CN111139228B - 一种抗草甘膦植物epsps酶双突变体及其克隆、表达与应用 - Google Patents
一种抗草甘膦植物epsps酶双突变体及其克隆、表达与应用 Download PDFInfo
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Abstract
本发明属于基因工程技术领域，具体涉及一种通过基因突变技术对来源于甘蓝型油菜的EPSPS酶进行定向进化，以对其草甘膦抗性进行改良，并公开该突变体及其编码基因在新型耐草甘膦作物培育领域中的应用。本发明所述抗草甘膦植物EPSPS酶双突变体，通过体外定向进化技术改造甘蓝型油菜EPSPS基因BnEPSPS，使之对草甘膦抗性的提高，进而获得具有草甘膦抗性的植物来源的EPSPS双突变体BnEV1和BnEV2。突变体BnEV1和BnEV2酶学性质测定表明草甘膦耐受性显著提升，并验证其应用到植物中提高植物的抗性，可为培育消费者更易接受的转基因抗草甘膦油菜新品种，提供重要的遗传资源，具有广泛的应用价值。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种通过基因突变技术对来源于甘蓝型油菜的5-烯醇丙酮酸莽草酸-3-磷酸合酶(EPSPS)进行定向进化，以对其草甘膦抗性进行改良，并进一步公开了该突变体及其编码基因在新型耐草甘膦作物培育领域中的应用。
背景技术
在作物种植时，由于杂草与农作物争夺有限的光照、水分和营养等物质，会严重影响农作物的产量，而日益攀升的人工除草的成本也使得农作物收益减少。草甘膦(商品名为Roundup)化学名N-磷酸亚甲基甘氨酸(N-(phosphonomethyl)glycine，GLP)，草甘膦与甘氨酸结构类似，属于甘氨酸的衍生物。草甘膦是一种非选择性、高效除草剂，具有高效、广谱和易降解的特点，是目前全球生产和使用最为广泛的广谱除草剂，也是目前全球使用量最大的农药。研究显示，草甘膦与抗草甘膦作物的联合使用可以有效解决除草难的问题，而如何有效提高作物的抗草甘膦性能也具有重要的作用。
在生物体内，由于芳香族氨基酸会参与维生素、生物碱和吲哚衍生物等多种物质的合成，并在蛋白质合成、细胞分裂等过程中发挥重要的生物功能。因此，生物体内芳香族氨基酸的减少会严重干扰植物的正常代谢，并进而影响植物的生长发育。研究表明，莽草酸途径是连接碳水化合物和芳香族氨基酸生物合成的重要生物代谢途径，对于植物芳香族氨基酸的生物合成具有极其重要的地位，该途径只存在于细菌、真菌、藻类和植物中。莽草酸途径的关键酶即是5-烯醇式丙酮酸莽草酸-3-磷酸合成酶(5-enolpyruvylshikimate-3-phosphate synthase，EPSPS)，该酶是莽草酸途径中第六步的关键酶，以PEP和3-磷酸莽草酸(shikimate-3-phosphate，S3P)为底物，催化合成5-烯醇式丙酮酸莽草酸-3-磷酸(EPSP)；随后分支酸合成酶催化EPSP形成芳香族氨基酸的前体物分支酸。草甘膦与EPSPS的作用底物之一PEP的过渡态具有类似的结构，因此具有竞争性地抑制EPSPS的作用。草甘膦的作用机理是以竞争磷酸烯醇式丙酮酸(PEP)和非竞争莽草酸-3-磷酸(S3P)的方式同植物体内5-烯醇式莽草酸-3-磷酸合酶(EPSPS)结合，形成稳定的EPSPS-S3P-草甘膦复合物，引起EPSPS活性的丧失，大量碳源流向S3P造成莽草酸在组织中快速积累。另一方面，蛋白质生物合成所必需的芳香族氨基酸合成则严重受阻，最终导致植物生长受到抑制直至死亡。草甘膦在发挥其除草功效的同时，随之带来的确是不得不面对的安全隐患，其中最重要的即是如何解决种植作物的抗草甘膦性能。
目前，国际上广泛应用的抗草甘膦基因为CP4-EPSPS，其来源为草甘膦极度污染的环境中分离得到的农杆菌CP4-Agrobacterium tumefaciens-[]。相对于跨物种的基因转移，利用作物自身的基因进行抗除草剂育种，理论上更容易消除消费者对转基因安全的顾虑。
甘蓝型油菜(Brassica napus)，十字花科芸苔属植物，是世界四大油料作物之一，具有重要的经济价值。目前，转基因抗草甘膦油菜在加拿大，美国及澳大利亚广发种植，取得了巨大的经济效益。目前所用的抗性基因均为Agrobacterium tumefaciens CP4来源的EPSPS，但甘蓝型油菜EPSPS对草甘膦敏感，抗草甘膦性能并不突出。因此，如何进一步提高甘蓝型油菜等作物的抗草甘膦性能具有重要的意义。
发明内容
为此，本发明所要解决的技术问题在于提供一种抗草甘膦植物EPSPS酶双突变体，所述双突变体通过体外定向进化技术改造油菜EPSPS基因获得，是一种具有草甘膦抗性的植物来源的EPSPS；
本发明解决的第二个技术问题在于提供上述抗草甘膦植物EPSPS酶双突变体的克隆、表达与应用。
为解决上述技术问题，本发明所述的一种抗草甘膦植物EPSPS酶双突变体，所述双突变体是在植物源EPSPS酶的基础上，将第167位的苏氨酸进行突变为异亮氨酸或亮氨酸，以及，将第171位的脯氨酸突变为丝氨酸或蛋氨酸。
具体的，所述的抗草甘膦植物EPSPS酶双突变体，所述双突变体是将第167位的苏氨酸进行突变为异亮氨酸，以及，将第171位的脯氨酸突变为丝氨酸，记为BnEV1；所述双突变体BnEV1包含如SEQ ID No：4所示的氨基酸序列。
具体的，所述的抗草甘膦植物EPSPS酶双突变体，所述双突变体是将第167位的苏氨酸进行突变为亮氨酸，以及，将第171位的脯氨酸突变为蛋氨酸，记为BnEV2；所述双突变体BnEV1包含如SEQ ID No：6所示的氨基酸序列。
具体的，所述植物源EPSPS酶包括甘蓝型油菜EPSPS酶。
本发明还公开了一种编码所述抗草甘膦植物EPSPS酶双突变体的基因。
具体的，所述的基因包含如SEQ ID No：3(BnEV1)或SEQ ID No：5(BnEV2)所示的核苷酸序列。
本发明还公开了一种含有所述抗草甘膦植物EPSPS酶双突变体编码基因的表达载体，即包括pGEX-6p-BnEV1和pGEX-6p-BnEV2重组质粒。
本发明还公开了一种含有所述抗草甘膦植物EPSPS酶双突变体编码基因的基因细胞系。
本发明还公开了一种含有所述抗草甘膦植物EPSPS酶双突变体编码基因的重组菌。
本发明还公开了所述抗草甘膦植物EPSPS酶双突变体在培育抗草甘膦转基因作物领域中的应用。
本发明还公开了一种培育抗草甘膦转基因作物的方法，即包括将所述抗草甘膦植物EPSPS酶双突变体转化入目的植物的步骤。优选的，所述植物包括甘蓝型油菜。
本发明所述抗草甘膦植物EPSPS酶双突变体，通过体外定向进化技术及饱和突变的方法改造甘蓝型油菜EPSPS基因BnEPSPS，进而获得具有草甘膦抗性的植物来源的EPSPS双突变体BnEV1和BnEV2，使之对草甘膦抗性的提高，并通过将所述双突变体BnEV1和BnEV2的重组质粒转入大肠杆菌aroA缺陷型菌株大肠杆菌Escherichia coli AB2829中，突变体BnEV1和BnEV2在重组的大肠杆菌菌株能够在含有300mM草甘膦的M9基础盐培养基中生长，突变体BnEV1酶学性质测定表明草甘膦耐受性显著提升，突变体BnEV1转基因烟草能够耐受5倍商业推荐剂量中量的农达，验证了所述双突变体对草甘膦的抗性水平，并验证其应用到植物中提高植物的抗性，可为培育消费者更易接受的转基因抗草甘膦油菜新品种，提供重要的遗传资源，具有广泛的应用价值。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明，其中，
图1为本发明基因突变的工艺流程图；
图2为表达载体pGEX-6p-BnEPSPS的质粒图谱；
图3为获得突变体BnEV1和BnEV2的抗性分析结果；其中，a为不含草甘膦的M9；b为含50mM草甘膦的M9；c为含100mM草甘膦的M9；d为含200mM草甘膦的M9；
图4为获得突变体BnEV1的诱导表达结果；其中，泳道1为IPTG诱导后的E.coliBL21(DE3)；泳道2为IPTG诱导后的含有pGEX-6p-1的E.coli BL21(DE3)；泳道3为IPTG诱导后含有pGEX-6p-BnEPSPS的E.coli BL21(DE3)；泳道4为纯化后的BnEPSPS；泳道5为IPTG诱导后含有pGEX-6p-BnEV1的E.coli BL21(DE3)；泳道6为纯化后的BnEV1 EPSPS；
图5为获得突变体BnEV1最适pH和最适温度的测定结果；其中，a为突变体BnEV1的最适pH；b为突变体BnEV1的最适温度；WT代表BnEPSPS，BnEV1代表BnEV1EPSPS；
图6为BnEPSPS和BnEV1的酶动力学曲线；其中，a为Km(PEP)，b为Km(S3P)；
