CN102286501B - 噻吩磺隆水解酶基因tsmE及其应用 - Google Patents
噻吩磺隆水解酶基因tsmE及其应用 Download PDFInfo
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Abstract
本发明属于应用环境微生物和农业领域,公开了噻吩磺隆水解酶基因tsmE及其应用。一种噻吩磺隆水解酶基因tsmE,其核苷酸序列为SEQ ID NO.1,全长为1194bp,G+C含量为51.09%,编码398个氨基酸,氨基酸序列为SEQ ID NO.2。本发明提供的噻吩磺隆水解酶TsmE能在1hr内完全降解100mg/L的噻吩磺隆,并将噻吩磺隆水解为无除草活性的产物噻吩磺酸;此外TsmE还能在1hr内完全降解100mg/L的除草剂高效盖草能。因此噻吩磺隆水解酶基因tsmE在构建抗噻吩磺隆的转基因作物中应用。噻吩磺隆水解酶蛋白质TsmE在降解噻吩磺隆和高效盖草能中应用。
Description
技术领域
本发明属于应用环境微生物和农业领域,涉及噻吩磺隆水解酶基因tsmE及其应用。
背景技术
除草剂的使用在减轻农业劳动强度、保证农业正常生产的同时,其残留也带来了严重的作物药害问题,据统计我国每年农田受除草剂药害面积达到3000万亩,其中严重药害面积达到500万亩,每年造成几十亿元的损失,而抗除草剂转基因是解决除草剂药害的最佳途径。磺酰脲类除草剂在中国使用量大,研究和应用发展迅速,已经成为继有机磷、乙酰胺类除草剂后的第三大除草剂,全球年销售额达到30亿美元以上,我国磺酰脲类除草剂每年的应用面积已超过200万公顷,并仍呈扩大的趋势,噻吩磺隆是使用非常广泛的一类磺酰脲类除草剂。磺酰脲类除草剂残留期较长,在土壤中积累会对下茬作物产生严重的药害作用,现已发现磺酰脲类除草剂的残留药害可伤及水稻、大豆、玉米、油菜、棉花、甜菜、亚麻和向日葵等多种重要作物。
获得磺酰脲类除草剂降解菌株和降解基因在治理除草剂残留,消除其药害技术研发中具有以下作用和功能,(一)通过现代生物技术将降解基因导入作物构建相应的除草剂抗性转基因作物,(二)通过现代微生物发酵技术将磺酰脲类除草剂降解菌株和基因制成降解菌剂或酶制剂将土壤中磺酰脲类除草剂残留降解。此外磺酰脲类除草剂降解基因还可用于有用化工产品及药物合成的生物转化。因此噻吩磺隆降解基因在消除该类除草剂药害及生物转化领域中具有非常重要的理论和应用价值。
磺酰脲类除草剂噻吩磺隆应用日益广泛,但其残留期较长,在土壤中积累会对下茬作物产生严重的药害作用,而构建和种植抗除草剂转基因是解决除草剂药害的最佳途径。目前能够降解噻吩磺隆的基因还未见报道。
发明内容
本发明的目的是针对现有技术的上述不足,提供一种噻吩磺隆水解酶基因,该基因可用于构建抗噻吩磺隆的转基因作物,也可用于土壤、水体中除草剂噻吩磺隆和高效盖草能残留的去除及药物合成的生物转化。
本发明的另一目的是提供该基因的应用。
本发明的目的通过如下技术方案实现:
一种噻吩磺隆水解酶基因tsmE,其核苷酸序列为SEQ ID NO.1。
本专利所用的出发菌株为一株能够降解噻吩磺隆的细菌菌株S113,在分类上属于嗜甲基菌(Methylophilus sp.),保存在中国普通微生物菌种保藏管理中心,保藏编号为CGMCC 1479,保藏日期为2005年10月12日。质谱分析结果表明菌株S113的粗酶液可以把噻吩磺隆水解为噻磺酸。
克隆噻吩磺隆水解酶基因采取的策略为鸟枪法(见图1)。首先提取菌株S113的总DNA,总DNA采用Sau3AI部分酶切后和用BamHI酶切的质粒pUC118酶连,酶连产物转化大肠杆菌DH10B感受态细胞构建菌株S113的总DNA文库,含有噻吩磺隆降解基因的克隆子能使培养基中的噻吩磺隆降解,生成的噻磺酸不抑制大肠杆菌生长,解除对大肠杆菌的抑制而生长。不含噻吩磺隆降解基因的克隆子则因受到噻吩磺隆的抑制而不能生长。利用这种克隆方法可以对文库进行高通量的筛选。
