CN104311648B - 一种杀虫蛋白及其编码基因与应用 - Google Patents
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Abstract
本发明涉及一种杀虫蛋白Cry1m7,所述杀虫蛋白为1)由SEQ ID NO:1所示的氨基酸序列组成的蛋白质,或2)在SEQ ID NO:1所示的氨基酸序列中经取代、缺失或插入一个或几个氨基酸序列所获得的具有同等活性的蛋白质。本发明还提供了所述杀虫蛋白的编码基因、表达载体、工程菌、所述蛋白在杀虫药剂中的应用以及所述蛋白的编码基因在转化植物中的应用。本发明提供了一种高毒力的杀虫蛋白Cry1m7及其编码基因;本发明所提供的杀虫蛋白可以用于杀死亚洲玉米螟等鳞翅目害虫,提高转基因作物的杀虫能力。
Description
技术领域
本发明涉及基因工程领域,特别涉及一种杀虫蛋及其编码基因与应用。
背景技术
虫害是造成农作物严重减产的一个主要因素,防治农业害虫最常见的手段就是使用广谱化学杀虫剂和一些生物杀虫剂。化学杀虫剂多具有广谱、高毒的特点,在杀死目标害虫的同时往往将很多益虫一起杀死,严重破坏生态平衡,并对环境造成严重污染;另一方面,农药残留对人类、牲畜的健康也造成严重威胁。生物杀虫剂具有易降解、与环境高度相容的特点,但在生产上需要重复施用,大大增加生产成本。为了弥补化学杀虫剂和生物杀虫剂在农业生产应用中的弊端,科学家们将能编码杀虫蛋白的基因导入到植物体内,培育出多种转基因植物。自1996年至今,已经有多种转Bt基因杀虫作物获得批准进行商业化生产,但这些抗虫转基因作物多使用Cry1A类杀虫蛋白基因,杀虫蛋白基因的单一化、大面积使用加速害虫抗性的产生,制约了转Cry1A类抗虫转基因作物的应用年限。因此,需要不断寻找高毒力、且与已经使用的杀虫蛋白基因无交互抗性的新的基因以延缓害虫抗性的产生。
目前,人们已经克隆了数百种Bt杀虫蛋白基因,但这些杀虫蛋白基因真正能应用于生产的却屈指可数,其原因主要是毒力较低,容易使害虫产生抗性;其次,多数基因存在不同程度的交互抗性。贺明霞等的研究结果表明Cry1Ie与Cry1Ab、Cry1Ac及Cry1Fa三种毒素不存在交互抗性,但Cry1Ab和Cry1Ac的交互抗性较高,Cry1Fa与Cry1Ab和Cry1Ac也存在一定程度的交互抗性(He,2013)。Gassmann等的研究结果表明孟山都公司培育的杀虫玉米转化事件MON88017中所使用的杀虫蛋白基因Cry3Bb1与先正达公司培育的杀虫玉米转化事件MIR604中所使用的杀虫蛋白基因mCry3A存在交互抗性,这使西方玉米根虫在两种玉米商业化使用三年内就对两种杀虫蛋白基因产生了严重的抗性,对玉米生产造成严重损失(Gassmann,2014)。除此之外,杀虫蛋白基因克隆资源的日渐匮乏也给基因克隆造成很大难度。以上因素都增加了具有应用潜力杀虫蛋白基因克隆的难度。
Cry1I类Bt蛋白对害虫有很好的杀虫活性。Cry1Ia1基因编码的杀虫晶体蛋白对鳞翅目和鞘翅目昆虫具有毒害作用,已用于培育抗马铃薯块茎蛾的转基因马铃薯如SpuntaG2及抗大豆食心虫的转基因大豆。Cry1Ie基因编码的杀虫晶体蛋白对鳞翅目害虫如小菜蛾、玉米螟有很高的抗性,且与商业化应用的一些抗虫基因不存在交互抗性。转Cry1Ie基因抗虫玉米高抗亚洲玉米螟,而且可以有效杀死对Cry1Ac蛋白产生抗性的棉铃虫,具有重大应用价值(Zhang,2013)。但Cry1Ie蛋白的毒力比杀虫蛋白Cry1Ac低,在某种程度上限制了其应用。因此,有必要对其进行改造,进一步提高Cry1Ie杀虫蛋白的毒力。
发明内容
本发明的目的在于克服现有研究中存在的上述缺陷,提供一种毒力显著高于Cry1Ie、Cry1Ia的一种新型杀虫蛋白基因及其编码蛋白。
为了实现本发明的目的,本发明首先提供一种杀虫蛋白Cry1m7,所述杀虫蛋白由SEQ ID NO:1所示的氨基酸序列组成。