图7为EPSPS的抑制动力学曲线；其中，a为BnEPSPS的Ki曲线；b为BnEV1的Ki曲线；c为草甘膦对EPSPS的IC50曲线；d为Kcat(gly)；
图8为pCAMBIA1300S-BnEV1转基因载体图谱；其中，a为植物载体构建示意图；b为EPSPS PCR扩增，泳道1为BnEPSPS，泳道2为BnEV1；c为农杆菌转化子验证，泳道1为BnEPSPS，泳道2为BnEV1；注：B代表空白对照，N代表阴性对照，P代表阳性对照；
图9为所述抗草甘膦转基因植物的转化流程；
图10为pCAMBIA1300S-BnEV1的转基因验证结果；
图11为转化植物验证抗性结果；注：左图为草甘膦未处理的BnEV1转基因烟草；中图为0.8％的农达处理野生型BnEPSPS转基因烟草；右图为4.0％的农达处理BnEV1转基因烟草。
具体实施方式
如图1所示给出了本发明所述基因突变的工艺流程图，即通过饱和突变的方法改造甘蓝型油菜EPSPS基因BnEPSPS，进而获得双突变体BnEV1和BnEV2。
本发明中草甘膦抗性基因BnEPSPS基因的核苷酸序列如SEQ ID NO：1所示，序列全长为1527bp，编码509个氨基酸残基序列如SEQ ID NO：2所示。
实施例1 BnEPSPS饱和突变体库构建与筛选
依据EPSPS保守序列LFLGNAGTAMRPL找到双突变T102/P106所对应的在BnEPSPS中的位置，分别位于BnEPSPS的167位和171位，设计引物如下：
BnS-F：
5’-NNNACGCATGGCNNNTCCAGCATTCCCAAGGTACAACTCGATATCACTC-3’；
BnS-R：
5’-GTACCTTGGGAATGCTGGANNNGCCATGCGTNNNCTTACCGCTGCAG-3’；
引物中的N代表A/G/C/T四种碱基的混合物，由武汉金开瑞生物技术有限公司合成。
饱和突变PCR 以pGEX-6p-BnEPSPS(将其编码基因通过pGEX-6p-1质粒载体上重组得到，谱图如附图2所示)重组质粒为模板，利用饱和突变引物进行环状PCR，PCR反应体系如下表1所示。
表1饱和突变的PCR反应体系
| 试剂 | 用量(μL) |
| 质粒DNA(30ng/μL) | 1 |
| dNTPs(10mM) | 4 |
| 5×FastPfu Buffer | 10 |
| BnS-F,10μM | 1 |
| BnS-F,10μM | 1 |
| FastPfu DNA polymerase | 1 |
| 加灭菌双蒸水至 | 50 |
PCR反应条件为：94℃预变性5min；94℃变性30s，58℃退火30s，72℃延伸3min30s；25个循环；72℃延伸10min，4℃保存5min结束。
PCR结束后，将PCR产物置于1.5mL的离心管中，加入3倍体积Binding buffer，将溶液加入到2mL吸附管12,000r/min，4℃离心1min，加入600μL洗脱液离心洗脱1min，再加入600μL洗脱液离心洗脱1min，将缓冲液弃去后再离心2min，室温晾置5min，加入30μL双蒸水洗脱收集DNA，-20℃保存，备用。
模板消化 因饱和突变模板是采用的质粒DNA，模板会对突变筛选产生干扰，不利于筛选。Dpn I能够切断来源于常用E.coli(dam+)的DNA，不能够切断PCR产物，遂采用Dpn I消化PCR产物，体系如下表2所示。
表2 PCR产物消化体系
| 试剂 | 用量(μL) |
| PCR产物(60ng/μL) | 30 |
| Takara Q cut Dpn I | 2 |
| 10×Q cut buffer | 5 |
| 加灭菌双蒸水至 | 50 |
37℃放置2h，采用美集生物的PCR回收试剂盒直接回收法消化后的DNA。将PCR产物置于1.5mL的离心管中，加入3倍体积Binding buffer(试剂盒自带)，将溶液加入到2mL吸附管12,000r/min，4℃离心1min，加入600μL洗脱液离心洗脱1min，再加入600μL洗脱液(试剂盒自带)离心洗脱1min，将缓冲液弃去后再离心2min，室温晾置5min，加入30μL双蒸水洗脱收集DNA，-20℃保存，备用。
电转化 取酶连产物1μL与50μL大肠杆菌感受态细胞E.coli DMT感受态细胞(购自TAKARA公司)混合，加入到预冷的1mm电转杯中，用2.1KV电压电击；迅速加入500μL液体LB培养基(胰蛋白胨10g，酵母粉5g，NaCl 10g，调pH至7.5，加ddH2O定容至1L，分装后121℃下高压蒸汽灭菌30min)，于37℃复苏60min；涂布在含有100μg/mL氨苄青霉素的LB平板上，37℃过夜。挑取转化子，接种到含有20mM草甘膦的M9液体(配方：Na2HPO4 6.8g，KH2PO4 3g，NaCl0.5g，NH4Cl 1g，20mL 1M葡萄糖，2mL 1M MgSO4，100μL 1M CaCl2加双蒸水至1L，灭菌前调pH至7.6，在121℃下高压蒸汽灭菌30min)培养基的96深孔板中于37℃震荡培养，测定OD600筛选有抗性的转化子。通过筛选10,000个转化子，筛选到两个突变体。
测序验证表明，上述操作共获得两个双突变体，其中，突变体BnEV1的核酸序列如序列表SEQ ID NO：3所示，序列全长为1527bp，编码509个氨基酸残基序列如序列表SEQ IDNO：4所示，其是将第167位的苏氨酸进行突变为异亮氨酸以及将第171位的脯氨酸突变为丝氨酸(T167I/P171S)；突变体BnEV2的核酸序列如序列表SEQ ID NO：5所示，序列全长为1527bp，编码509个氨基酸残基序列如序列表SEQ ID NO：6所示，其是将第167位的苏氨酸进行突变为亮氨酸以及将第171位的脯氨酸突变为蛋氨酸(T167L/P171M)。
突变体质粒的提取 挑取抗性突变体菌株单菌落，用装有10mL含100μg/mL的氨苄青霉素的LB液体培养基的试管内，于37℃震荡培养过夜。采用AXYGEN小量质粒抽提试剂盒提取质粒。用4mL离心管收集菌体，以12,000r/min离心1min，弃上清，加入250μL溶液Ⅰ(试剂盒自带)重悬菌体；混匀，加入250μL溶液Ⅱ(试剂盒自带)，轻轻颠倒混匀，放置时间不超过5min；加入350μL溶液Ⅲ(试剂盒自带)，颠倒混匀；于12,000r/min离心10min，取上清，转换至制备管，以12,000r/min离心1min，弃滤液；加入500μL Buffer W1(试剂盒自带)，以12,000r/min离心1min，弃滤液；加700μL Buffer W2(试剂盒自带)，于12,000r/min离心1min；以同样的方法再用700μL Buffer W2洗涤一次，弃滤液；将制备管置回2mL离心管中，于12,000r/min离心2min；室温晾置5min，加30μL去离子水洗脱，得到基因突变后的重组质粒，并进行琼脂糖凝胶电泳验证，将成功突变的质粒于-20℃保存备用。
实施例2突变体BnEV1和BnEV2抗性分析
分别将所得双突变体BnEV1和BnEV2的编码基因通过pGEX-6p-1质粒载体上重组得到pGEX-6p-BnEV1和pGEX-6p-BnEV2质粒。
分别将pGEX-6p-BnEPSPS、pGEX-6p-BnEV1和pGEX-6p-BnEV2质粒转化至E.coliAB2829感受态，挑取转化子单菌落置于氨苄青霉素(Ampicillin)的抗性LB中，于37℃过夜培养，洗涤2遍除去LB营养成分，调整菌体浓度OD600至1.0，按2％转接量转接于含有不同浓度(0mM、50mM、100mM、200mM)草甘膦M9基础盐培养基中，每组设置3个平行。于37℃、200r/min摇床上培养，每隔3h利用分光光度计测定OD600数值。测定生长曲线如图3所示，可见，双突变体BnEV1和BnEV2的抗性水平明显提升，且BnEV1比BnEV2抗性要好。
实施例3突变体BnEV1表达纯化
分别将pGEX-6p-BnEV1、pGEX-6p-BnEPSPS质粒转化至E.coli BL21(DE3)，将鉴定阳性克隆子过夜活化，以体积比5％的接种量转接至10mL含100μg/mL Ampicillin的LB液体培养基中，于37℃培养3h-5h，直至OD600达到0.6左右，加入诱导剂IPTG使终浓度至0.1mM，于18℃、180r/min下诱导培养16h。离心收集菌体，然后用Hepes缓冲液(50mM 4-羟乙基哌嗪乙磺酸(Hepes)，调pH至7.6，补足双蒸水至1L)将菌体洗涤2遍，再用1mL Hepes缓冲液悬浮后用超声波破碎仪破碎细胞。将细胞破碎液于4℃、12,000r/min离心10min，分别收集上清和沉淀，沉淀用Hepes缓冲液将菌体洗涤2遍，用100μL Hepes缓冲液悬浮。采用SDS-PAGE法检测目的蛋白的表达，SDS-PAGE配方如下表3所示。
