CN111197052B - 一种适冷的i型5-烯醇丙酮酰莽草酸-3-磷酸合酶基因 - Google Patents
一种适冷的i型5-烯醇丙酮酰莽草酸-3-磷酸合酶基因 Download PDFInfo
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- CN111197052B CN111197052B CN202010178189.9A CN202010178189A CN111197052B CN 111197052 B CN111197052 B CN 111197052B CN 202010178189 A CN202010178189 A CN 202010178189A CN 111197052 B CN111197052 B CN 111197052B
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Abstract
本发明属于微生物基因工程技术领域,具体涉及一种适冷的I型5‑烯醇丙酮酰莽草酸‑3‑磷酸合酶基因。所述的I型5‑烯醇丙酮莽草酸‑3‑磷酸合酶(EPSPS)基因的核苷酸序列如序列表SEQ ID NO:1所述,该基因编码的蛋白质序列如SEQ ID NO:2所述。所述适冷的I型5‑烯醇丙酮莽草酸‑3‑磷酸合酶基因是根据Genbank中报导的同属嗜盐白蚁菌Isoptericola sp.GR1TH_0001的aroA基因保守序列克隆得到的,经过生物学试验验证,证明该酶对草甘膦具有较高的耐受性,且具有独特的低温适应性。
Description
技术领域
本发明属于微生物基因工程技术领域,具体涉及一种分离的适冷的I型5-烯醇丙酮酰莽草酸-3-磷酸合酶基因。本发明对来源于微生物对草甘膦具有抗性的5-烯醇丙酮酰莽草酸-3-磷酸合酶(EPSPS)基因的进行了分离与抗性鉴定,证明本发明克隆的基因编码的5-烯醇丙酮酰莽草酸-3-磷酸合酶的核苷酸序列对草甘膦具有较高的抗性,含有该基因的大肠杆菌细胞能够耐受浓度为200mM/L的草甘膦。同时,该酶在10℃下仍具有较高的活性,该酶是首个鉴定的具有低温适应性且抗草甘膦的5-烯醇丙酮酰莽草酸-3-磷酸合酶,其编码基因在培育越冬抗除草剂作物方面具有极大的应用潜力。
背景技术
杂草一直是干扰作物栽培产量的主要问题,人工成本的攀升使得人工除草日益被化学除草剂灭杀所替代。
草甘膦(glyphosate,中文名称:农达)是目前全球生产、使用最为广泛的广谱除草剂,对大多数植物具有灭生性。
莽草酸途径是连接碳水化合物和芳香族化合物生物合成的重要生物代谢途径。该途径只存在于细菌、真菌、藻类、和植物中[1-3]。5-烯醇丙酮酸莽草酸-3-磷酸合成酶(EPSPS)是莽草酸途径中第六步的关键酶,催化底物磷酸烯醇式丙酮酸(PEP)和莽草酸-3-磷酸(S3P)生成5-烯醇丙酮酰莽草酸-3-磷酸[4]。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[5],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[6]。
草甘膦及其衍生物能够通过莽草酸代谢途径杀死大多数植物或抑制大多数细菌生长。但在自然界中仍存在一些细菌的EPSPS酶能够耐受较高浓度的草甘膦。通过转化外源抗性基因到植物细胞,使植物对草甘膦获得耐受性。早期研究根据EPSPS对草甘膦的敏感性分为Ⅰ型和Ⅱ型。Ⅰ型EPSPS对草甘膦敏感,大多数微生物和植物来源的EPSPS都属于这一类,典型代表有大肠杆菌Escherichia coli、鼠伤寒沙门氏菌Salmonella typhimurium、烟草Nicotiana tabacum、拟南芥Arabidopsis thaliana等。Ⅱ型EPSPS对草甘膦具有天然抗性,与Ⅰ型同源性较低,典型代表有根癌农杆菌Agrobacterium sp.CP4、假单胞菌Pseudomonassp.PG2982和可变盐单胞菌Haloviborio variabilis等。目前广泛应用于转基因草甘膦作物培育的基因主要为来源于根癌农杆菌Agrobacterium sp.CP4的5-烯醇丙酮酸莽草酸-3-磷酸合成酶(CP4-EPSPS)编码基因,该酶虽对草甘膦具有很高的抗性,但低温环境下,其活性较低,限制了其应用范围。对于冬油菜、小麦等越冬作物,通过转基因技术导入低温下具备较高酶活性且耐受草甘膦的EPSPS酶编码基因,可实现对草甘膦抗性,培育越冬作物抗草甘膦新品种。但从现有研究看,全球缺乏既具有高草甘膦抗性且具有低温环境适应性EPSPS酶编码基因。
本发明从抗草甘膦的微生物菌种中分离了一个对草甘膦具有高抗性和适冷的EPSPS基因,其编码的蛋白与目前已报道的对草甘膦的耐受的EPSPS序列相似性较低,属于新的抗草甘膦的EPSPS酶。生化鉴定表明,该酶在10℃保留70%的活性,在20℃保留80%的活性,这一低温适应性的特点,突破了现有EPSPS酶温度适应性瓶颈,具有极大的应用潜力。
发明内容
本发明的目的在于克服现有技术的缺陷,分离和通过抗性鉴定来源于微生物对草甘膦具有抗性的和适冷的5-烯醇丙酮酰莽草酸-3-磷酸合酶(EPSPS)基因。该基因编码的5-烯醇丙酮酰莽草酸-3-磷酸合酶的核苷酸序列对草甘膦具有较高的抗性和适冷性,含有该基因的重组大肠杆菌细胞能够耐受浓度为200mM/L的草甘膦。
