Genetic Transformation And Expression Study Of Multiple Insect-resistant And Salt-Tolerance Genes On Populus Nigra L.

Pest and soil salinization are important factors that restrict the growth of trees.By transgenic technology,it is possible to improve the resistance of trees and promote the growth of trees.In this study,transgenic plants were obtained by Agrobacterium-mediated transformation of the double Bt genes,Cry1Ac gene,Cry3A gene and two salt-tolerance genes,mtl D gene and BADH gene.The expression of exogenous genes were detected by PCR,fluorescence quantitative PCR,enzyme-linked immunosorbent assay,insect resistance test and salt tolerance test.The main results were obtained in 10 transgenic lines from DNA level,transcription level and translation level respectively.The main results were as follows:（1）Genetic transformation of Populus nigra L.was carried out by Agrobacterium-mediated transformation,and 28 resistant strains were obtained by the screening of Kanamycin sulfate.The DNA of tentransgenic lines and non-transgenic control were extracted,the results of PCR showed that there were four exogenous gene bands in 10 transgenic lines,but no bands were amplified from the non-transgenic control.The results showed that the exogenous genes were inserted into the genome of Populus nigra L..（2）Fluorescence quantitative PCR showed that the fluorescence signals of Cry1Ac,Cry3A,mtl D and BADH genes were detected in the transgenic lines,while which were not detected in the control Populus nigra L..The transcriptional abundance of Cry1Ac gene was on the order of 10⁵-10⁸,and the transcriptional abundance of the transgenic lines were significantly different.The transcriptional abundance of Cry3A gene was on the order of10⁶-10⁷,and there weren’t significant differences in the transcriptional abundance among transgenic lines.The transcriptional abundance of mtl D gene was on the order of 10⁵-10⁸,and the transcriptional abundance of transgenic lines were significantly different.The transcriptional abundance of the BADH gene was on the order of 10⁴-10⁷,and the transcriptional abundance among the transgenic lines were significantly different.There was aextremely significant positive correlation between the transcription abundance of the double Bt genes.The transcription abundance of double Bt genes were not associated with the transcription abundance of two salt-tolerance genes.The transcriptional relationship between the two salt-tolerant genes mtl D and BADH was not big.（3）The protein expression of transgenic lines was detected by enzyme-linked immunosorbent assay（ELISA）.The results showed that 10 transgenic lines had Bt protein expression,but the transgenic control hadn’t.The average expression level of Cry1Ac protein in 10 transgenic lines was 9.93 ng·g^-1,there was no significant difference in the expression level among the lines.The average expression level of Cry3A protein in 10transgenic lines was 7346.18 ng·g^-1,and there were significant differences in the expression levels among the lines.The correlation analysis showed that there was a significant negative correlation between the expression of Cry1Ac protein and the expression of Cry3A protein,there was utual inhibition between the two expressions.（4）The results of laboratory test showed that the transgenic lines showed good insect resistance.The mortality rate of the larvae of the Hyphantria cunea（Drury）was 60.00%-100%,and the mortality among the transgenic lines was significantly different,and it was significantly higher than that of the non-transgenic control.The larvae mortality of the Plagiodera versicolora Laicharting was 66.67%-100%,there was significant difference in mortality among the transgenic lines,and it was significantly higher than the control.（5）A preliminary salt tolerance test was carried out for seed plants of transgenic lines1 and 3 with better insect resistance,the preliminary results showed that the two transgenic lines showed no obvious tolerance to salt stress,comparing with the non-transgenic control.The decrease of plant height,photosynthetic characteristics,fluorescence kinetics parameters,chlorophyll content and relative conductivity was lower than that of non-transgenic control under salt stress,but the difference was not significant.The results of biomass measurement showed that the decrease of biomass,under 3‰Na Cl condition,in the underground part,No.3 strain was less than non-transgenic control,and non-transgenic control was less than No.1strain,in the aboveground part,No.3 strainand non-transgenic control decreased the same,non-transgenic control decreased less than No.1 strain;under6‰Na Cl,the decrease of underground and aboveground biomass of transgenic lines was higher than that of the control,the difference was not significant.