应当理解的是,本领域技术人员可以根据SEQ ID NO:1所示的氨基酸序列在不影响其活性的条件下,对该序列进行取代、缺失或插入一个或几个氨基酸序列以获得具有同等活性的蛋白质。
本发明的发明人以Cry1Ie(GenBank:AF211190.1)和Cry1Ia1(GenBank:X62821.1)为参考序列,进行结构域改造,得到重组杀虫蛋白Cry1m7。
本发明还提供了编码所述蛋白的基因,所述基因的核苷酸序列如SEQ ID NO:2所示。
本发明所提供的基因包括所述蛋白相应的编码核酸序列。应当理解的是,由于密码子具有简并性以及不同物种密码子所具有的偏爱性,本领域技术人员可以根据需要使用适合特定物种表达的密码子。在本发明中,所述核苷酸序列采用了玉米偏爱性密码子。
本发明还提供了所述基因的表达载体,其中,所述表达载体包括诱导表达载体和植物表达载体。
本发明还提供了含有所述基因的工程菌。
本发明还提供了基因Cry1m7在转化植物中的应用。
进一步地,所述应用是在植物体内表达所述杀虫蛋白的编码基因,提高植物的抗害虫能力。
进一步地,所述植物包括玉米、水稻、棉花、大豆、高粱。
进一步地,所述害虫为鳞翅目害虫,包括但不限于亚洲玉米螟、棉铃虫和粘虫。
本发明还提供了杀虫蛋白Cry1m7在杀虫药剂中的应用。
本发明所获得的有益效果在于:提供了一种高毒力的杀虫蛋白Cry1m7及其编码基因;本发明所提供的杀虫蛋白可以用于杀死亚洲玉米螟等鳞翅目害虫,提高转基因作物的杀虫能力。
附图说明
图1为pET30a酶切载体与基因Cry1m7连接产物转大肠杆菌不同菌斑的PCR产物;
M:DL2000plus DNA marker;1-6:pET30a酶切载体与基因Cry1m7连接产物转大肠杆菌不同菌斑的PCR产物;图中箭头所指的位置为2525bp。
图2为质粒pET30a-Cry1m7经BamHI和SacI双酶切产物的琼脂糖电泳图;
M:DL2000plus DNA marker;1:质粒pET30a-Cry1m7双酶切产物。
图3为含有Cry1m7或Cry1Ie抗虫基因的pET30a载体图;
其中pET30a-Bt中Bt示意杀虫基因名字Cry1m7或Cry1Ie。
图4为Cry1m7杀虫蛋白基因蛋白诱导纯化后SDS-PAGE电泳检测;
M:PageRuler Prestained Protein Ladder;1:Cry1Ie;2:Cry1m7。
图5为植物表达载体p3301Ubi-Cry1m7构建时Cry1m7基因PCR扩增产物的琼脂糖电泳图;
M:DL2000plus DNA marker;1:Cry1m7。
图6为含有Cry1m7杀虫蛋白基因的p3301Ubi-Cry1m7植物表达载体图。
具体实施方式
下面将通过具体实施方式对本发明进行详细说明。
实施例1
Cry1m7诱导表达载体的构建。
Cry1m7的基因序列委托上海生工合成并构建到pUC57载体上,构成具有氨苄青霉素抗性的质粒pUC57-Cry1m7。
将质粒pUC57-Cry1m7及质粒pET30a(购自美国Novagen公司)各取20ng加入到50μL大肠杆菌感受态细胞Trans5α中,冰浴30min;42℃热激90sec,冰浴2min;加入300μL LB液体培养基,37℃低速振荡恢复培养1hr后,将菌液涂于含相应抗生素的平板上,晾干;37℃培养箱倒置培养15hr。挑取单克隆菌斑于10ml含氨苄青霉素的LB液体培养基中,37℃摇床震荡过夜培养,次日,依照天根质粒小提中量提取试剂盒操作步骤提取质粒,并用NanoDrop2000C微量紫外分光光度计对提的质粒定量。
选择在质粒上有单一酶切位点且恰位于基因两端的限制性内切酶BamHI和SacI双酶切质粒pUC57-Cry1m7及pET30a,使基因与载体的两端具有相同的粘性末端。pUC57-Cry1m7酶切后,经琼脂糖胶电泳,对照Marker分别切取约2.1kb大小左右的片段,用天根胶回收试剂盒对核酸片段进行回收;质粒pET30a经双酶切后用天根纯化试剂盒进行纯化回收。然后,按照T4连接酶的使用说明将切胶回收的核酸片段与纯化回收的载体片段进行连接。连接反应结束,取5μL连接产物转化大肠杆菌Trans5α,方法同上。PCR扩增筛选阳性克隆,具体引物信息如下:
上游引物:5’-TAATACGACTCACTATAGGG-3’(SEQ ID NO:3)
下游引物:5’-GCTAGTTATTGCTCAGCGG-3’(SEQ ID NO:4)
扩增片段长度为2525bp。
PCR扩增体系及反应程序如下:
PCR反应体系:
PCR反应程序:
95℃预变性5min;94℃变性30sec,55℃退火30sec,72℃延伸2min,共30个循环后再在72℃下延伸7min,25℃保温。PCR扩增产物琼脂糖电泳结果见图1。
挑取PCR扩增筛选的阳性克隆菌斑于10ml含相应抗生素的LB液体培养基中,37℃摇床震荡过夜培养,次日,提质粒并定量。一部分质粒用于对pET30a载体中杀虫蛋白基因测序;另一部分质粒,用BamHI和SacI双酶切质粒,经琼脂糖电泳验证杀虫蛋白基因与pET30a酶切位点连接的正确性。电泳验证酶切结果见图2,酶切验证体系如下:
测序、酶切验证均正确的pET30a-Cry1m7及实验室保存的pET30a-Cry1Ie诱导表达载体见图3。
实施例2
Cry1m7和Cry1Ie蛋白的诱导与纯化。
将测序并酶切验证正确的质粒pET30a-Cry1m7转入购自全式金公司的菌株Transetta(DE3)中,挑取单克隆,PCR扩增验证阳性菌斑。将检测阳性的菌斑接种于10mL的LB液体培养基中(含适宜抗生素),于37℃过夜振荡培养。含有pET30a-Cry1Ie质粒的大肠杆菌菌株Transetta(DE3)为实验室-70℃冻存,按1:100接种到10mL LB液体培养基中(含适宜抗生素),同样于37℃过夜振荡培养。
次日,按1:200接种到200mL LB液体培养基中(含适宜抗生素),37℃,200rpm震荡培养至OD600为0.4-0.6,加入IPTG至终浓度0.5mM,16℃摇床,160rpm,震荡培养20hr左右,诱导目的蛋白的表达。
分别收集已诱导的菌体,5000rpm,低温下离心5min,弃上清液,加入1/20体积比的重悬buffer(25mM Tris-Cl,150mM NaCl,15mM imidazole),加入10mg/mL的溶菌酶至终浓度为100μg/mL,振荡之后,冰上放置10min,超声波破碎,每隔3s超声4s,300w,15-20min,使菌体充分破碎,破碎过程冰上操作。
12000rpm,4℃,离心15min,取上清,用0.45μm滤膜过滤。加入1mL混匀的50%的Ni-NTA树脂,室温下于摇床上轻微振荡90min,使目的蛋白充分结合到Ni-NTA树脂上。将结合有目的蛋白的Ni-NTA树脂装柱纯化。新打开包装的镍柱用70%乙醇平衡,之后用5倍镍柱体积的重悬buffer平衡(含咪唑15mM)。
低速向柱子中加入混匀好的蛋白上清和树脂的混合物,收集流出液,标记为L15。
25mM Tris-Cl,150mM NaCl,30mM imidazole,8mL洗涤,收集流出液,标记为L30。
25mM Tris-Cl,150mM NaCl,100mM imidazole,4mL洗涤,收集流出液,标记为L100。
25mM Tris-Cl,150mM NaCl,250mM imidazole,10mL洗涤,收集流出液,标记为L250。
以上纯化过程均在4℃环境中操作,并严格避免交叉污染。
纯化蛋白分别转入透析袋中,用透析液(25mM Tris-Cl,150mM NaCl)在4℃环境中,转子搅拌下透析24hr,每8hr更换一次透析液。透析处理后,采用考马斯亮蓝蛋白定量试剂盒对纯化的L250蛋白进行定量。同时,将杀虫蛋白L250收集的流出液进行SDS-PAGE电泳检测,电泳结果见图4。
实施例3
Cry1Ie纯化蛋白的室内玉米螟虫试。