本发明的技术方案如下所述:
本发明通过草甘膦压力筛选出一株能够在含500mM草甘膦的M9基础盐培养基(配方见实施例)中生长的菌株。经鉴定,该菌株属于Isoptericola属,申请人将该菌株鉴定为Isoptericola sp.GR1,进一步,申请人克隆了该菌株EPSPS酶基因,将该EPSPS酶基因被命名为IS-EPSPS基因。本发明将IS-EPSPS基因克隆到表达载体pGEX-6p-1[7]上,将所得的质粒命名为pGEX-6p-1-IS.EPSPS重组质粒(图5)。
进一步,将本发明将pGEX-6p-1-IS.EPSPS重组质粒转入大肠杆菌aroA缺陷型菌株大肠杆菌Escherichia coli AB2829[8]中,分析了IS-EPSPS对草甘膦的抗性。含有IS.EPSPS的Escherichia coli AB2829能够在含有浓度高达200mM/L草甘膦的培养基上生长,表明IS-EPSPS对草甘膦有较高的抗性和适冷性。
本发明分离克隆的草甘膦抗性基因IS-EPSPS的核苷酸序列如序列表SEQ ID NO:1所示,序列全长为1326bp,编码442个氨基酸残基,将所得的氨基酸序列与典型的I类aroA氨基酸序列(E.coli-EPSPS)和典型的II类aroA氨基酸序列(CP4-EPSPS)进行比较,结果显示,IS-EPSPS氨基酸序列与两个代表型EPSPS基因的氨基酸序列同源性最高值仅为32.81%和27%。与其他已鉴定的抗草甘膦的EPSPS也具有较低的相似性(表5)。
对IsEPSPS进行了生化鉴定,结果表明其最适反应温度为30℃,20℃时能保持约80%的活性,0℃时亦能保持50%以上的活性,表明该酶具有独特的低温适应性能。
酶促动力学分析表明,IsEPSPS的亲和常数Km(PEP)值为0.1607mM、Km(S3P)值为0.1095mM、半抑制浓度IC50值为3.022mM、草甘膦抑制常数Ki值为5.352mM,Ki/Km值为48。抑制常数Ki是草甘膦抗性的重要参数,该值越大,意味着酶对草甘膦的抗性越高。本发明中IsEPSPS对草甘膦的Ki值为5.352mM,远高于GR79-EPSPS的0.171mM(US2011173716A1)、A15-EPSPS的0.0106mM(CN200810036732)、RD-EPSP的0.146mM(CN200710039338)和I.variabilis EPSPS的1.15mM(CN2014101085952)。
申请人将本发明得到的重组菌株大肠杆菌LZD002命名为大肠杆菌pGEX-6p1-IS.EPSPS,Escherichia coli pGEX-6p1-IS.EPSPS,于2019年6月18日送交中国典型培养物保藏中心(CCTCC)保藏,保藏编号为CCTCC NO:M2019463。
附图说明
图1:本发明的技术流程图。
图2:原始菌株Isoptericola sp.能够在500mM草甘膦的2216平板生长情况。
图3:Isoptericola sp.进化树分析。
图4:IS-EPSPS进化树分析。
图5:本发明构建的重组质粒pGEX-6p-1-IS.EPSPS的图谱。
图6:将pGEX-6p-1-IS.EPSPS导入E.coli AB2829后的生长曲线。附图标记说明:A为0mM草甘膦下,E.coli生长曲线;B为100mM草甘膦下,E.coli生长曲线;C为200mM下,E.coli生长曲线。
图7:pGEX-6p-1-IS.EPSPS导入E.coli AB2829蛋白表达SDS-PAGE检测。
图8:无机磷标准曲线。
图9:pH对GST-IS.EPSPS酶活性的影响。
图10:温度对GST-IS.EPSPS酶活性的影响。
图11:离子对GST-IS.EPSPS酶活性的影响。
图12:Km(PEP)的测定。
图13:Km(S3P)的测定。
图14:草甘膦半抑制剂量IC50值的测定。
图15:草甘膦抑制常数Ki的测定。
具体实施方式
对序列表的说明:
序列表SEQ ID NO:1是本发明克隆的IS-EPSPS基因的核苷酸序列。
序列表SEQ ID NO:2是IS-EPSPS基因编码的蛋白质序列。
实施例1
1.原始菌株的筛选
从实验室保存的从深海海泥(山东青岛)里分离出来的海洋菌(Isoptericolasp.GR1,实施本发明并不限于该海洋菌)在含20mM草甘膦的2216培养基(蛋白胨10g,酵母粉5g,牛肉浸膏2g,NaCH2COOH 1g,NH4NO3 0.2g,NaCl 19.45g,MgCl2 0.5g,MgSO4 0.5g,CaCl20.5g,KCl 0.55g,NaHCO3 0.16g,加琼脂粉18g,补充双蒸水至1L,灭菌前调pH至7.6,在121℃下高压蒸汽灭菌30min;)上划线培养,多次提高草甘膦浓度进行复筛,将复筛后获得纯化后的单菌落在含有500mM草甘膦的2216培养基中培养,筛选出一株能够在高草甘膦浓度的2216平板上生长的菌株(图2)。
2.细菌基因组DNA提取
