Cloning And Function Analysis Of DsGSTU1 Gene Of Digitaria Sanguinalis Resistance To Haloxyfop-P-Methyl

Digitaria sanguinalis is one of the most serious gramineous weeds,which seriously affects the yield and quality of crops.The research group found that Digitaria sanguinalis developed glutathione S-transferase(GSTs)mediated metabolic resistance to haloxyfop-P-methyl,the dominant herbicide in cotton field,and the candidate gene Ds GSTU1 was discovered by transcriptome second-generation sequencing and third-generation sequencing technology.In this paper,based on the results of transcriptome sequencing,the code sequences(CDS)of this gene was amplified and sequenced by PCR.Through sequence analysis and phylogenetic tree analysis,the classification of this gene in GSTs gene family was clarified.The difference of expression levels of this gene in sensitive and resistant Digitaria sanguinalis was compared by real-time quantitative PCR,and the vector p ET32a(+)/Ds GSTU1 was expressed in E.coli.Its metabolic effect on haloxyfop-P-methyl was determined in vitro to verify its function.The research results are as follows.(1)Based on the sequencing results of the third generation and the second generation transcriptome,the partial base sequence of the up-regulated gene Ds GSTU1 was obtained,and the CDS region of the gene was cloned by PCR technology,with a length of 717 bp.(2)Sequence analysis and phylogenetic tree showed that Ds GSTU1 belongs to the GSTs of Tau family,has no signal peptide,is a cytoplasmic GSTs,and is a stable hydrophobic protein,there is no transmembrane domain in its peptide chain,and its phosphorylation sites reach 11.Its tertiary structure is mainly α-helix and β-turn,which are connected with each other by irregular curly structure.(3)The expression level of Ds GSTU1 gene in resistant and sensitive Digitaria sanguinalis before and after haloxyfop-P-methyl treatment was analyzed by real-time quantitative PCR.It was found that Ds GSTU1 gene was expressed in Digitaria Sanguinalis before and 12,24 and 36 h after treatment with haloxyfop-P-methyl,the expression of Ds GSTU1 gene in resistant biotype was 3.86 times higher than that in sensitive biotype before treatment,but 36 h after treatment,the expression of Ds GSTU1 in resistant biotype was 14.92 times higher than that in sensitive biotype,which indicated that the resistance of Digitaria sanguinalis to haloxyfop-P-methyl might be related to the increased expression of Ds GSTU1 gene.(4)The constructed p ET32a(+)/Ds GSTU1 vector was expressed in Escherichia coli,and the GSTs activity of the strain protein extract of the recombinant vector induced expression by IPTG(isopropyl-β-D-thiogalactoside)was determined.The results showed that the GSTs activity of the recombinant vector p ET32a(+)/Ds GSTU1 was 7.17 times than that of recombinant vector without IPTG and 5 times that of the unloaded p ET32a(+)protein.Through in vitro enzyme activity determination,it was found that it had metabolic activity on haloxyfop-P-methyl,and compared with p ET32a(+),the specific activity is 4.87 nmol/min/mg.