表3 SDS-PAGE配方
取20μL细胞破碎液，加5μL 5×蛋白上样缓冲液混合均匀，沸水浴10min，离然后点样10μL于梳齿孔中，采用80V电压电泳直至溴酚蓝移至上下层胶分界线处，调整为120V电压电泳，溴酚蓝离胶底部1cm时关闭电源。剥离上层胶，加染色液染色1.5h，洗去残留加脱色液脱色，于室温、75r/min脱色直至能看到清晰条带。
大量纯化是在小量诱导表达后进行的，挑取含有重组质粒的E.coli BL21(DE3)单菌落，接种到10mL含有100μg/mL氨苄青霉素的LB液体培养基中，180r/min，37℃过夜震荡培养。以1％的接种量转接至含500mL LB液体培养基的大型三角瓶，37℃培养2-3h，当OD600达0.6左右时，加入IPTG至终浓度0.1mM/L，18℃、180r/min，诱导16h左右。诱导完成后，7,500r/min离心10min收集菌体，用事先预冷的Hepes缓冲液(pH 7.0)洗涤菌体，离心去上清，洗涤两遍，再用100mL新鲜Hepes buffer充分悬浮菌体，然后用低温高压破碎仪破碎细胞，收集细胞破碎液后于12,000r/min，4℃离心60min，收集上清，进行GST亲和层析纯化。吸取lmL GSH Sepharose 4B柱材料，加入到层析柱中，先用预冷ddH2O洗去乙醇，再用Hepes缓冲液洗涤柱材料约100mL左右，使柱材料充分平衡；平衡后的柱材料加入到细胞破碎液上清中，冰上振荡75r/min孵育2h,将孵育过后的细胞破碎液上清加入到层析柱中，过2-3遍，尽量使柱材料能够结合更多的蛋白；上样完成后，往层析柱中加入Hepes缓冲液约1L，充分洗涤柱材料；取一定浓度的3C蛋白酶与1mL Hepes buffer混匀，加入层析柱，尽量使3C蛋白酶与柱材料混合均匀，4℃过夜酶解；收集酶解后的蛋白，必要时可再次加入1mL Hepes缓冲液进行洗脱；SDS-PAGE电泳检测纯化后的BnEPSPS蛋白质浓度和纯度。SDS-PAGE法检测目的蛋白表达、纯化情况如图4所示。结果表明，BnEPSPS及突变体BnEV1在上清中均有可溶性表达，且经3C蛋白酶酶切后，目的蛋白大小与预测酶切后的55.3kDa大小一致。
蛋白浓度测定采用Braford快速蛋白定量试剂盒(购自庄盟生物)测定。按Bradford稀释液：Bradford储存液＝47：3的比列新鲜配制混匀；然后采用离心的办法(或滤纸过滤)将粗杂质去掉备用。将试剂盒附带的蛋白标准品1mg/mL牛血清白蛋白(BSA)稀释成不同的浓度：200μg/mL、175μg/mL、150μg/mL、15μg/mL、125μg/mL、100μg/mL、75μg/mL、50μg/mL、25μg/mL、0μg/mL，分别取20μL与200μL新鲜配制的Bradford显色液混合均匀，室温放置5min，取200μL于96浅孔板测定OD595，制作标准曲线。将20μL蛋白样品与Bradford显色液混合测定OD595，根据标准曲线计算蛋白浓度。BnEPSPS蛋白浓度为0.12mg/mL、BnEV1蛋白浓度为0.14mg/mL。
实施例4 BnEV1酶活测定
EPSPS活性的检测是通过测定反应产生的无机磷的量的多少来判定，无机磷可以通过孔雀石绿显色法间接测定，在660nm波长处具有最大吸收峰。EPSPS每催化PEP和S3P生成一分子EPSP时，就会生成一分子无机磷Pi。目的蛋白EPSPS活性测定参照文献(Lanzettaet al 1979)报道的无机磷的测定方法。所有酶活测定反应均设计1组对照、3组平行，酶学数据处理均采用GraphPad Software公司的GraphPad Prism 7.0。
无机磷标准曲线的制作：配制10mM的无机磷标准溶液，分别取0-200μL于21个1.5mL离心管中，加入适量Hepes缓冲液补足至1mL，使20个离心管中无机磷终浓度分别为0mM、0.010mM、0.02mM……0.19mM、0.2mM。重新准备21个离心管，每管取20μL上述稀释后无机磷溶液，于28℃水浴锅静置4min后，加入800μL MAT溶液，室温放置1min后迅速加入100μL34％柠檬酸三钠溶液，放置30min后取200μL于96孔板中测定OD660值。设计1个对照组(不加莽草酸-3-磷酸S3P)、3个平行实验组。以无机磷浓度为横坐标，以不同浓度无机磷的OD660值为纵坐标作图得到无机磷标准曲线图。
酶活测定时，将实验组与对照的OD660值相减后，即可利用标准曲线图求得反应过程中释放出的无机磷浓度。酶的反应速度为单位时间内增加的产物浓度，测定时固定S3P和PEP的浓度均为1mM。
最适pH和最适温度测定：20μL反应体系：1mM莽草酸-3-磷酸(S3P)，1mM磷酸烯醇式丙酮酸(PEP)，加入2μL纯化后的BnEPSPS蛋白，加不同pH(pH3、pH4、pH5、pH6、pH7、pH8、pH9、pH10、pH11、pH12)的Hepes缓冲液补足到20μL。28℃反应4min，加入800μL MAT溶液，1min后加入100μL 34％柠檬酸三钠(SC)溶液，室温放置30min后，取200μL于96孔板中，用预热的酶标仪测定OD660值。对照组不加莽草酸-3-磷酸，每个实验组设计3个平行。野生型最适pH为8，双突变体最适pH为7，说明突变对最适pH产生了影响(如图5中a)。以pH值为横坐标，以活性为纵坐标作图，得到最适pH测定曲线；与上述20μL体系相同的反应体系不同温度(0℃、10℃、20℃、30℃、40℃、50℃、60℃)下反应4min，加入800μL MAT溶液，1min后加入100μL 34％柠檬酸三钠溶液，室温放置30min后，取200μL于96孔板中，用预热的酶标仪测定OD660值(对照组不加莽草酸-3-磷酸(S3P)，设计3个实验组)。以温度为横坐标，以活性为纵坐标作图，得到最适温度测定曲线。野生型、双突变体最适温度都是40℃，野生型、双突变体在30℃和50℃时，基本都能保持80％的活性(如图5中b)。
Km(PEP)测定：将体系中莽草酸-3-磷酸(S3P)的浓度固定为1mM，在不同PEP浓度(0mM、0.02mM、0.05mM、0.1mM、0.2mM、0.4mM、0.8mM、1.0mM)按上述20μL反应体系测定酶反应速度，以PEP浓度为横坐标，以反应速度为纵坐标作图，得到Km(PEP)的测定结果。
Km(S3P)测定：将体系中磷酸烯醇式丙酮酸(PEP)的浓度固定为1mM，在不同莽草酸-3-磷酸(S3P)浓度(0mM、0.02mM、0.05mM、0.1mM、0.2mM、0.4mM、0.8mM、1.0mM)下按上述20μL反应体系测定酶反应相对速度，以S3P浓度为横坐标，以反应速度为纵坐标作图，得到Km(S3P)的测定结果。
测定Km，根据公式V＝Vmax[S]/(Km+[S])，其中V是反应速度，Vmax是最大反应速度，[S]是测定相应Km时的底物浓度。催化常数Kcat为Vmax除以酶浓度。
设定不同浓度的底物，测定EPSPS对另一底物的亲和性，Km(PEP)、Km(S3P)的动力学曲线如图6所示。野生型的Km(PEP)为0.1659mM,突变体Km(PEP)为0.1838mM，Km(PEP)值增大，表明双突变后该酶对底物PEP的亲和性降低了；野生型的Km(S3P)值为0.2309mM，突变体Km(S3P)为0.5259mM，表明双突变后该酶对底物S3P亲和性也变小了。
BnEPSPS和BnEV1的酶动力学具体数值如表4所示。BnEV1的催化常数Kcat是野生型的一半，BnEV1的催化效率Kcat/Km是野生型的45％，BnEV1的(Kcat/Km)×Ki值是野生型的2倍以上。
表4 EPSPS动力学测定结果
为了评估突变体BnEV1对抑制剂草甘膦的耐受性，进行了抑制常数Ki(glyphosate)的测定：将体系中莽草酸-3-磷酸(S3P)的浓度固定为1mM，采用不同PEP浓度(0.05mM、0.067mM、0.1mM、0.2mM、0.5mM、1.0mM)配制20μL反应体系，不同PEP浓度的体系中分别添加终浓度为50μM的草甘膦，测定酶反应速度，以底物的浓度为横坐标，以相对活性为纵坐标作图，得到Ki的测定结果如图7中a和图7中b所示，野生型Ki值为10.36mM,BnEV1的Ki值为55mM，比野生型提高了4倍，证明BnEV1对草甘膦的耐受性明显提高。
为进一步评估突变体对草甘膦的耐受性，还进行了半抑制剂量(IC50)的测定：在上述反应体系中添加不同浓度(10-5mM、10-4mM、10-3mM、10-2mM、10-1mM、100mM、101mM、67mM)的草甘膦，所得数据以草甘膦浓度为横坐标，采用对数坐标，以相对活性速度为纵坐标作图，得到半抑制剂量IC50值的测定结果。
