CN105936644A - 一种杀虫蛋白及其核苷酸序列和应用 - Google Patents
一种杀虫蛋白及其核苷酸序列和应用 Download PDFInfo
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Abstract
本发明涉及一种杀虫蛋白，特别涉及一种对蚊类有杀虫活性的杀虫蛋白。其氨基酸序列为如SEQ ID No.2所示的氨基酸序列。本发明还提供了能够翻译为如本发明的杀虫蛋白的核苷酸序列，以及包括该核苷酸序列的转基因微生物和/或转基因植物。
Description
技术领域
本发明涉及一种杀虫蛋白，特别涉及一种对蚊类有杀虫活性的杀虫蛋白。
背景技术
蚊类属于昆虫纲、双翅目，蚊总科。其主要分为卫生害虫和农业害虫，例如，重要的卫生害虫蚊类有按蚊(Anopheles)、库蚊(Culex)、伊蚊(Aedes)；重要的农业害虫蚊类有眼蕈蚊(Sciaridae)，其在全国各地均有分布。
卫生害虫可传播疾病的病原体以及传播鼠疫、伤寒、疟疾等流行病，严重威胁人们的生命安全，因此其对人类危害毋庸置疑。
而农业害虫也深刻影响着人类的生活，以韭菜迟眼蕈蚊(Bradysia odoriphaga)为例，其又名韭蛆、黄脚蕈蚊，属双翅目，眼蕈蚊科，主要危害韭菜、大葱、洋葱、小葱、大蒜等百合科蔬菜，偶尔也危害莴苣、青菜、芹菜等，是葱蒜类蔬菜的主要害虫之一，在全国各地均有分布。韭菜迟眼蕈蚊的幼虫生活在土壤表层，群集在韭菜地下部的鳞茎和柔嫩的茎部蛀食为害。初孵幼虫先为害韭菜叶鞘基部和鳞茎的上端；春、秋季主要为害嫩茎，导致根茎腐烂。受害韭菜地上部分生长细弱，叶片发黄萎蔫下垂，最后韭叶枯黄死亡。夏季气温高时，幼虫向下移动，为害韭菜鳞茎，致使整个鳞茎腐烂，严重时整墩韭菜枯死。对于韭菜迟眼蕈蚊的防治方法主要有物理防治(糖醋液、臭氧等)和化学防治。由于物理防治存在操作繁琐、防治效果受环境影响大等缺点，生产上主要采用农药灌根等化学手段进行防治，但是由于韭蛆繁殖快、隐蔽性强、防治难度大，导致菜农不规范或过量、甚至违规使用禁用的高毒农药进行防治，导致农药残留超标、污染环境、危害人类健康以及杀害有益鸟类等问题。另一方面，长期大量使用化学农药增强害虫抗药性、污染环境、杀伤鸟类、造成害虫再度猖獗的恶性循环，不能达到对害虫种群的有效控制，同时造成韭菜农残超标，使消费者健康受到威胁。近年来毒韭菜事件频发，如：2004年“香河毒韭菜事件”、2010年“青岛毒韭菜”和2011年河南南阳“毒韭菜事件”，连续的毒韭菜曝光事件，使得消费者“谈韭色变”，严重影响了菜农的经济利益和消费者的食品安全。
为了减少化学农药对环境的污染及对人民健康的威胁，筛选对双翅目害虫，特别是蚊类有高致病性的生防微生物是防治该类害虫的有效方法和重要措施。
发明内容
本发明之一提供了一种杀虫蛋白，其氨基酸序列为如SEQ ID No.2所示的氨基酸序列。
本发明之二提供了一种杀虫蛋白，其氨基酸序列为与如SEQ ID No.2所示的氨基酸序列的一致性在78％以上，且与如SEQ ID No.2所示的氨基酸序列具有相同功能的氨基酸序列；优选其氨基酸序列为与如SEQ ID No.2所示的氨基酸序列的一致性在95％以上，且与如SEQ ID No.2所示的氨基酸序列具有相同功能的氨基酸序列；特别优选其氨基酸序列为与如SEQ ID No.2所示的氨基酸序列的一致性在99％以上，且与如SEQ ID No.2所示的氨基酸序列具有相同功能的氨基酸序列。
本发明之三提供了一种核苷酸序列，所述核苷酸序列为能够翻译为如本发明之一和/或本发明之二所述的杀虫蛋白的核苷酸序列。
在一个具体实施例中，所述核苷酸序列为如SEQ ID No.1所示的核苷酸序列。
本发明之四提供了一种组合物，其包括本发明之一和/或本发明之二所述的杀虫蛋白。在本发明中，所述组合物可以不包括保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.9763的野生菌株。
在一个具体实施例中，能够翻译为如所述组合物中的本发明之一的杀虫蛋白的核苷酸序列为如SEQ ID No.1所示的核苷酸序列。