用300mM草甘膦2216液体培养基培养海洋菌(Isoptericola sp.GR1),在28℃过夜培养,使用天根生化(北京)科技有限公司生产的细菌基因组DNA提取试剂盒(按试剂盒说明书操作),提取Isoptericola sp.GR1基因组DNA。具体步骤为:取细菌培养液3ml,在10,000rpm离心机上离心1min,尽量吸净上清。向菌体沉淀中加入180μl缓冲液(配方:20mMTris,pH 8.0;2的mM Na2-EDTA;1.2%Triton;终浓度为20mg/ml的溶菌酶),37℃处理30min;加入4μl RNase A溶液(试剂盒自带,),振荡15s,室温下放置5min。向管中加入20μlProteinase K溶液(试剂盒自带),混匀。加入220μl缓冲液GB(试剂盒自带),振荡15s,70℃放置10min,溶液变清亮后简短离心以去除管盖内壁的水珠。加220μl无水乙醇,充分振荡混匀15s,简短离心以去除管盖内壁的水珠。将上一步所得溶液和絮状沉淀都加入一个吸附柱CB3中(吸附柱放入收集管中),以12,000rpm离心30s,倒掉废液,将吸附柱CB3放入收集管中。向吸附柱CB3中加入500μl缓冲液GD(试剂盒自带),12,000rpm离心30s,倒掉废液,将吸附柱CB3放入收集管中;向吸附柱CB3中加入600μl漂洗液PW(试剂盒自带),12,000rpm离心30s,倒掉废液,吸附柱CB3放入收集管中;重复操作上一步骤;将吸附柱CB3放回收集管中,以12,000rpm离心2min,倒掉废液。将吸附柱CB3置于室温放置数分钟,以彻底晾干吸附材料中残余的漂洗液。将吸附柱CB3转入一个干净的离心管中,向吸附膜的中间部位悬空滴加30μl去离子水,室温放置5min,以12,000rpm离心2min,将溶液收集到离心管中,-20℃保存待用。
3. 16SrDNA鉴定
将提取的基因组DNA进行16SrDNA扩增,扩增用的正向引物27F序列为5’-AGAGTTTGATCCTGGCTCAG-3’,反向引物1492R序列为5’-TACGGCTACCTTGTTACGACTT-3’,PCR体系见表1:
表1基因组DNA16SrDNA扩增的PCR程序
基因组DNA(60ng/μl) | 1μL |
正向引物27F(10mM) | 1μL |
反向引物1492R(10mM) | 1μL |
dNTPs(10mM) | 1μL |
FastPfu DNA polymerase | 1μL |
5×FastPfu Buffer | 10μL |
加灭菌双蒸水至 | 50μL |
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。
将PCR产物送至武汉擎科生物技术有限公司测序。将获得的16s RNA基因序列通过NCBI的blastn程序进行比对(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome),比对数据库为rRNA/ITSdatabases,比对结果(见图3)表明以上分离的抗草甘膦菌株属于Isoptericola属。
4.克隆5-烯醇丙酮莽草酸-3-磷酸合酶基因
(1)根据NCBI(https://blast.ncbi.nlm.nih.gov)同源性最高菌株Isoptericolasp.BJGMM-B20的aroA基因(Genbank Accession Number:JQ726226.1)设计引物如下:
正向引物(5’-IS.EPSPS-BamH I):5’-TAGGATCCATGACCACCGCGCCGGCGCCGACCG-3’,其中:下划线部分为酶切位点;
反向引物(3’-IS.EPSPS-Xho I):5’-CCGCTCGAGTTACGCTTGCGCTTCCGCACCCTG-3’,其中:下划线部分为酶切位点。由南京金斯瑞生物科技有限公司合成。
以基因组DNA为模板,PCR扩增EPSPS。PCR反应体系见表2。
表2扩增EPSPS基因的PCR反应体系
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。PCR产物采用OMEGA DNA GEL试剂盒,按照说明书直接回收。具体步骤为:收集PCR产物,加入与PCR产物等量的Binding buffer,将混合液加入到2mL吸附管,以12000rpm离心1min,加入500μl洗脱液离心洗脱1min,再加入700μl洗脱液离心洗脱1min,将缓冲液弃去后再离心2min,晾置5min,加入20μL双蒸水洗脱收集DNA。
(2)PCR产物和载体pGEX-6p-1双酶切及重组质粒构建
将获得的PCR产物和质粒载体pGEX-6p-1分别用限制性内切酶BamH I/XhoI(购自宝生物工程大连有限公司)进行双酶切,酶切体系如表3。质粒载体(100μl体系)见表4。
表3酶切体系PCR产物(50μl体系)
PCR产物(100ng/μl) | 25μl |
Takara Q cut BamH I | 2.5μl |
Takara Q cut Xho I | 2.5μl |
10×Q cut buffer | 5μl |
Add双蒸水至 | 50μl |
表4质粒载体(100μl体系)
pGEX-6p-1(120ng/μl) | 50μl |
Takara Q cut BamH I | 5μl |
Takara Q cut Xho I | 5μl |