IC50的计算按照公式V＝Vmin+(Vmax-Vmin)/(1+([I]/IC50)n)，其中，[I]是抑制物的浓度，即草甘膦的浓度；n是曲线在IC50值处的斜率；IC50值越大证明对草甘膦的耐受性越好，图7中c中野生型IC50值为0.2249mM，双突变体IC50值为30.7mM,是野生型的136倍，证明BnEV1的草甘膦耐受型显著提升。
为模拟植物胞内环境，设计以BnEPSPS的Km值为底物浓度，草甘膦浓度设为植物分生组织所能获取的草甘膦浓度1mM，并添加乙二醇和KCl辅助模拟，具体为：0.2mM PEP，0.2mM PEP，1mM glyphosate，100mM KCl，5％乙二醇。采用20μL酶活反应体系，上述反应体系中缓冲液用模拟缓冲液替代，其他成分及反应条件不变，测定转换数Kcat(gly)。在模拟环境中图7中d中，BnEV1的转换数116s-1显著高于野生型13s-1，BnEV1比野生型提高了8倍。
EPSPS抑制动力学测定结果如表5所示。总的来说，BnEV1草甘膦耐受性较野生型WT要好。
表5 EPSPS抑制动力学测定结果
| Enzyme | K<sub>i</sub>(glyphosate)(mM) | K<sub>i</sub>/K<sub>m(PEP)</sub> | IC<sub>50</sub>(mM) | K<sub>cat</sub>(gly)(s<sup>-1</sup>) |
| BnEPSPS | 10.36±1.077 | 62.45 | 0.2249 | 13.25 |
| BnEV1EPSPS | 55.39±16.12 | 301.40 | 30.7 | 116.7 |
实施例5转化植物验证抗性
为了分析BnEV1在植物中的草甘膦抗性，我们以pCAMBIA1300S为载体，岩97为受体，通过叶盘转化法，培养了了含有突变体BnEV1的转基因烟草。
选择植物载体pCAMBIA1300S，利用潮霉素抗性基因作为筛选标记，构建示意图如图8中a所示。
pCAMBIA1300S载体和pGEX-6p-BnEV1的扩增的准备：pCAMBIA1300S E.coli DH5α从-80℃保存的甘油管里划线培养，挑取单菌落进行液体培养，提取质粒保存备用；根据pCAMBIA1300S和BnEPSPS的序列信息选择KpnⅠ和SalⅠ酶切位点，设计引物如下：
BnEPSPS-KpnⅠ-F：5’-GGGGTACCATGGCGCAAGCTAGCAG-3’；
BnEPSPS-SalⅠ-R：5’-ACGCGTCGACTTAGTGCTTTGTGA-3’；
引物由南京金斯瑞合成。
目的基因的扩增 以pGEX-6p-BnEPSPS、pGEX-6p-BnEV1为模板，分别进行PCR。反应体系如表6所示。
表6 BnEPSPS的PCR反应体系
| 试剂 | 用量(μL) |
| 质粒DNA(40ng/μL) | 1 |
| dNTPs(10mM) | 1 |
| 5×FastPfu Buffer | 10 |
| 正向引物BnEPSPS-KpnⅠ-F,10μM) | 1 |
| 反向引物BnEPSPS-SalⅠ-R,10μM) | 1 |
| FastPfu DNA polymerase | 1 |
| 加灭菌双蒸水至 | 50 |
PCR反应条件：94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸40s；30个循环；72℃延伸10min，4℃保存5min结束。采用PCR产物回收试剂盒回收。构建过程中，BnEPSPS基因PCR产物如图8中b所示。
pCAMBIA1300S和PCR产物双酶切及酶连 用KpnⅠ和Sa lⅠ对pCAMBIA1300S质粒载体和PCR产物37℃双酶切6h，酶切体系如表7、8。
表7 PCR产物双酶切体系(50μL体系)
| 试剂 | 用量(μL) |
| PCR产物(120ng/μL) | 25 |
| Takara Q cut KpnⅠ | 2.5 |
| Takara Q cut SalⅠ | 2.5 |
| 10×Q cut buffer | 5 |
| 加灭菌双蒸水至 | 50 |
表8 pCAMBIA1300S质粒载体双酶切体系(100μL体系)
酶切后的载体用胶回收法回收，酶切后的PCR产物直接回收，回收后进行连接。10μL连接体系中含有：经上述步骤构建好的含SalⅠ/KpnⅠ双酶切位点的pGEX-6p-1质粒载体0.03nmol；经SalⅠ/KpnⅠ双酶切的目的基因0.20nmol；T4 DNA ligase 1μL；10×T4 DNAligase buffer 1μL；加双蒸水补至10μL，于4℃酶连过夜。
电转化 取酶连产物1μL与50μL大肠杆菌感受态细胞DH5α混合，加入到预冷的1mm电转杯中，用2.1KV电压电击；迅速加入500μL液体LB培养基，于37℃复苏60min；涂布在含有卡纳霉素的LB平板上，37℃过夜。挑取转化子，进行菌落PCR验证。最后选取阳性转化子送至武汉擎科生物技术有限公司测序验证。抽提重组质粒提取备用。
转化农杆菌GV3101 取1μL构建好的植物重组载体质粒和2μL PJIC质粒，与50μL农杆菌感受态细胞GV3101混合，加入到预冷的1mm电转杯中，用2.1KV电压电击；迅速加入600μL液体LB培养基，于28℃复苏3h；涂布在含有卡纳霉素、四环素和利福平的LB平板上，28℃培养。四天后挑取转化子，进行菌落PCR验证，体系如表9，分别蘸取转化子菌体于体系中。
表9菌落PCR验证(20μL体系)
| 试剂 | 用量(μL) |
| 2×Taq mix | 10 |
| 正向引物(P1,10μM) | 0.5 |
| 反向引物(P2,10μM) | 0.5 |
| 加灭菌双蒸水至 | 20 |
PCR反应条件：94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸1min20s；30个循环；72℃延伸10min，4℃保存5min结束。DNA凝胶电泳检测，农杆菌转化子菌落PCR验证结果如图8中c所示，保存含有目的基因的农杆菌菌株备用。
农杆菌介导转化烟草。培养烟草无菌苗，选择岩97成熟种子浸入清水中保持5min，采用纱布过滤，清水再次冲洗。70％的酒精侵泡8s，2％次氯酸钠侵泡10min，灭菌水清洗5遍。接种于MS发芽培养基，25℃、光强1000-1500lux、光照16h、黑暗8h条件下交替培养。种子一周后萌发，无菌苗两周后长出，待小苗的叶片长至5-6片时即可用于转化。使用无菌打孔器取0.5cm左右的叶片(避开叶脉)，转入预培养基中，上表皮朝上，18h光照、黑暗6h条件下下培养1d。在利福平、四环素、卡那霉素液体小量含有目的基因的根癌农杆菌，28℃培养3天后转接至50mL培养基中继续培养。液体共培养培养基悬浮农杆菌，将OD600值调至0.5-1.0，放入冰中备用。使用农杆菌菌液侵染预培养后的叶盘，大约30min,用镊子轻轻地一个一个取出叶盘，去除多余的菌液，放置到MS共培养基中，表皮朝上，共培养基表面放一张滤纸。上表皮朝上，并用封口膜密封，23℃黑暗中培养3d。先用无菌水清洗共培养后的叶盘，再用含有头孢霉素的水溶液清洗叶盘，并用滤纸吸干叶盘表面水分。转移至筛选培养基，轻压叶盘边缘到培养基中，24℃、光强1000-1500lux、光照12h、黑暗12h交替培养2周左右。在筛选培养基中，外植体边缘长出愈伤，芽点从愈伤中长出。芽点长到3mm长度时，转至生根培养基中，每瓶放置2-3株，待根长至3cm-4cm，即可炼苗移栽。
转基因苗PCR检测 剪取再生苗植株叶片，采用CTAB法提取DNA，利用潮霉素抗性基因特异性引物如下：
hpt557-F：5’-ACACTACATGGCGTGATTTCAT-3’；
hpt557-R：5’-TCCACTATCGGCGAGTACTTCT-3’；
进行PCR扩增，体系如表10所示。
表10 PCR检测体系(50μL体系)
PCR反应条件：94℃预变性5min；94℃变性30s，55℃退火30s，72℃延伸30s；30个循环；72℃延伸10min，4℃保存5min结束。DNA凝胶电泳检测。采用根癌农杆菌介导的方式将BnEPSPS和BnEV1转化烟草，整个过程包括发芽、预培养、共培养、分化、生根、移栽等过程(如图9所示)。
