The Molecular Mechanism Of Glyphosate Tolerence In Field Bindweed (Convolvulus Arvenisis L.)

Glyphosate is the nonselective, broad-spectrum herbicide for the control of annual and perennialweeds. It inhibits the enzyme5-enolpyruvylshikimate-3phosphate synthase (EPSPS). This inhibitionprevents the biosynthesis of the aromatic amino acids and results in a substantial accumulation ofshikimic acid, and then disrupts the normal nitrogen metabolism and causes plants to die. Fieldbindweed (Convolvulus arvensis L.) is a kind of perennial troublesome weed threatening wheat、maize、soybean and cotton production in China. Meanwhile, field bindweed is a naturally glyphosate toleranceweed. This study focuses on glyphosate target EPSPS to investigate the glyphosate tolerance mechanismin field bindweed. The results showed as follows:1. The full-length cDNA of1,707nucleotides from Convolvulus arvensis was cloned. The results ofhomology analysis revealed that EPSPS showed highly homologous with EPSPS proteins fromother14plant species. However, comparison of the EPSPS gene sequence reaveled that valine atposition141was substituted by alanine in field bindweed. Moreover, cysteine at position155wassubstituted by serine in field bindweed and calystegia hederacea. The typical EPSP-synthasestructure was found in field bindweed EPSPS.2. EPSPS gene was cloned with the template of cDNA from field bindweed and inserted into plasmidpBI121which contains promoter35S and GUS gene. A plant super expressed vector ofpBI-EPSPS was constructed and transferred to Arabidopsis by the Agrobacterium mediatedtransformation system. Fifteen transgenic plants were finally obtained. Tteated with glyphosate,transgenic plants showed more tolerance to glyphosate.3. Quantitative RT-PCR was employed to analysize the expression of EPSPS gene in field bindweedat different stages, different organs and the induction of glyphosate to EPSPS mRNA. The resultsshowed that the expression of EPSPS was different at different leaf stages. The highest expressionlevel was found at12leaf stage and was2.1times than3leaf stage. The expression of EPSPS inleaves was higher than stems, cotyledon and roots. After glyphosate treatment, EPSPS expressionlevel increased fist and then decreased, the peak induction was found at24h after glyphosatetreatment. EPSPS expression level increased higher with the higher glyphosate concentration.4. According to the EPSPS gene cDNA from Field bindweed already cloned, a1142bp promotersequence (Genbank accession number: KC107822) of EPSPS was obtained by genome walking.Sequence analysis indicated that A/T base、TATA-box、CAAT-box and other cis-acting elementswere found within the promoter sequence, such as, GATA-motif, TC-rich repeats, spl and so on.5. A GUS expression construction driven by EPSPS-P was introduced into Arabidopsis with thefloral dip method. Histochemical analysis of transgenic Arabidopsis showed that GUS enzymeactivity was present in leaves, stems and roots. However, it was relatively higher in leaves and stems than roots. After treated with glyphosate, histochemical and GUS enzyme activity analysisindicated that GUS activity in transgenic Arabidopsis was improved after glyphosate treatments.