10×Q cut buffer | 10μl |
Add双蒸水至 | 100μl |
酶切产物的回收使用胶回收试剂盒(购自OMEGA公司),按照该试剂盒的说明书回收电泳产物。具体步骤为:在紫外灯下将目的条带切下后置于1.5mL的离心管中,每100mg加入100μL Binding buffer,于55℃水浴锅温浴10min,直到凝胶全部融化。将溶胶液加入到2mL吸附管12000rpm,4℃离心1min,加入500μl洗脱液离心洗脱1min,再加入700μl洗脱液离心洗脱1min,将缓冲液弃去后再离心2min,晾置5min,加入20μL双蒸水洗脱收集DNA。
将上述回收的IS-EPSPS基因和pGEX-6p-1质粒载体进行连接。10μL连接体系中含有:经上述步骤构建好的含BamH I/Xho I双酶切位点的pGEX-6p-1质粒载体0.03nmol;经BamH I/Xho I双酶切的目的基因0.21nmol;T4 DNA ligase(购自NEB公司)1μL;10×T4DNAligase buffer 1μL;加双蒸水补至10μL,于4℃酶连过夜。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(购自Bio Rad公司)中,用2.1KV电压电击;迅速加入600μL液体LB培养基,于37℃复苏60min;涂布在含有100μg/mL氨苄青霉素(Ampicillin,购自北京Transgene公司)的LB平板上,37℃过夜。挑取转化子,接种到液体LB培养基于37℃震荡培养,快速抽取质粒并进行电泳检测,并送至武汉擎科生物技术有限公司测序验证。对测序结果与已知EPSPS基因序列进行比对,采用MEGA方法构建进化树,根据进化关系,可以判断IS-EPSPS基因属于Ⅰ型EPSPS(图4)。IS-EPSPS与目前广泛鉴定的EPSPS的同一性分析表明,在氨基酸水平上与CP4-EPSPS的相似性仅为27%(具体见表5)。
表5 IS-EPSPS基因与代表性EPSPS一致性的比较
(4)质粒的提取
从划线平板上挑取经验证后正确的单菌落,用装有10mL含100μg/mL的氨苄青霉素(Ampicillin)的LB液体培养基的试管内,于37℃震荡培养过夜。采用AXYGEN公司的小量质粒抽提试剂盒提取质粒。用2mL离心管收集菌体,以12000rpm离心1min,弃上清,加入250μL溶液Ⅰ重悬菌体;混匀,加入250μl溶液Ⅱ,轻轻颠倒混匀,放置时间不超过5min。加入350μl溶液Ⅲ,颠倒混匀。于12000rpm离心10min,取上清,转换至制备管,以12000rpm离心1min,弃滤液;加入500μl Buffer W1,以12000rpm离心1min,弃滤液。加700μl Buffer W2,于12000rpm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12000rpm离心1min。室温晾置5min,加40μl去离子水洗脱,得到的阳性克隆子即含有IS-EPSPS基因的重组质粒pGEX-6p-1-IS.EPSPS。
5.生长曲线测定
将重组质粒电转入aroA基因缺陷型菌株Escherichia coli AB2829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,洗涤除去LB营养成分,调整OD600至1.0,按2%转接量转接于不同浓度草甘膦M9基础盐培养基(配方:Na2HPO46.8g,KH2PO4 3g,NaCl 0.5g,NH4Cl 1g,20mL 1mol/L葡萄糖,2mL 1mol/L MgSO4,100μL 1mol/L 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)中,于37℃、200rpm摇床上培养,每隔3h利用分光光度计测定OD600数值。得到如图6所示的生长曲线,由此说明与对照相比,本发明克隆的IS-EPSPS基因显示出有明显的抗草甘膦水平。
6.蛋白质重组表达
将重组质粒pGEX-6p-1-IS.EPSPS转化到表达宿主细胞大肠杆菌aroA缺陷型菌株大肠杆菌Escherichia coli AB2829。将鉴定阳性克隆子过夜活化,以体积比1%的接种量转接至10mL含100μg/mL Ampicillin的LB液体培养基中,于37℃培养3-5h,直至OD600达到0.6左右,加入诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)终浓度至0.1mM,于18℃,180rpm下培养16h。离心收集菌体,然后用Hepes缓冲液(配方:50mM 4-羟乙基哌嗪乙磺酸(Hepes),调pH至7.0,补足双蒸水至1L,备用)将菌体洗涤一遍,再用2mL Hepes缓冲液悬浮后用超声波破碎仪(购自宁波新芝公司型号:JY92-IIDN)破碎细胞。将细胞破碎液于4℃、12000rpm离心10min,分别收集上清和沉淀。用SDS-PAGE法检测目的蛋白的表达。
SDS-PAGE配方如下:
浓缩胶(5%聚丙烯酰胺),配方如下:
H2O 1.4mL;
30%Acr-Bis(29:1) 0.33mL;
1M Tris-HCl,pH6.8 0.02mL;