用上面的策略筛选鸟枪法构建的基因文库获得一个能在加入10ppm噻吩磺隆的基础盐培养基(葡萄糖为碳源)上生长的阳性克隆子,进一步的降解实验表明该阳性克隆子能降解噻吩磺隆。测序结果表明该阳性克隆子含有5143个碱基对,其中包含有18个潜在的ORF(大于150bp),对这些潜在的ORF分别进行亚克隆和序列比对分析,最后确定编码噻吩磺隆水解酶的基因的大小为1194kb,命名为tsmE。这是首次克隆到能降解磺酰脲类除草剂的水解酶基因。
所述的噻吩磺隆水解酶酶基因tsmE核苷酸序列所编码的噻吩磺隆水解酶蛋白质TsmE,其氨基酸序列为:SEQ ID NO.2。
含有所述的噻吩磺隆水解酶基因tsmE的重组表达载体。
所述的重组表达载体优选将所述的噻吩磺隆水解酶基因tsmE插入pET-29a(+)的NdeI和HindIII位点之间所得。
含有所述的噻吩磺隆水解酶基因tsmE的基因工程菌。
所述的基因工程菌优选以大肠杆菌BL21(DE3)为出发菌株。
所述噻吩磺隆水解酶基因tsmE在构建抗噻吩磺隆的转基因作物中的应用。
所述噻吩磺隆水解酶基因tsmE在降解噻吩磺隆和高效盖草能中的应用。
所述噻吩磺隆水解酶蛋白质TsmE在降解噻吩磺隆和高效盖草能中的应用。
所述噻吩磺隆水解酶蛋白质TsmE在去除土壤、水体中除草剂噻吩磺隆残留和高效盖草能中的应用。
本发明的有益效果如下:
1.本发明用鸟枪法成功的从菌株S113(CGMCC 1479)中克隆出噻吩磺隆水解酶基因tsmE。在GenBank比对结果表明该基因为一个新的基因,全长(从起始密码子到终止密码子)为1194bp,G+C含量为51.09%,编码398个氨基酸。
2.本发明提供的噻吩磺隆水解酶TsmE能在1hr内完全降解100mg/L的噻吩磺隆,并将噻吩磺隆水解为无除草活性的产物噻吩磺酸(见图4和图5),此外TsmE还能在1hr内完全降解100mg/L的除草剂高效盖草能(见图6和图7)。tsmE可用于构建抗噻吩磺隆的转基因作物,也可用于土壤、水体中除草剂噻吩磺隆和高效盖草能残留的去除及药物合成的生物转化,具有非常重要的理论和应用价值。
附图说明
图1噻吩磺隆水解酶基因tsmE克隆的策略图。
图2噻吩磺隆水解酶基因tsmE在BL21(pET-29a(+))中表达策略图。
图3噻吩磺隆水解酶TsmE蛋白电泳图谱;
其中泳道1为蛋白质marker,泳道2为纯化的噻吩磺隆水解酶TsmE蛋白。
图4噻吩磺隆和高效盖草能水解酶TsmE降解噻吩磺隆LC-MS图;
A:噻吩磺隆和高效盖草能水解酶TsmE降解噻吩磺隆的液相色谱图;
B:噻吩磺隆和高效盖草能水解酶TsmE降解噻吩磺隆的一级质谱图;
C:噻吩磺隆和高效盖草能水解酶TsmE降解噻吩磺隆的子离子二级质谱图。
图5噻吩磺隆和高效盖草能水解酶TsmE降解噻吩磺隆的途径。
图6噻吩磺隆和高效盖草能水解酶TsmE降解高效盖草能MS/MS图。
A:噻吩磺隆和高效盖草能水解酶TsmE降解高效盖草能的一级质谱图。
B:噻吩磺隆和高效盖草能水解酶TsmE降解高效盖草能的二级质谱图。
图7噻吩磺隆和高效盖草能水解酶TsmE降解高效盖草的途径。
生物材料保藏信息
嗜甲基菌S113(Methylophilus sp.),保存在中国普通微生物菌种保藏管理中心(CGMCC),地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为CGMCC 1479,保藏日期为2005年10月12日。
具体实施方式
实施例1.噻吩磺隆水解酶基因的克隆
1.1细菌基因组总DNA的提取
S113(CGMCC 1479)大量培养后,采用CTAB法提取高纯度、大片段的S113的基因组总DNA,溶于TE缓冲液(pH8.0)中,置于-20℃保藏,具体方法参考F·奥斯伯等编的《精编分子生物学实验指南》。
