Identification Of Tobacco BRI1 Gene Family And Study Of Drought Resistance Of BRL3 Gen

Tobacco(Nicotiana tabacum L.)is an important economic crop and plays a pivotal role in the national economy.Guizhou is the second largest tobacco growing area in the country.In 2014,the planting area of K326 increased significantly,accounting for 23.72% of the total planting area in the country,second only to Yunyan87.Under drought stress,the yield of K326 is low,and the drought resistance is not obvious,which will cause serious economic losses.To this end,this study analyzed the response mode of Nt BRI1 family genes in K326 under drought stress and performed bioinformatics analysis,constructed an overexpression vector of tobacco Nt BRL3 gene,genetically transformed it into tobacco K326,and combined metabolome and transcriptome sequencing to explore the The response mechanism of Nt BRL3 gene to drought stress.The main research results obtained are as follows:1、In this study,four member genes of the tobacco Nt BRI1 family were identified and named Nt BRI1,Nt BRL2-1,Nt BRL2-2,and Nt BRL3 according to the genes in the same subline as the Arabidopsis BRI1 family in the phylogenetic tree.In the tobacco Nt BRI1 family,Nt BRL2-1 is the smallest protein with 1133 amino acids(aa),while Nt BRI1 is the largest protein with 1213 amino acids(aa).The molecular weight of the protein ranged from 121.07 KDa(Nt BRI1)to 132.68 KDa(Nt BRL3),with an average of 126.23 KDa.The p I ranged from 5.75(Nt BRL2-2)to 11.17(Nt BRI1),with a mean of 5.96.According to the prediction of subcellular localization,the members of tobacco Nt BRI1 gene were all localized on the cell membrane.2、The expression pattern of Nt BRI1 gene family in K326 showed that the expression levels of Nt BRI1 gene and Nt BRL3 gene were higher in roots.After 2hours of drought stress treatment,Nt BRI1 and Nt BRL3 genes were significantly up-regulated in roots,reached the maximum at 12 hours,and down-regulated at 24hours;Nt BRI1 and Nt BRL3 genes showed a trend of up-regulation in stems and leaves,but the up-regulation was not significant.After 2 hours of drought stress,the expression levels of Nt BRL2-1 and Nt BRL2-2 were slightly increased in roots,and down-regulated in stems and leaves,and the down-regulation of Nt BRL2-2 was most significant in stems and leaves.It indicated that Nt BRI1 and Nt BRL3 were induced by drought.3、Under drought stress,compared with wild-type plants,the plants overexpressing the Nt BRL3 gene had enhanced GST,POD,SOD and CAT enzyme activities,increased chlorophyll content,soluble sugar content and proline content,and malondialdehyde oxidation Substance content decreased,indicating that overexpression of Nt BRL3 gene can effectively improve the drought resistance of overexpression tobacco.4、The results of metabolomics analysis showed that a total of 528 differentially accumulated metabolites were detected in the roots of Nt BRL3 ox and WT plants,and85 metabolites were significantly different,of which 33 were up-regulated and 52 were down-regulated.Excessive accumulation of maltose and sucrose in Nt BRL3 ox compared with wild type.Among the free amino acids,proline,methionine,arginine,tyrosine,isoleucine,and asparagine were found to accumulate in excess.It shows that Nt BRL3 can improve the drought resistance of tobacco by promoting the accumulation of free amino acids in plants.5、The results of transcriptome analysis showed that there were 106 differential genes between overexpressed Nt BRL3 ox and wild-type plants,of which 49 differential metabolites were up-regulated and 57 differential metabolites were down-regulated.Drought resistance-related genes among the significantly different genes were obtained,and these genes were involved in the response of plants to drought stress and their expression induced by drought.Further analysis revealed that there were four genes that were induced by JA and two genes that were related to ABA-induced response.At the same time,a carotenoid-cleaving dioxygenase(CCD8)involved in strigolactone(SL)biosynthesis,two UDP-glycosyltransferase(UGT)genes and a glutathione S-transfer enzyme(GST)gene.6、The results of correlation analysis between transcriptome and metabolome showed that in Nt BRLox,UDP-glycosyltransferase was significantly positively correlated with all sugars except maltotetraose,indicating that the high expression of UDP-glycosyltransferase gene may be related to carbohydrate metabolism.related to the upregulation of the product.The BURP domain protein RD22-like gene(RD22)is highly related to a variety of sugar and amino acid metabolites,suggesting that RD22 genes may be involved in the regulation of abiotic stress responses.The results showed that Nt BRL3 not only affected the accumulation of metabolites,but also induced the up-regulation of drought-related genes,and these up-regulated differential metabolites showed a significant positive correlation with differential genes.It shows that Nt BRL3 may regulate the accumulation of metabolites by affecting the expression level of genes,and finally improve the drought resistance of plants.In summary,under drought stress,we identified and bioinformatically analyzed the Nt BRI1 gene family in tobacco,and detected the phenotype and related enzyme activity changes of Nt BRL3 gene overexpressed plants under drought conditions by analyzing gene structure and expression.Combined with the analysis of metabolome and transcriptome data,the response mechanism of Nt BRL3 gene in tobacco K326 in the face of drought stress was studied,and it was proved that Nt BRL3 overexpression can enhance the drought resistance of tobacco,providing a theoretical basis for guiding tobacco stress-resistant cultivation and breeding.