10%SDS 0.02mL;
10%过硫酸铵 0.02mL;
四甲基乙二铵(TEMED) 0.002mL。
分离胶(12%聚丙烯酰胺),配方如下:
H2O 2.45mL;
30%Acr-Bis(29:1) 3mL;
1M Tris-HCl,pH8.8 2.5mL;
10%SDS 0.075mL;
10%过硫酸铵 0.075mL;
四甲基乙二铵(TEMED) 0.003mL。
取20μL细胞破碎液,加5μL 5×蛋白上样缓冲液(6mL 1mol/L Tris-HCl,pH8.8,20mL10%SDS,50mL50%甘油,5mLβ-巯基乙醇,1mL1%溴酚蓝,加双蒸水至100mL,备用),沸水浴10min,然后点样10μL电泳检测,SDS-PAGE电泳检测结果见图7。图7表明,目的蛋白EPSPS在大肠杆菌中主要成包涵体形式表达,上清中含有微量目的蛋白。含GST标签和目的蛋白的融合蛋白,申请人将此融合蛋白命名为GST-IS.EPSPS,其大小约为71kDa,符合预测结果。
7.目的蛋白活性的检测
目的蛋白EPSPS活性测定参照文献[9]报道的无机磷的测定方法。具体步骤如下:
(1)无机磷标准曲线的制作:配制10mM的无机磷标准溶液(称取0.2282g K2HPO4·3H2O,用Hepes缓冲液溶解定容至100mL),分别取0-20μL于20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0-0.2mM.每管取100μL,于28℃静置4min后,加入0.8mL MAT溶液(现配现用:0.045%孔雀石绿75mL,4.2%钼酸铵用4N盐酸溶解于25mL,混合后采用3层滤纸过滤,备用),室温放置1min后加入0.1mL 34%柠檬酸钠溶液,放置30min后取200μL于96孔板中测定OD660值。设计一个对照组、三个实验组[10]。以无机磷浓度为横坐标,以OD660为纵坐标作图得到无机磷标准曲线图(见图8)。
(2)最适pH和最适温度测定::20μL反应体系:1mM莽草酸三磷酸(S3P),1mM磷酸烯醇式丙酮酸(PEP),加入2uL GST-IS.EPSPS粗酶上清液,加不同pH(4、5、6、7、8、9、10、11)的Hepes缓冲液补足到20μL。28℃反应4min,加入800μL MAT溶液,1min后加入100μL34%柠檬酸钠(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。对照组不加莽草酸三磷酸,设计3个实验组。以pH值为横坐标,以相对活性为纵坐标作图,得到图9所示的最适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0.8mL MAT溶液,1min后加入0.1mL34%柠檬酸钠溶液,室温放置30min后,取200μL于96孔板中,用预热的酶标仪测定OD660值(对照组不加莽草酸三磷酸(S3P),设计3个实验组)。以温度为横坐标,以相对活性的平均值为纵坐标作图,得到最适温度测定曲线(见图10)。
(3)阳离子对酶活性的影响:20μL反应体系1mM莽草酸三磷酸(S3P),1mM磷酸烯醇式丙酮酸(PEP),加入2uL GST-IS.EPSPS粗酶上清液,分别添加KCl、KBr、NaCl、MgSO4、NH4Cl、K2SO4、CaCl2、MgCl2、CaCO3的盐溶液至终浓度为10mM后测定酶相对活性。离子对酶活性的影响见图11。
(4)Km(PEP)测定:将体系中莽草酸三磷酸(S3P)的浓度固定为1mM,在不同PEP浓度(0.05、0.067、0.1、0.2、0.5、1.0mM)下按上述20μL反应体系测定酶反应相对速度,以PEP浓度为横坐标,以相对活性为纵坐标作图,得到如图12所示的Km(PEP)的测定结果。
(5)Km(S3P)测定:将体系中磷酸烯醇式丙酮酸(PEP)的浓度固定为1mM,在不同莽草酸三磷酸(S3P)浓度(0、0.02、0.05、0.1、0.2、0.4、0.8、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,以反应相对活性为纵坐标作图,得到如图13所示的Km(S3P)的测定结果。
(6)半抑制剂量(IC50)测定:在上述反应体系中添加不同浓度(10-5、10-4、10-3、10-2、10-1、100、101、100、1000mM)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,得到如图14所示的半抑制剂量IC50值的测定结果。
(7)抑制常数Ki(glyphosate)测定:将体系中莽草酸三磷酸(S3P)的浓度固定为1mM,采用不同PEP浓度(0.067、0.1、0.2、0.5、1.0mM)配制20ul反应体系,不同PEP浓度的体系中分别添加0.5mM、1mM、2mM、5mM的草甘膦,测定酶相对活性,以底物的浓度为横坐标,以相对活性为纵坐标作图,得到如图15所示的Ki(PEP)的测定结果。
主要参考文献
1.Keeling,P.J.,et al.,Shikimate pathway in apicomplexanparasites.Nature,1999.397(6716):p.219-20.