利用潮霉素抗性基因特异性引物PCR扩增，检测BnEPSPS转基因株系，其中25株为阳性转化植株，株系号为1、2、3、4、5、6、8、9、10、11、12、13、14、15、16、17、19、20、22、24、25、26、28、29和30(图10中上图)；BnEV1转基因植物中29株为阳性转化植株(如图10中下图)，株系号为1、2、3、4、5、6、7、8、9、10、11、12、13、14、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30。将检测后的转基因植株移栽至培养箱，待生长至6叶期-9叶期时，验证草甘膦抗性水平。
转基因烟草生长至6叶期-9叶期时，在BnEV1转基因叶片上表皮喷洒4.0％的农达(41％草甘膦异丙胺盐，孟山都)，7天后观察现象。BnEPSPS转基因植株“WT-2”株系在0.8％的农达处理时，出现了萎蔫，枯黄现象，甚至死亡等现象；BnEV1转基因株系“BnEV1-12”在4.0％的农达处理时，生长良好，长势与草甘膦未处理的BnEV1转基因株系“BnEV1-23”相当(见图11所示)。抗性验证表明BnEV1转基因烟草能够耐受5倍商业推荐剂量中量(一般杂草商业推荐剂量0.5％-1％，本研究以0.8％为推荐剂量中量)的农达，且长势与草甘膦未处理组相当。表明BnEV1抗性水平较高，具有抗草甘膦转基因重大应用价值。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
序列表
<110>
<120> 一种抗草甘膦植物EPSPS酶双突变体及其克隆、表达与应用
<130> PI19B0752CN
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 1527
<212> DNA
<213> BnEPSPS
<400> 1
atggcgcaag ctagcagaat ctgccagaac cagaacccat gtgttatctc caatctctcc 60
aaatcaaacc aacgcaaatc gcccttctct gtttctctga agacgcacca gatttcttcg 120
tgggggttga agaagagtaa caacgggtct gtgattcgtc cggttcgggt aacggcgtct 180
gtttccacgg ctgagaaatc ttcggagatt gtgcttcagc ccattagaga aatctcgggt 240
ctgatcaagc tacccggatc caaatctctg tccaatcgaa tccttcttct agcagctcta 300
tccgagggaa ccactgtagt tgacaacttg ttgaacagtg atgacatcaa ttacatgctt 360
gatgcgttga agaaattggg gcttaatgtg gaacgtgaca gtgagaataa ccgtgcggtt 420
gttgaaggat gtggcgggat attcccagct tctttagatt ccaagagtga tatcgagttg 480
taccttggga atgctggaac agccatgcgt ccacttaccg ctgcagttac tgctgcaggt 540
ggcaacgcaa gttatattct tgatggggtg cctagaatga gggaaaggcc tataggagat 600
ttggttgttg gtcttaagca gcttggtgct gatgttgaat gtactcttgg aactaactgc 660
cctcctgttc gcgtcaatgc taatggtggc cttcccggtg gaaaggtgaa gctatctggt 720
tcaatcagta gtcaatactt gactgctctg ctcatggcag ctcctttagc tcttggagac 780
gttgagattg agatcgttga taaactgatc tctgttccgt atgttgaaat gacattgaag 840
ttgatggaac gttttggtgt tagtgccgag catagtgaca gttgggatcg tttctttgtc 900
aagggcggtc agaaatacaa gtcgcctggt aatgcttacg tagaaggtga tgcttctagt 960
gctagttatt tcttggctgg tgctgccatt actggtgaaa ccgtcactgt tgaaggttgt 1020
ggaacaacca gcctgcaggg agatgtgaag ttcgctgagg tttttgagaa aatgggatgt 1080
aaagtgtcat ggacagagaa cagtgtgact gtgactggac catctagaga tgcttttgga 1140
atgagacact tgcgcgctgt tgatgtcaac atgaacaaaa tgcctgatgt agccatgact 1200
cttgccgttg ttgctctctt tgcagatggt ccaaccacca ttagagatgt ggctagctgg 1260
agagtaaagg agacagaaag gatgattgcc atttgcacag agcttaggaa gcttggagct 1320
acagtggaag agggttcaga ttattgtgtg ataactccac cagcaaagct gaaaccgtcg 1380
gagattgaca catatgatga tcatagaatg gcaatggcat tctcccttgc agcttgtgct 1440
gatgttccag taaccatcaa agatcctggt tgcaccagga aaactttccc tgactacttc 1500
caggtccttg aaagtatcac aaagcac 1527
<210> 2
<211> 509
<212> PRT
<213> BnEPSPS
<400> 2
Met Ala Gln Ala Ser Arg Ile Cys Gln Asn Gln Asn Pro Cys Val Ile
1 5 10 15
Ser Asn Leu Ser Lys Ser Asn Gln Arg Lys Ser Pro Phe Ser Val Ser
20 25 30
Leu Lys Thr His Gln Ile Ser Ser Trp Gly Leu Lys Lys Ser Asn Asn
35 40 45
Gly Ser Val Ile Arg Pro Val Arg Val Thr Ala Ser Val Ser Thr Ala
50 55 60
Glu Lys Ser Ser Glu Ile Val Leu Gln Pro Ile Arg Glu Ile Ser Gly
65 70 75 80
Leu Ile Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu
85 90 95
Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu Asn
100 105 110
Ser Asp Asp Ile Asn Tyr Met Leu Asp Ala Leu Lys Lys Leu Gly Leu
115 120 125
Asn Val Glu Arg Asp Ser Glu Asn Asn Arg Ala Val Val Glu Gly Cys
130 135 140
Gly Gly Ile Phe Pro Ala Ser Leu Asp Ser Lys Ser Asp Ile Glu Leu
145 150 155 160
Tyr Leu Gly Asn Ala Gly Thr Ala Met Arg Pro Leu Thr Ala Ala Val
165 170 175
Thr Ala Ala Gly Gly Asn Ala Ser Tyr Ile Leu Asp Gly Val Pro Arg
180 185 190
Met Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu
195 200 205
Gly Ala Asp Val Glu Cys Thr Leu Gly Thr Asn Cys Pro Pro Val Arg
210 215 220
Val Asn Ala Asn Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly
225 230 235 240