在室内温度为28±1℃、光周期(L:D)16:8h、相对湿度70%~80%的条件下,采用人工饲料混合法进行室内玉米螟虫试,即分别将Cry1m7和Cry1Ie的纯化蛋白添加到人工饲料中配成0μg/g,0.025μg/g,0.05μg/g,0.25μg/g,0.5μg/g,2.5μg/g,5μg/g,25μg/g,50μg/g共9个浓度梯度的饲养饲料,将每个蛋白对应浓度配制的饲料均分到三个48孔细胞培养板中,每孔接玉米螟初孵幼虫一头,每个浓度共接虫144头,7天后统计虫子死亡率及平均虫重Cry1m7和Cry1Ie纯化蛋白室内生测数据分析LC50的结果详见表1。从表1的数据可以看出,Cry1m7的杀虫效果比Cry1Ie的杀虫效果提高了49.24%,比Cry1Ia1的杀虫效果提高了49.38%。
表1
蛋白种类 | LC50(μg/g) |
Cry1Ie | 5.395 |
Cry1Ia1 | 5.400 |
Cry1m7 | 3.615 |
实施例4
植物转化载体p3301Ubi-Cry1m7的构建。
植物表达载体的骨架为改造的pCAMBIA3301,p3301Ubi-Cry1m7载体的构建采用Clontech公司的infusion酶进行构建。首先,设计infusion引物对Cry1m7基因进行PCR扩增,引物设计时下游引物补上翻译终止所需的终止密码子TAG,具体引物信息如下:
上游引物:
5’-CGACTCTAGAGGATCCATGAAGCTGAAGAACCCGGA-3’(SEQ ID NO:5)
下游引物:
5’-GCTGGTCACCGAGCTCGAGCTCTCACATGTTCCTCT-3’(SEQ ID NO:6)
扩增片段长度为2175bp。
PCR扩增体系及反应程序如下:
PCR反应体系:
PCR反应程序:95℃预变性2min;95℃变性20sec,58℃退火20sec,72℃延伸1.5min,共28个循环;再在72℃下继续延伸7min,25℃保温。
以上述体系及程序进行PCR反应,获得Cry1m7基因的扩增片段,PCR扩增产物电泳结果见图5。然后,取5μl PCR扩增产物,加2μl Cloning Enhancer于PCR仪中37℃反应15min,80℃继续反应15min,将反应产物置于冰上备用。
同时用BamHI和SacI将改造的pCAMBIA3301载体双酶切,37℃水浴锅中酶切1hr,酶切体系如下:
取1μl Cloning Enhancer处理的PCR反应产物,3μl上述双酶切的pCAMBIA3301载体,加1μl 5×In-Fusion HD Enzyme Premix,50℃水浴锅中反应18min,取1ul反应产物加入到50μl大肠杆菌感受态Trans5a中,冰浴30min;42℃热激90sec,冰浴2min;加入300μL LB液体培养基,37℃低速振荡恢复培养1hr后,将菌液涂于含相应抗生素的平板上,晾干;37℃培养箱倒置培养10~16hr。挑取单克隆菌斑,Cry1m7的infusion引物进行阳性筛选,并对阳性克隆的菌斑测序,测序正确后即完成植物转化载体p3301Ubi-Cry1m7的构建,其结构如图6所示。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (7)
1.一种杀虫蛋白Cry1m7,其特征在于:
其为由SEQ ID NO:1所示的氨基酸序列组成的蛋白质。
2.权利要求1所述的杀虫蛋白的编码基因,其特征在于,核苷酸序列如SEQ ID NO:2所示。
3.含有权利要求2所述基因的表达载体。
4.含有权利要求2所述基因的工程菌。
5.权利要求2所述的基因在转化植物中的应用。
6.根据权利要求5所述的应用,其特征在于,所述应用是在植物体内表达所述杀虫蛋白的基因,提高植物的抗害虫能力;所述害虫为鳞翅目害虫。
7.权利要求1所述的蛋白在杀鳞翅目害虫药剂中的应用。
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