2.Herrmann,K.M.and L.M.Weaver,The Shikimate Pathway,in Annu Rev PlantPhysiol Plant Mol Biol.1999.p.473-503.
3.Krekel,F.,et al.,Substrate and inhibitor-induced conformationalchanges in the structurally related enzymes UDP-N-acetylglucosamineenolpyruvyl transferase(MurA)and 5-enolpyruvylshikimate 3-phosphate synthase(EPSPS).Biochemistry,1999.
38(28):p.8864-78.
4.Bentley,R.,The shikimate pathway--a metabolic tree with manybranches.Crit Rev Biochem Mol Biol,1990.25(5):p.307-84.
5.Alibhai,M.F.and W.C.Stallings,Closing down on glyphosateinhibition--with a new structure for drug discovery.Proc Natl Acad Sci U S A,2001.98(6):p.2944-6.
6.Dill,G.M.,Glyphosate-resistant crops:history,status and future.PestManag Sci,2005.61(3):p.219-24.
7.Yi,S.Y.,et al.,Characterization of a new type of glyphosate-tolerant 5-enolpyruvyl shikimate-3-phosphate synthase from Isoptericolavariabilis.Journal of Molecular Catalysis B-Enzymatic,2015.111:p.1-8.
8.Pittard,J.and B.J.Wallace,Distribution and Function of GenesConcerned with Aromatic Biosynthesis in Escherichia Coli.Journal ofBacteriology,1966.91(4):p.1494-&.
9.Lanzetta,P.A.,et al.,An improved assay for nanomole amounts ofinorganic phosphate.Analytical Biochemistry,1979.100(1):p.95-97.
10.Jin,D.,et al.,Identification of a new geneencoding EPSPS with highglyphosate resistance fromthe metagenomic library.Current Microbiology,2007.55(4):p.350-355。
序列表
<110> 华中农业大学
<120> 一种适冷的I型5-烯醇丙酮酰莽草酸-3-磷酸合酶基因
<141> 2020-03-14
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1326
<212> DNA
<213> 草甘膦(glyphosate)
<220>
<221> gene
<222> (1)..(1326)
<220>
<221> CDS
<222> (1)..(1326)
<400> 1
atg acc acc gcg ccg gcg ccg acc gac ctg ccg ctg tgg gat gcg ccg 48
Met Thr Thr Ala Pro Ala Pro Thr Asp Leu Pro Leu Trp Asp Ala Pro
1 5 10 15
gtt gcg gac ggt gtg ctg gat gcg acc gtt gag gtg ccg ggc agc aag 96
Val Ala Asp Gly Val Leu Asp Ala Thr Val Glu Val Pro Gly Ser Lys
20 25 30
agc ctg acc aac cgt ctg ctg gtt ctg gcg gcg ctg gcg gac ggt ccg 144
Ser Leu Thr Asn Arg Leu Leu Val Leu Ala Ala Leu Ala Asp Gly Pro
35 40 45
ggt acc ctg cac ggt gcg ctg cgt agc cgt gac gcg gat ctg atg atc 192
Gly Thr Leu His Gly Ala Leu Arg Ser Arg Asp Ala Asp Leu Met Ile
50 55 60
gcg gcg ctg cgt agc ctg ggt gcg acc gtt acc gag ggt gaa gcg ccg 240
Ala Ala Leu Arg Ser Leu Gly Ala Thr Val Thr Glu Gly Glu Ala Pro
65 70 75 80
agc acc ctg cac gtg gcg ccg ggt cgt att acc ggt gac gtg gcg gtt 288
Ser Thr Leu His Val Ala Pro Gly Arg Ile Thr Gly Asp Val Ala Val
85 90 95
gat tgc ggt ctg gcg ggc acc gtt atg cgt ttc ctg ccg ccg gtt gcg 336
Asp Cys Gly Leu Ala Gly Thr Val Met Arg Phe Leu Pro Pro Val Ala
100 105 110
gcg ctg gcg gat ggt gat gtt cgt ttt gac ggc gat ccg gaa gcg cgt 384
Ala Leu Ala Asp Gly Asp Val Arg Phe Asp Gly Asp Pro Glu Ala Arg
115 120 125
gtt cgt ccg atg cag ccg gtg ctg gcg ggt ctg cac acc ctg ggc gtg 432