Ser Ile Ser Ser Gln Tyr Leu Thr Ala Leu Leu Met Ala Ala Pro Leu
245 250 255
Ala Leu Gly Asp Val Glu Ile Glu Ile Val Asp Lys Leu Ile Ser Val
260 265 270
Pro Tyr Val Glu Met Thr Leu Lys Leu Met Glu Arg Phe Gly Val Ser
275 280 285
Ala Glu His Ser Asp Ser Trp Asp Arg Phe Phe Val Lys Gly Gly Gln
290 295 300
Lys Tyr Lys Ser Pro Gly Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser
305 310 315 320
Ala Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Glu Thr Val Thr
325 330 335
Val Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys Phe Ala
340 345 350
Glu Val Phe Glu Lys Met Gly Cys Lys Val Ser Trp Thr Glu Asn Ser
355 360 365
Val Thr Val Thr Gly Pro Ser Arg Asp Ala Phe Gly Met Arg His Leu
370 375 380
Arg Ala Val Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr
385 390 395 400
Leu Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Thr Ile Arg Asp
405 410 415
Val Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Ile Ala Ile Cys
420 425 430
Thr Glu Leu Arg Lys Leu Gly Ala Thr Val Glu Glu Gly Ser Asp Tyr
435 440 445
Cys Val Ile Thr Pro Pro Ala Lys Leu Lys Pro Ser Glu Ile Asp Thr
450 455 460
Tyr Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala
465 470 475 480
Asp Val Pro Val Thr Ile Lys Asp Pro Gly Cys Thr Arg Lys Thr Phe
485 490 495
Pro Asp Tyr Phe Gln Val Leu Glu Ser Ile Thr Lys His
500 505 509
<210> 3
<211> 1527
<212> DNA
<213> BnEV1
<400> 3
atggcgcaag ctagcagaat ctgccagaac cagaacccat gtgttatctc caatctctcc 60
aaatcaaacc aacgcaaatc gcccttctct gtttctctga agacgcacca gatttcttcg 120
tgggggttga agaagagtaa caacgggtct gtgattcgtc cggttcgggt aacggcgtct 180
gtttccacgg ctgagaaatc ttcggagatt gtgcttcagc ccattagaga aatctcgggt 240
ctgatcaagc tacccggatc caaatctctg tccaatcgaa tccttcttct agcagctcta 300
tccgagggaa ccactgtagt tgacaacttg ttgaacagtg atgacatcaa ttacatgctt 360
gatgcgttga agaaattggg gcttaatgtg gaacgtgaca gtgagaataa ccgtgcggtt 420
gttgaaggat gtggcgggat attcccagct tctttagatt ccaagagtga tatcgagttg 480
taccttggga atgctggaat agccatgcgt tcacttaccg ctgcagttac tgctgcaggt 540
ggcaacgcaa gttatattct tgatggggtg cctagaatga gggaaaggcc tataggagat 600
ttggttgttg gtcttaagca gcttggtgct gatgttgaat gtactcttgg aactaactgc 660
cctcctgttc gcgtcaatgc taatggtggc cttcccggtg gaaaggtgaa gctatctggt 720
tcaatcagta gtcaatactt gactgctctg ctcatggcag ctcctttagc tcttggagac 780
gttgagattg agatcgttga taaactgatc tctgttccgt atgttgaaat gacattgaag 840
ttgatggaac gttttggtgt tagtgccgag catagtgaca gttgggatcg tttctttgtc 900
aagggcggtc agaaatacaa gtcgcctggt aatgcttacg tagaaggtga tgcttctagt 960
gctagttatt tcttggctgg tgctgccatt actggtgaaa ccgtcactgt tgaaggttgt 1020
ggaacaacca gcctgcaggg agatgtgaag ttcgctgagg tttttgagaa aatgggatgt 1080
aaagtgtcat ggacagagaa cagtgtgact gtgactggac catctagaga tgcttttgga 1140
atgagacact tgcgcgctgt tgatgtcaac atgaacaaaa tgcctgatgt agccatgact 1200
cttgccgttg ttgctctctt tgcagatggt ccaaccacca ttagagatgt ggctagctgg 1260
agagtaaagg agacagaaag gatgattgcc atttgcacag agcttaggaa gcttggagct 1320
acagtggaag agggttcaga ttattgtgtg ataactccac cagcaaagct gaaaccgtcg 1380
gagattgaca catatgatga tcatagaatg gcaatggcat tctcccttgc agcttgtgct 1440
gatgttccag taaccatcaa agatcctggt tgcaccagga aaactttccc tgactacttc 1500
caggtccttg aaagtatcac aaagcac 1527
<210> 4
<211> 509
<212> PRT
<213> BnEV1
<400> 4
Met Ala Gln Ala Ser Arg Ile Cys Gln Asn Gln Asn Pro Cys Val Ile
1 5 10 15
Ser Asn Leu Ser Lys Ser Asn Gln Arg Lys Ser Pro Phe Ser Val Ser
20 25 30
Leu Lys Thr His Gln Ile Ser Ser Trp Gly Leu Lys Lys Ser Asn Asn
35 40 45
Gly Ser Val Ile Arg Pro Val Arg Val Thr Ala Ser Val Ser Thr Ala
50 55 60
Glu Lys Ser Ser Glu Ile Val Leu Gln Pro Ile Arg Glu Ile Ser Gly
65 70 75 80
Leu Ile Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu
85 90 95
Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu Asn
