phosphorus in the southern part of China,which affected the growth of Pinus massoniana.In our previous work,Pinus massoniana phosphorus transporter gene PmMYB169 were cloned and their expression in transcription level was analyzed under phosphorus stress.To further validate the function of the gene in low phosphorus stress,PmMYB169 was genetically transformed into tobacco(Nicotiana tabacum)and Arabidopsis thaliana.Transgenic plants were subjected to different low-phosphorus stresses,subsequently,their growth,physiological and biochemical characteristics were quantified,and the main results are as follows:1.Gene over-expression vector pCAMBIA1301-PmMYB169 was constructed and genetically transformed into Arabidopsis thaliana and tobacco.Totally,two transgenic lines of Arabidopsis thaliana and 22 transgenic lines of tobacco were obtained.After qRT-PCR detection,three transgenic tobacco lines(Line-2,Line-11 and Line-16)with high expression level were selected for further experiments.2.The transgenic Arabidopsis lines and their wild type were subjected to different concentrations of low-phosphorus stresses.The activity of acid phosphatase,the contents of inorganic phosphorus and total phosphorus in the roots,as well as the biomass of the transgenic plants were higher in comparison with those of the wild type,whereas the content of anthocyanin was lower in the trangenic lines.Interestingly,no significant differences were obtained between the transgenic lines and the wild type under normal phosphorus conditions.The available evidences suggested that the over-expression of PmMYB169 could significantly improve the phosphorus utilization ability of plants under low effective phosphorus,leading to the enhancement of plant tolerance to low-phosphorus stress.3.The transgenic and wild tobacco were were subjected to different concentrations of low-phosphorus stresses.Except that the content of MDA,the content of phosphorus,biomass,Apase activity,POD activity and SOD activity in transgenic tobacco were significantly higher in comparison with tthose of the wild type,under low-phosphorus stress.4.The expression of exogenous gene in transgenic Arabidopsis and tobacco under different concentrations of low-phosphorus stresses showed that theover-expression of PmMYB169 gene in transgenic plants was significantly increased under the condition of phosphorus deficiency in Arabidopsis,and the expression in the root was more obvious;The expression of PmMYB169 gene in transgenic plants after low-phosphorus stress in tobacco was significantly increased under phosphate-free and low-phosphorus stress,and the opposite of high phosphorus treatment.Combining with the results of physiological and biochemical determination,the PmMYB169 gene was used to further improve the reuse efficiency of phosphorus in the body.5.Expression analysis of Arabidopsis on Pht1 family gene PHT1;1 and PHT1;4after over-expression of PmMYB169.The results showed that the expression of PHT1;1 in the roots of transgenic plants was significantly higher in comparison with tthose of the wild type under low-phosphorus stress,although PHT1;4 was higher in the roots than the upper part of the plant,but there was no significant difference in the expression of the gene in the wild type and the transgenic plant.This means that PmMYB169 has a significant effect on PHT1;1 in Arabidopsis thaliana,which may promote PHT1;1 expression by positive regulation,thus promoting the plant's phosphorus absorption capacity and improving plant tolerance to low-phosphorus stress.6.Expression analysis of tobacco on Pht1 family gene NtPT1 and NtPT4 after over-expression of PmMYB169.Overexpression of PmMYB169 gene enhanced the expression of NtPT1 and NtPT4 of tobacco.Although the expression of NtPT4 gene has been upregulated,but there is no significant difference between transgenic plants and wild type plants.7.The pBWA(V)HS-PmMYB169-GLosgfp and pBWA(V)HS-GFP expression vector was transformed into tobacco leaves for transient expression,and observed by laser confocal microscopy.The results showed that PmMYB169 was expressed on nucleus in tobacco leaf.The subcellular localization of tobacco is more effective to determining the role of the PmMYB169 gene in the nucleus,which provides new information for further understanding the function of the PmMYB169 gene.