田旋花对草甘膦耐药性分子机制
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摘要
草甘膦是一种广谱灭生性、内吸传导型除草剂，是国内外防除一年生及多年生杂草的主要药剂。草甘膦的作用机制是抑制植物体内5-烯醇式丙酮酸-3-磷酸合成酶（5-enolpyruvylshikimate-3-phosphate synthase，EPSPS）的活性，导致莽草酸大量积累，并抑制芳香族氨基酸的生物合成，进而扰乱正常的氮代谢致使植物死亡。田旋花（Convolvulus arvensis L.）属旋花科，是一种多年生恶性杂草，其繁殖和再生能力极强，是我国小麦、玉米、大豆、棉花等旱作物田的重要杂草，也是对草甘膦具有天然耐药性的杂草之一。本研究以田旋花为对象，以草甘膦的靶标EPSPS为主线，运用分子生物学等技术，研究田旋花对草甘膦的耐药性机制。主要结论如下：
1.克隆的田旋花EPSPS基因片段长度为1707bp，将EPSPS基因推导出氨基酸序列与其它14种植物进行比对，结果显示田旋花EPSPS保守性较高，与其它植物相比田旋花在141位为丙氨酸，而其它植物为缬氨酸；此外，田旋花和打碗花在155位为丝氨酸，而其它植物为半胱氨酸。田旋花EPSPS氨基酸序列具有典型的EPSP-synthase结构。
2.以田旋花cDNA为模板，合成EPSPS基因并连接到含有35S启动子和GUS基因的pBl l21载体上，构建植物超表达载体pBI-EPSPS。采用农杆菌介导法转化拟南芥，共获得15株转EPSPS基因拟南芥。对转EPSPS基因拟南芥进行草甘膦处理发现，转基因拟南芥草甘膦耐受性增强。
3.采用实时荧光定量PCR测定了田旋花EPSPS基因在不同叶龄、不同器官的相对表达量以及草甘膦对田旋花EPSPS基因表达的影响。结果表明：田旋花EPSPS基因在不同叶龄表达有差异，在9叶期的表达量最高，是3叶期的2.1倍；EPSPS基因在叶的表达量高于茎、子叶和根；用草甘膦对田旋花进行茎叶喷雾处理，EPSPS基因的表达量先升高后降低，在处理后24小时达最大值。随着草甘膦浓度的提高，EPSPS基因表达呈增强趋势。
4.根据已克隆的田旋花EPSPS基因cDNA序列设计三个嵌套引物，通过染色体步移技术获得了1142bp的EPSPS-P启动子片段(GenBank登录号: KC107822)，对启动子序列分析显示该片段富含A/T碱基、TATA-box、CAAT-box及其它顺势作用元件如GATA-motif、TC-rich repeats、spl等。
5.将EPSPS-P与GUS报告基因连接构建植物表达载体，利用农杆菌介导法获得转EPSPS-P拟南芥。对转基因拟南芥进行组织化学染色分析表明，EPSPS基因在根、茎、叶中都有表达，表达量的高低顺序是叶中最高，其次是茎和根。对转EPSPS-P拟南芥进行草甘膦处理，组织化学染色和GUS酶活性检测结果表明草甘膦处理可以引起EPSPS基因表达升高。
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.
1.克隆的田旋花EPSPS基因片段长度为1707bp，将EPSPS基因推导出氨基酸序列与其它14种植物进行比对，结果显示田旋花EPSPS保守性较高，与其它植物相比田旋花在141位为丙氨酸，而其它植物为缬氨酸；此外，田旋花和打碗花在155位为丝氨酸，而其它植物为半胱氨酸。田旋花EPSPS氨基酸序列具有典型的EPSP-synthase结构。
2.以田旋花cDNA为模板，合成EPSPS基因并连接到含有35S启动子和GUS基因的pBl l21载体上，构建植物超表达载体pBI-EPSPS。采用农杆菌介导法转化拟南芥，共获得15株转EPSPS基因拟南芥。对转EPSPS基因拟南芥进行草甘膦处理发现，转基因拟南芥草甘膦耐受性增强。
3.采用实时荧光定量PCR测定了田旋花EPSPS基因在不同叶龄、不同器官的相对表达量以及草甘膦对田旋花EPSPS基因表达的影响。结果表明：田旋花EPSPS基因在不同叶龄表达有差异，在9叶期的表达量最高，是3叶期的2.1倍；EPSPS基因在叶的表达量高于茎、子叶和根；用草甘膦对田旋花进行茎叶喷雾处理，EPSPS基因的表达量先升高后降低，在处理后24小时达最大值。随着草甘膦浓度的提高，EPSPS基因表达呈增强趋势。
4.根据已克隆的田旋花EPSPS基因cDNA序列设计三个嵌套引物，通过染色体步移技术获得了1142bp的EPSPS-P启动子片段(GenBank登录号: KC107822)，对启动子序列分析显示该片段富含A/T碱基、TATA-box、CAAT-box及其它顺势作用元件如GATA-motif、TC-rich repeats、spl等。
5.将EPSPS-P与GUS报告基因连接构建植物表达载体，利用农杆菌介导法获得转EPSPS-P拟南芥。对转基因拟南芥进行组织化学染色分析表明，EPSPS基因在根、茎、叶中都有表达，表达量的高低顺序是叶中最高，其次是茎和根。对转EPSPS-P拟南芥进行草甘膦处理，组织化学染色和GUS酶活性检测结果表明草甘膦处理可以引起EPSPS基因表达升高。
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.
引文
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