本发明之五提供了一种含有本发明之三的核苷酸序列，且能产生本发明之一和/或本发明之二的杀虫蛋白的转基因微生物。例如所述杀虫蛋白的氨基酸序列为如SEQ IDNo.2所示的氨基酸序列。或所述杀虫蛋白氨基酸序列为与如SEQ ID No.2所示的氨基酸序列的一致性在78％以上，且与如SEQ ID No.2所示的氨基酸序列具有相同功能的氨基酸序列；优选其氨基酸序列为与如SEQ ID No.2所示的氨基酸序列的一致性在95％以上，且与如SEQ ID No.2所示的氨基酸序列具有相同功能的氨基酸序列；特别优选其氨基酸序列为与如SEQ ID No.2所示的氨基酸序列的一致性在99％以上，且与如SEQ ID No.2所示的氨基酸序列具有相同功能的氨基酸序列。
在一个具体实施例中，所述转基因微生物包括芽孢杆菌(Bacillus)、假单胞菌(Pseudomonas)、肠杆菌(Escherichia)和酵母(Saccharomyces)中的至少一种。
在一个优选的具体实施例中，所述芽孢杆菌包括苏云金芽孢杆菌(Bacillusthuringiensis)、枯草芽孢杆菌(Bacillus subtilis)、萎缩芽孢杆菌(Bacillusatrophaeus)和蜡样芽孢杆菌(Bacillus cereus)中的至少一种；所述假单胞菌包括荧光假单胞杆菌(Pseudomonas fluorescens)；所述肠杆菌包括大肠杆菌(Escherichiacoli)。
本发明之六提供了根据本发明之三的核苷酸序列在制备转基因植物中的应用，其中，所述转基因植物能够产生根据本发明如上的杀虫蛋白。例如其氨基酸序列为如SEQ ID No.2所示的氨基酸序列。或其氨基酸序列为与如SEQ ID No.2所示的氨基酸序列的一致性在78％以上，且与如SEQ ID No.2所示的氨基酸序列具有相同功能的氨基酸序列；优选其氨基酸序列为与如SEQ ID No.2所示的氨基酸序列的一致性在95％以上，且与如SEQ ID No.2所示的氨基酸序列具有相同功能的氨基酸序列；特别优选其氨基酸序列为与如SEQ ID No.2所示的氨基酸序列的一致性在99％以上，且与如SEQID No.2所示的氨基酸序列具有相同功能的氨基酸序列。
在一个优选的具体实施例中，所述转基因植物包括百合科(Liliaceae)、姜科(Zingiberaceae)、菊科(Compositae)、葫芦科(Cucurbitaceae)和伞形科(Umbelliferae)中的至少一种。
在一个优选的具体实施例中，所述百合科植物包括葱属(Allium)；更优选包括韭菜(Allium tuberosum)、大葱(Allium fistulosum)、小香葱(Alliumascalonicum)、洋葱(Allium cepa)和大蒜(Allium sativum)中的至少一种。
在一个优选的具体实施例中，所述姜科包括姜属(Zingiber)、山姜属(Alpinia)、豆蔻属(Amomum)、大豆蔻属(Hornstedtia)、偏穗姜属(Plagiostachys)、直唇姜属(Pommereschea)、喙花姜属(Rhynchanthus)和茴香砂仁属(Achasma)；更优选包括姜(Zingiber officinale)和/或山姜(Alpinia japonicaMiq)。
在一个优选的具体实施例中，所述菊科包括莴苣属(Lactuca)；优选包括结球生菜(Lactuca sativa L.var.capitata L)、直立生菜(Lactuca sativa var.c romanaHort)和莴苣(Lactuca sativa)中的至少一种。
在一个优选的具体实施例中，所述葫芦科包括葫芦属(Lagenaria)、黄瓜属(Cucumis)、冬瓜属(Benincasa)、南瓜属(Cucurbita)、丝瓜属(Luffa)、西瓜属(Citrullus)和甜瓜属(Cucumis)中的至少一种；优选包括葫芦(Lagenariasiceraria)、黄瓜(Cucumis sativus L.var.sativus)、冬瓜(Benincasa hispida)、南瓜(Cucurbita moschata)、丝瓜(Luffa cylindrica)、西瓜(Citrullus lanatus)和甜瓜(Cucumis melo)中的至少一种；
