T-DNA Integration Information Of Double Bt Gene Populus×euramericana “Neva” And Its Effect On Gene Expression

Populus × euramericana “Neva” has gained widespread attention for its advantages of high quality,rapid growth and abundant yield.As the most effective way of genetic improvement,transgenic technology has shown great potential in the genetic improvement of forest trees.In order to clarify the T-DNA insertion sites and unintended effects of transgenic lines,and to make the new transgenic poplar varieties enter a higher stage of evaluation,the molecular response mechanism of Populus × euramericana“Neva” to Bt gene was explored.In this study,six transgenic double Bt gene 107 lines(DB1,DB7,DB9,DB11,DB14,DB15)screened in the laboratory at the stage of environmental release were selected as research objects,measurement of growth and physiological and other indicators for each line.Based on NGS sequencing technology,the T-DNA integration information of the transgenic lines was clarified and the preference of T-DNA insertion was preliminarily investigated.Probing the effect of T-DNA insertion on 107 popular gene expressions using RNA-seq sequencing to reveal the molecular mechanism of unintended effects.The response mechanism of 107 popular to Bt gene was investigated based on weighted gene co-expression network analysis(WGCNA).The main findings are as follows:1.Compared with the control,the transgenic lines showed no significant differences in growth indicators.While the insect resistance was enhanced,the net photosynthetic rate of the transgenic lines increased,and the leaf pigment content,especially the content of chlorophyll a,increased,and the leaf color difference red-green value a *It becomes smaller,the green becomes lighter,the red becomes deeper,and the leaf area of some plants increases.The content of starch and sucrose increased,and some strains reached a significant level(p<0.05).2.A total of 15 insertion sites were detected in six transgenic lines based on NGS technology,with DB1,DB7,DB9,DB11,DB14 and DB15 lines having 5,2,1,4 and 1T-DNA copies,respectively.T-DNA insertion resulted in the deletion of small fragments of Populus 107 genome,and the deletion of vector boundary was serious,and only a single end of T-DNA could be detected at most sites.T-DNA preferred to insert into regions rich in AT bases,with an average AT content of 68.9% upstream and downstream of the 15 insertion sites,and the highest site reached 76.5%;The insertion of T-DNA did not result in significant changes in the expression of disrupted genes and upstream and downstream efficiently expressed genes,but activated the trace expression of some genes.3.Leaf RNA-seq analysis showed that the integration of exogenous genes resulted in significant changes in gene expression in Populus 107.A total of 4342 DEGs were identified in the six comparison groups,among which the DB1 vs CK comparison group had the most DEGs(1055),the DB11 vs CK comparison group had the least DEGs(419).Compared with insertion position and copy number,the expression of exogenous Bt gene has a greater impact on the gene expression of Populus 107,and the Cry1 Ac toxin protein plays a major role.Insertion of the Bt gene resulted in significant enrichment of the transgenic line in GO terms of lignin synthesis,redox response salt stress response,cell wall,hydrolase activity and polysaccharide binding capacity.The metabolic pathways most affected by the Bt gene were starch and sucrose metabolism,pentose and gluconate conversion,with most of the associated DEGs down-regulated in expression and reduced enzyme activity.4.WGCNA was performed based on the gene expression level in RNA-seq results,and 21 co-expression modules were identified by consensus.Module phenotype association showed,Brown module had a high correlation with Cry1 Ac transcript abundance(r2=0.51,r=0.71,p=0.02);The Turquoise module had a negative correlation with Cry1 Ac toxin content(r2=0.48,r=0.69,p=0.03);Tan module,green module and copy number were negatively correlated;No module with significant correlation with Cry3 A gene transcript abundance and Cry3 A toxin content was detected;12 hub genes with modules significantly correlated with Cry1 Ac transcription,expression and copy number were screened.The functional annotation of HUB gene showed that these genes were related to starch and sucrose conversion,catalysis,material transfer membrane transport and sugar metabolism.