100 105 110
Ser Asp Asp Ile Asn Tyr Met Leu Asp Ala Leu Lys Lys Leu Gly Leu
115 120 125
Asn Val Glu Arg Asp Ser Glu Asn Asn Arg Ala Val Val Glu Gly Cys
130 135 140
Gly Gly Ile Phe Pro Ala Ser Leu Asp Ser Lys Ser Asp Ile Glu Leu
145 150 155 160
Tyr Leu Gly Asn Ala Gly Ile Ala Met Arg Ser Leu Thr Ala Ala Val
165 170 175
Thr Ala Ala Gly Gly Asn Ala Ser Tyr Ile Leu Asp Gly Val Pro Arg
180 185 190
Met Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu
195 200 205
Gly Ala Asp Val Glu Cys Thr Leu Gly Thr Asn Cys Pro Pro Val Arg
210 215 220
Val Asn Ala Asn Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly
225 230 235 240
Ser Ile Ser Ser Gln Tyr Leu Thr Ala Leu Leu Met Ala Ala Pro Leu
245 250 255
Ala Leu Gly Asp Val Glu Ile Glu Ile Val Asp Lys Leu Ile Ser Val
260 265 270
Pro Tyr Val Glu Met Thr Leu Lys Leu Met Glu Arg Phe Gly Val Ser
275 280 285
Ala Glu His Ser Asp Ser Trp Asp Arg Phe Phe Val Lys Gly Gly Gln
290 295 300
Lys Tyr Lys Ser Pro Gly Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser
305 310 315 320
Ala Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Glu Thr Val Thr
325 330 335
Val Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys Phe Ala
340 345 350
Glu Val Phe Glu Lys Met Gly Cys Lys Val Ser Trp Thr Glu Asn Ser
355 360 365
Val Thr Val Thr Gly Pro Ser Arg Asp Ala Phe Gly Met Arg His Leu
370 375 380
Arg Ala Val Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr
385 390 395 400
Leu Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Thr Ile Arg Asp
405 410 415
Val Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Ile Ala Ile Cys
420 425 430
Thr Glu Leu Arg Lys Leu Gly Ala Thr Val Glu Glu Gly Ser Asp Tyr
435 440 445
Cys Val Ile Thr Pro Pro Ala Lys Leu Lys Pro Ser Glu Ile Asp Thr
450 455 460
Tyr Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala
465 470 475 480
Asp Val Pro Val Thr Ile Lys Asp Pro Gly Cys Thr Arg Lys Thr Phe
485 490 495
Pro Asp Tyr Phe Gln Val Leu Glu Ser Ile Thr Lys His
500 505 509
<210> 5
<211> 1527
<212> DNA
<213> BnEV2
<400> 5
atggcgcaag ctagcagaat ctgccagaac cagaacccat gtgttatctc caatctctcc 60
aaatcaaacc aacgcaaatc gcccttctct gtttctctga agacgcacca gatttcttcg 120
tgggggttga agaagagtaa caacgggtct gtgattcgtc cggttcgggt aacggcgtct 180
gtttccacgg ctgagaaatc ttcggagatt gtgcttcagc ccattagaga aatctcgggt 240
ctgatcaagc tacccggatc caaatctctg tccaatcgaa tccttcttct agcagctcta 300
tccgagggaa ccactgtagt tgacaacttg ttgaacagtg atgacatcaa ttacatgctt 360
gatgcgttga agaaattggg gcttaatgtg gaacgtgaca gtgagaataa ccgtgcggtt 420
gttgaaggat gtggcgggat attcccagct tctttagatt ccaagagtga tatcgagttg 480
taccttggga atgctggact ggccatgcgt atgcttaccg ctgcagttac tgctgcaggt 540
ggcaacgcaa gttatattct tgatggggtg cctagaatga gggaaaggcc tataggagat 600
ttggttgttg gtcttaagca gcttggtgct gatgttgaat gtactcttgg aactaactgc 660
cctcctgttc gcgtcaatgc taatggtggc cttcccggtg gaaaggtgaa gctatctggt 720
tcaatcagta gtcaatactt gactgctctg ctcatggcag ctcctttagc tcttggagac 780
gttgagattg agatcgttga taaactgatc tctgttccgt atgttgaaat gacattgaag 840
ttgatggaac gttttggtgt tagtgccgag catagtgaca gttgggatcg tttctttgtc 900
aagggcggtc agaaatacaa gtcgcctggt aatgcttacg tagaaggtga tgcttctagt 960
gctagttatt tcttggctgg tgctgccatt actggtgaaa ccgtcactgt tgaaggttgt 1020
ggaacaacca gcctgcaggg agatgtgaag ttcgctgagg tttttgagaa aatgggatgt 1080
aaagtgtcat ggacagagaa cagtgtgact gtgactggac catctagaga tgcttttgga 1140
atgagacact tgcgcgctgt tgatgtcaac atgaacaaaa tgcctgatgt agccatgact 1200
cttgccgttg ttgctctctt tgcagatggt ccaaccacca ttagagatgt ggctagctgg 1260
agagtaaagg agacagaaag gatgattgcc atttgcacag agcttaggaa gcttggagct 1320
acagtggaag agggttcaga ttattgtgtg ataactccac cagcaaagct gaaaccgtcg 1380
gagattgaca catatgatga tcatagaatg gcaatggcat tctcccttgc agcttgtgct 1440