1.2总DNA的酶切S113(CGMCC 1479)总DNA采用Sau3AI部分酶切。
1.3DNA的回收
酶切后的总DNA通过电泳(TAE缓冲液)进行纯化,采用axygen biosciences(China)回收试剂盒进行回收,回收的DNA溶于10mmol/L的Tris·Cl(pH8.0)中,置于-20℃保藏。
1.4酶连
建立如下反应体系:
1.5制备大肠杆菌DH10B高效感受态细胞
大肠杆菌DH10B购自上海英骏生物技术有限公司。高效感受态细胞制备的具体方法参照F.奥斯伯等编的《精编分子生物学实验指南》P 22-23。
1.6转化
取10μl酶连产物转化200μl大肠杆菌DH10B感受态细胞,具体方法参照F.奥斯伯等编的《精编分子生物学实验指南》P 23。涂布含有10mg/kg的噻吩磺隆,100mg/kg氨苄青霉素的基础培养基平板,培养24h后挑取生长的菌落,进一步验证获得一个能将噻吩磺隆转化为噻磺酸的转化子。基础盐培养基配方为5.0 glucose,1.0 NH4NO3,1.0 NaCl,1.5 K2HPO4,0.5 KH2PO4,0.2 MgSO4·7H2O,pH 7.0。
1.7基因核苷酸序列测定
将1.6中获得的能将噻吩磺隆转化为噻磺酸的转化子送交上海英骏生物技术有限公司进行序列测定,噻吩磺隆水解酶基因的核苷酸序列为SEQ ID NO.1,根据噻吩磺隆水解酶基因核苷酸序列所推到的398个氨基酸序列为SEQ ID NO.2。
实施例2噻吩磺隆水解酶基因在BL21(pET-29a(+))中的高效表达(图2)
2.1噻吩磺隆水解酶基因的PCR扩增
以正向引物:5’-TGCAGACATATGGAAACCGATAAAAAAAC-3’(SEQ ID NO.3)和反向引物:5’-TGCAGAGAATTCCCTTCCATAAGAGCGCCGAT-3’(SEQ ID NO.4)为引物,用PCR从S113(CGMCC 1479)基因组DNA中扩增出噻吩磺隆水解酶基因片段。
扩增体系:
PCR扩增程序:
a.95℃变性3min;
b.95℃变性1.5min,53℃退火0.5min,72℃延伸1.5min,进行25个循环;
c.72℃延伸10min,冷却到室温。
2.2PCR产物用NdeI和HindIII双酶切。
酶切体系:
在37℃水浴中,反应3h以上。酶切产物进行2%的琼脂糖凝胶电泳切胶回收。2.3pET-29a(+)用NdeI和HindIII双酶切(参考2.2)。
2.4转化
2.2中的回收片段和2.3中酶切好的pET-29a(+)进行酶连(参考1.5)。酶连好的含噻吩磺隆水解酶基因的pET-29a(+)重组质粒转化到表达宿主菌BL21(DE3)获得重组微生物BL21(TsmE)。
2.5TsmE的表达、纯化和功能验证
BL21(TsmE)在LB培养基中培养至OD600nm为0.6到0.8之间,加IPTG至浓度1mM,30℃培养4个小时。100ml菌液离心,用10ml(50mM,pH 7.0)PBS缓冲液重悬菌体,超声破碎(AutoScience,UH-650B ultrasonic processor,30% intensity)5分钟,离心,收集上清,用镍离子亲和层析柱对TsmE进行了纯化,纯化后的酶进行蛋白质电泳,见图3。
2.6TsmE活力测定
酶活反应体系:50mM磷酸缓冲液(pH 7.0),0.2mM噻吩磺隆或0.2mM高效盖草能、反应酶量(2.5中纯化所得)50μl,30℃反应20min。每个反应以加入酶开始计时,用3ml二氯甲烷终止反应,分层后有机相经无水硫酸钠脱水,噻吩磺隆或高效盖草能含量用反向HPLC测定(具体方法见2.7)。一个酶活力单位(U)定义为:在pH 7.0,温度30℃条件下,每分钟催化减少1μmol噻吩磺隆或高效盖草能所需的酶量。降解试验表明纯化后的TsmE能在1hr内降解100mg/kg的噻吩磺隆或高效盖草能,酶学试验表明TsmE对噻吩磺隆和高效盖草能的比酶活分别为67和55U/mg protein。