Val Arg Pro Met Gln Pro Val Leu Ala Gly Leu His Thr Leu Gly Val
130 135 140
cgt atc agc ggt ccg gat ggt acc ccg ccg agc cac ctg ccg ttc acc 480
Arg Ile Ser Gly Pro Asp Gly Thr Pro Pro Ser His Leu Pro Phe Thr
145 150 155 160
atc cac ggt cgt ggt cgt gtt gcg ggt ggc gcg gtg gac att gat gcg 528
Ile His Gly Arg Gly Arg Val Ala Gly Gly Ala Val Asp Ile Asp Ala
165 170 175
agc ggt agc agc caa ttc gtg agc gcg ctg ctg ctg gcg gcg gcg cgt 576
Ser Gly Ser Ser Gln Phe Val Ser Ala Leu Leu Leu Ala Ala Ala Arg
180 185 190
ttt gag cgt ccg ctg acc ctg cgt cac atc ggt acc acc ctg ccg agc 624
Phe Glu Arg Pro Leu Thr Leu Arg His Ile Gly Thr Thr Leu Pro Ser
195 200 205
ctg ccg cac att gac atg acc gtt gcg acc ctg cgt gaa gtg ggc gtt 672
Leu Pro His Ile Asp Met Thr Val Ala Thr Leu Arg Glu Val Gly Val
210 215 220
gcg gtg gac gat agc cgt gat ggt atc tgg cag gtg acc ccg ggt ccg 720
Ala Val Asp Asp Ser Arg Asp Gly Ile Trp Gln Val Thr Pro Gly Pro
225 230 235 240
att gcg gcg cgt gac gtt cgt gtg gag ccg gat ctg agc aac gcg gcg 768
Ile Ala Ala Arg Asp Val Arg Val Glu Pro Asp Leu Ser Asn Ala Ala
245 250 255
ccg ttc ctg gcg gcg gcg ctg gtt gcg ggt ggc acc gtt cgt gtg ccg 816
Pro Phe Leu Ala Ala Ala Leu Val Ala Gly Gly Thr Val Arg Val Pro
260 265 270
ggt tgg ccg acc agc acc acc caa ccg ggt gcg atg gtg ccg gag ctg 864
Gly Trp Pro Thr Ser Thr Thr Gln Pro Gly Ala Met Val Pro Glu Leu
275 280 285
ctg gaa cgt ctg ggt gcg acc acc gcg ctg gac gat ggt gtt ctg agc 912
Leu Glu Arg Leu Gly Ala Thr Thr Ala Leu Asp Asp Gly Val Leu Ser
290 295 300
gtg acc ggt acc ggc gag atc cgt ggt att gac gtt gat ctg cac gcg 960
Val Thr Gly Thr Gly Glu Ile Arg Gly Ile Asp Val Asp Leu His Ala
305 310 315 320
gcg ggt gaa ctg gcg ccg acc ttt gcg gtg ctg gcg gcg ctg gcg gat 1008
Ala Gly Glu Leu Ala Pro Thr Phe Ala Val Leu Ala Ala Leu Ala Asp
325 330 335
agc ccg agc cgt ctg cgt ggt atc gcg cac ctg cgt ggt cac gag acc 1056
Ser Pro Ser Arg Leu Arg Gly Ile Ala His Leu Arg Gly His Glu Thr
340 345 350
gat cgt ctg gcg gcg ctg gcg gcg gaa att acc cgt ctg ggt ggc cgt 1104
Asp Arg Leu Ala Ala Leu Ala Ala Glu Ile Thr Arg Leu Gly Gly Arg
355 360 365
tgc gag gaa acc cgt gac ggt ctg gtg gtt acc ccg cgt ccg ctg cac 1152
Cys Glu Glu Thr Arg Asp Gly Leu Val Val Thr Pro Arg Pro Leu His
370 375 380
ggt ggc gtt ttc cgt acc tac gcg gat cac cgt atg gcg acc agc gcg 1200
Gly Gly Val Phe Arg Thr Tyr Ala Asp His Arg Met Ala Thr Ser Ala
385 390 395 400
gcg ctg ctg ggt ctg cgt gtg ccg gac ctg cag gtt gag gat gtg gcg 1248
Ala Leu Leu Gly Leu Arg Val Pro Asp Leu Gln Val Glu Asp Val Ala
405 410 415
acc acc gcg aaa acc ctg ccg ggt ttt gac cgt att tgg gcg cgt atg 1296
Thr Thr Ala Lys Thr Leu Pro Gly Phe Asp Arg Ile Trp Ala Arg Met
420 425 430
ctg gcg cag ggt gcg gaa gcg caa gcg taa 1326
Leu Ala Gln Gly Ala Glu Ala Gln Ala
435 440