gatgttccag taaccatcaa agatcctggt tgcaccagga aaactttccc tgactacttc 1500
caggtccttg aaagtatcac aaagcac 1527
<210> 6
<211> 509
<212> PRT
<213> BnEV2
<400> 6
Met Ala Gln Ala Ser Arg Ile Cys Gln Asn Gln Asn Pro Cys Val Ile
1 5 10 15
Ser Asn Leu Ser Lys Ser Asn Gln Arg Lys Ser Pro Phe Ser Val Ser
20 25 30
Leu Lys Thr His Gln Ile Ser Ser Trp Gly Leu Lys Lys Ser Asn Asn
35 40 45
Gly Ser Val Ile Arg Pro Val Arg Val Thr Ala Ser Val Ser Thr Ala
50 55 60
Glu Lys Ser Ser Glu Ile Val Leu Gln Pro Ile Arg Glu Ile Ser Gly
65 70 75 80
Leu Ile Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu Leu
85 90 95
Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu Asn
100 105 110
Ser Asp Asp Ile Asn Tyr Met Leu Asp Ala Leu Lys Lys Leu Gly Leu
115 120 125
Asn Val Glu Arg Asp Ser Glu Asn Asn Arg Ala Val Val Glu Gly Cys
130 135 140
Gly Gly Ile Phe Pro Ala Ser Leu Asp Ser Lys Ser Asp Ile Glu Leu
145 150 155 160
Tyr Leu Gly Asn Ala Gly Leu Ala Met Arg Met Leu Thr Ala Ala Val
165 170 175
Thr Ala Ala Gly Gly Asn Ala Ser Tyr Ile Leu Asp Gly Val Pro Arg
180 185 190
Met Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu
195 200 205
Gly Ala Asp Val Glu Cys Thr Leu Gly Thr Asn Cys Pro Pro Val Arg
210 215 220
Val Asn Ala Asn Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly
225 230 235 240
Ser Ile Ser Ser Gln Tyr Leu Thr Ala Leu Leu Met Ala Ala Pro Leu
245 250 255
Ala Leu Gly Asp Val Glu Ile Glu Ile Val Asp Lys Leu Ile Ser Val
260 265 270
Pro Tyr Val Glu Met Thr Leu Lys Leu Met Glu Arg Phe Gly Val Ser
275 280 285
Ala Glu His Ser Asp Ser Trp Asp Arg Phe Phe Val Lys Gly Gly Gln
290 295 300
Lys Tyr Lys Ser Pro Gly Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser
305 310 315 320
Ala Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Glu Thr Val Thr
325 330 335
Val Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys Phe Ala
340 345 350
Glu Val Phe Glu Lys Met Gly Cys Lys Val Ser Trp Thr Glu Asn Ser
355 360 365
Val Thr Val Thr Gly Pro Ser Arg Asp Ala Phe Gly Met Arg His Leu
370 375 380
Arg Ala Val Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr
385 390 395 400
Leu Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Thr Ile Arg Asp
405 410 415
Val Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Ile Ala Ile Cys
420 425 430
Thr Glu Leu Arg Lys Leu Gly Ala Thr Val Glu Glu Gly Ser Asp Tyr
435 440 445
Cys Val Ile Thr Pro Pro Ala Lys Leu Lys Pro Ser Glu Ile Asp Thr
450 455 460
Tyr Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala
465 470 475 480
Asp Val Pro Val Thr Ile Lys Asp Pro Gly Cys Thr Arg Lys Thr Phe
485 490 495
Pro Asp Tyr Phe Gln Val Leu Glu Ser Ile Thr Lys His
500 505 509
Claims (8)
1.一种抗草甘膦植物EPSPS酶双突变体，其特征在于，所述双突变体是在甘蓝型油菜EPSPS酶的基础上，将第167位的苏氨酸进行突变为异亮氨酸或亮氨酸，以及，将第171位的脯氨酸突变为丝氨酸或蛋氨酸。
2.根据权利要求1所述的抗草甘膦植物EPSPS酶双突变体，其特征在于，所述双突变体是将第167位的苏氨酸进行突变为异亮氨酸，以及，将第171位的脯氨酸突变为丝氨酸，记为BnEV1；所述双突变体BnEV1包含如SEQ ID No：4所示的氨基酸序列。
3.根据权利要求1所述的抗草甘膦植物EPSPS酶双突变体，其特征在于，所述双突变体是将第167位的苏氨酸进行突变为亮氨酸，以及，将第171位的脯氨酸突变为蛋氨酸，记为BnEV2；所述双突变体BnEV2包含如SEQ ID No：6所示的氨基酸序列。
4.一种编码权利要求1-3任一项所述抗草甘膦植物EPSPS酶双突变体的基因。
5.根据权利要求4所述的基因，其特征在于，包含如SEQ ID No：3或SEQ ID No：5所示的核苷酸序列。
6.一种含有权利要求4或5所述抗草甘膦植物EPSPS酶双突变体编码基因的表达载体。
7.一种含有权利要求4或5所述抗草甘膦植物EPSPS酶双突变体编码基因的重组菌。
8.权利要求1-3任一项所述抗草甘膦植物EPSPS酶双突变体在培育抗草甘膦转基因作物领域中的应用。
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| WO2014151255A1 (en) * | 2013-03-15 | 2014-09-25 | Monsanto Technology Llc | Methods and compositions for weed control |
| CN104202969A (zh) * | 2012-02-01 | 2014-12-10 | 陶氏益农公司 | 新的草甘膦抗性基因类别 |
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| WO2014151255A1 (en) * | 2013-03-15 | 2014-09-25 | Monsanto Technology Llc | Methods and compositions for weed control |
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