2.7代谢产物的确定
2.7.1噻吩磺隆降解代谢产物的确定
2.6中的噻吩磺隆的酶反应液过滤,取20μL滤液进行LC-MS,液相色谱条件:色谱柱:Agilent Zorbax XDB-C18柱(2.1×50mm,3.5μm),流动相:甲醇∶水=80∶20,流速0.25ml/min;紫外检测波长255nm。一级质谱条件:离子检测方式为多反应离子检测;离子极性为负离子;离子化方式为电喷雾离子化;毛细管电压为4000伏;干燥气温度:330℃;干燥气流速:10.0L/min,雾化气压力:35psi,碰撞电压:135伏;质量扫描范围(m/z):300-500。二级子离子质谱条件:碰撞电压:90伏;质量扫描范围(m/z):30-400。
LC-MS的液谱图(见图4A)表明,其产物的保留时间为1.95min,一级质谱图(见图4B)显示其有m/z为372.30分子负离子峰,m/z为372.30的分子负离子其二级质谱图(见图4C)中有m/z为162.10,188.10,206.20的片段,这与噻磺酸相符。因此,TsmE水解噻吩磺隆的生化反应是就噻吩磺隆转化成噻磺酸(见图5)。
2.7.2高效盖草能降解代谢产物的确定
利用串联质谱测定高效盖草能降解代谢产物,2ml 2.6中的高效盖草能酶反应液二氯甲烷提取物经氮气吹干后溶于100μL甲醇,进行串联质谱测定,串联质谱条件:MS/MS(FinniganTSQ Quantum Ultra AM,Thermal,U.SA.),采用电喷雾形式离子化,正负离子同时检测,质量扫描范围(m/z):30-1200。
MS/MS的一级质谱图(见图6A)中有m/z为359.87的分子负离子峰,m/z为359.87的分子负离子其二级质谱(见图6B)中有m/z为287.60的片段,这与TsmE水解高效盖草能的代谢产物2-[4-(3-氯-5-三氟甲基-2-吡啶氧基)苯氧基]丙酸相符。因此,TsmE水解高效盖草能的生化反应是就将高效盖草能转化成2-[4-(3-氯-5-三氟甲基-2-吡啶氧基)苯氧基]丙酸(见图7)。以上实施例中使用的微生物来源如下:
pUC118(BamHI) 购自宝生物工程(大连)有限公司,
大肠杆菌DH10B 购自上海英骏生物技术有限公司,
大肠杆菌高表达载体pET-29a(+) 购自Novegen公司,
表达宿主菌大肠杆菌BL21(DE3) 购自上海英骏生物技术有限公司。
Claims (9)
1.一种噻吩磺隆水解酶基因tsmE,其核苷酸序列为SEQ ID NO.1。
2. 权利要求1所述的噻吩磺隆水解酶基因tsmE核苷酸序列所编码的噻吩磺隆水解酶蛋白质TsmE,其氨基酸序列为:SEQ ID NO.2。
3. 含有权利要求1所述的噻吩磺隆水解酶基因tsmE的重组表达载体。
4.根据权利要求3所述的重组表达载体,其特征在于是将权利要求1所述的噻吩磺隆水解酶基因tsmE插入pET-29a(+)的NdeI和HindIII位点之间所得。
5. 含有权利要求1所述的噻吩磺隆水解酶基因tsmE的基因工程菌。
6. 根据权利要求5所述的基因工程菌,其特征在于所述的基因工程菌的出发菌株为大肠杆菌BL21(DE3)。
7. 权利要求1所述噻吩磺隆水解酶基因tsmE在降解噻吩磺隆和高效盖草能中的应用。
8. 权利要求2所述噻吩磺隆水解酶蛋白质TsmE在降解噻吩磺隆和高效盖草能中的应用。
9. 权利要求2所述噻吩磺隆水解酶蛋白质TsmE在去除土壤、水体中除草剂噻吩磺隆和高效盖草能残留中的应用。
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