<210> 2
<211> 441
<212> PRT
<213> 草甘膦(glyphosate)
<400> 2
Met Thr Thr Ala Pro Ala Pro Thr Asp Leu Pro Leu Trp Asp Ala Pro
1 5 10 15
Val Ala Asp Gly Val Leu Asp Ala Thr Val Glu Val Pro Gly Ser Lys
20 25 30
Ser Leu Thr Asn Arg Leu Leu Val Leu Ala Ala Leu Ala Asp Gly Pro
35 40 45
Gly Thr Leu His Gly Ala Leu Arg Ser Arg Asp Ala Asp Leu Met Ile
50 55 60
Ala Ala Leu Arg Ser Leu Gly Ala Thr Val Thr Glu Gly Glu Ala Pro
65 70 75 80
Ser Thr Leu His Val Ala Pro Gly Arg Ile Thr Gly Asp Val Ala Val
85 90 95
Asp Cys Gly Leu Ala Gly Thr Val Met Arg Phe Leu Pro Pro Val Ala
100 105 110
Ala Leu Ala Asp Gly Asp Val Arg Phe Asp Gly Asp Pro Glu Ala Arg
115 120 125
Val Arg Pro Met Gln Pro Val Leu Ala Gly Leu His Thr Leu Gly Val
130 135 140
Arg Ile Ser Gly Pro Asp Gly Thr Pro Pro Ser His Leu Pro Phe Thr
145 150 155 160
Ile His Gly Arg Gly Arg Val Ala Gly Gly Ala Val Asp Ile Asp Ala
165 170 175
Ser Gly Ser Ser Gln Phe Val Ser Ala Leu Leu Leu Ala Ala Ala Arg
180 185 190
Phe Glu Arg Pro Leu Thr Leu Arg His Ile Gly Thr Thr Leu Pro Ser
195 200 205
Leu Pro His Ile Asp Met Thr Val Ala Thr Leu Arg Glu Val Gly Val
210 215 220
Ala Val Asp Asp Ser Arg Asp Gly Ile Trp Gln Val Thr Pro Gly Pro
225 230 235 240
Ile Ala Ala Arg Asp Val Arg Val Glu Pro Asp Leu Ser Asn Ala Ala
245 250 255
Pro Phe Leu Ala Ala Ala Leu Val Ala Gly Gly Thr Val Arg Val Pro
260 265 270
Gly Trp Pro Thr Ser Thr Thr Gln Pro Gly Ala Met Val Pro Glu Leu
275 280 285
Leu Glu Arg Leu Gly Ala Thr Thr Ala Leu Asp Asp Gly Val Leu Ser
290 295 300
Val Thr Gly Thr Gly Glu Ile Arg Gly Ile Asp Val Asp Leu His Ala
305 310 315 320
Ala Gly Glu Leu Ala Pro Thr Phe Ala Val Leu Ala Ala Leu Ala Asp
325 330 335
Ser Pro Ser Arg Leu Arg Gly Ile Ala His Leu Arg Gly His Glu Thr
340 345 350
Asp Arg Leu Ala Ala Leu Ala Ala Glu Ile Thr Arg Leu Gly Gly Arg
355 360 365
Cys Glu Glu Thr Arg Asp Gly Leu Val Val Thr Pro Arg Pro Leu His
370 375 380
Gly Gly Val Phe Arg Thr Tyr Ala Asp His Arg Met Ala Thr Ser Ala
385 390 395 400
Ala Leu Leu Gly Leu Arg Val Pro Asp Leu Gln Val Glu Asp Val Ala
405 410 415
Thr Thr Ala Lys Thr Leu Pro Gly Phe Asp Arg Ile Trp Ala Arg Met
420 425 430
Leu Ala Gln Gly Ala Glu Ala Gln Ala
435 440
Claims (3)
1.一种适冷的I型5-烯醇丙酮莽草酸-3-磷酸合酶基因,其核苷酸序列如序列表SEQ IDNO:1所示。
2.一种适冷的I型5-烯醇丙酮莽草酸-3-磷酸合酶基因,该基因编码的蛋白质序列如序列表SEQ ID NO:2所示。
3.权利要求1所述的适冷的I型5-烯醇丙酮莽草酸-3-磷酸合酶基因在提高大肠杆菌(Escherichia coli)抗草甘膦能力中的应用。
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CN110272880A (zh) * | 2019-05-22 | 2019-09-24 | 华中农业大学 | 一种突变型草甘膦降解酶及其克隆、表达与应用 |
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CN103834674B (zh) * | 2014-03-21 | 2016-03-23 | 华中农业大学 | 一种分离的5-烯醇丙酮莽草酸-3-磷酸合酶基因 |
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CN110272880A (zh) * | 2019-05-22 | 2019-09-24 | 华中农业大学 | 一种突变型草甘膦降解酶及其克隆、表达与应用 |
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