在一个优选的具体实施例中，所述伞形科包括芹属(Apium)，优选包括欧芹(Apium petroselinum)和/或香芹(Apium graveolens)。
本发明之七提供了一种制备本发明之一和/或本发明之二所述的杀虫蛋白的方法，其包括利用本发明之五的转基因微生物，和/或利用本发明之六所述的应用中的转基因植物来生产所述杀虫蛋白；优选还包括在利用所述转基因微生物和/或转基因植物生产所述杀虫蛋白后，纯化所述杀虫蛋白使所述杀虫蛋白的纯度达到80％以上，更优选使所述杀虫蛋白的纯度达到90％以上，甚至是使所述杀虫蛋白的纯度达到99％以上。
本发明之八提供了本发明之一和/或本发明之二所述的杀虫蛋白，本发明之四的组合物，本发明之五的转基因微生物，以及本发明之六中的转基因植物中的至少一种在防治双翅目害虫；优选防治蚊类害虫；特别优选防治眼蕈蚊(Sciaridae)中的应用。
具体实施方式
从河北省南宫市韭菜迟眼蕈蚊害虫严重的韭菜田中采集韭菜迟眼蕈蚊，其饲养方法的参照文献为：“慕卫，刘峰，贾忠明，何茂华，相冠锋；韭菜迟眼蕈蚊简便人工饲养技术，华东昆虫学报，2003，12(2)：87-89”。
苏云金芽孢杆菌(Bacillus thuringiensis)，菌株JQD117于2014年10月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址：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 No.9763。
基因的克隆与表达：
以JQD117菌株基因组DNA为模板，利用引物cry39AF：AACTTTAAGAAGGAGATATACATATGAATTCATACGAGAATAAAAA，cry39AR：TCAGTGGTGGTGGTGGTGGTGCTCGAGTTAATTGGTAAACAGATCGTTCA，进行PCR扩增，94℃预变性5min，94℃1min，54℃1min，72℃2min，30个循环，72℃，10min。PCR产物纯化，克隆cry39A型(SEQ ID No.1)基因至pET21b载体，命名pET21cry39A。并最后将该重组质粒转化之大肠杆菌Rosetta(DE3)。
经测序分析，cry39A基因长度1986bp，其编码蛋白为Cry39A，共661个氨基酸(SEQ ID No.2)，表达72.82KD蛋白。
将上述基因在大肠杆菌Rosetta(DE3)中进行表达，SDS-PAGE电泳结果发现，cry39A基因在可溶和不可溶性组份中均表达；其中表达产物约75kDa左右蛋白，与预期结果相符。阴性对照(即不含有cry39A基因的大肠杆菌Rosetta(DE3))在可溶和不可溶性组份中都没有相应蛋白的表达条带。
LB固体培养基配方：胰蛋白胨10g，酵母提取物5g，氯化钠10g，琼脂粉15g，水1L，pH7.2。
LB液体培养基配方：胰蛋白胨10g，酵母提取物5g，氯化钠10g，水1L，pH7.2。
上述培养基菌在121℃的条件下灭菌20-30min后备用。
Cry39A杀虫蛋白对韭蛆二龄幼虫的活性测定
I.含有Cry39A蛋白的菌液制备
将携带cry39A基因基因的大肠杆菌Rosetta(DE3)菌株以1％的接种量接种于LB液体培养基中，37℃培养至OD600值达到0.5-1.0之间时，加入诱导物50mM IPTG，在150rpm，20℃低温下诱导12h。然后在4℃下，8000rpm离心3min。离心收集菌体，加入50mM Tris·Cl(pH 8.0)悬浮；破碎菌体(利用超声波常规完全破碎)，将超声破碎后的菌液在4℃下12,000rpm离心15min；然后收集上清液。按照常规方法对所述上清液进行SDS-PAGE定量分析。具体参照蛋白定量试剂盒(DC Protein Assay)的使用说明，以牛血清蛋白(BSA)为标准蛋白制作蛋白浓度标准曲线，测定大肠杆菌Rosetta(DE3)重组菌株中总蛋白的浓度。结合ImageMaster VDS软件分析SDS-PAGE中目的蛋白所占比例，计算目的蛋白，即Cry39A的浓度。
II.活性测定
采用浸渍法进行生测：将步骤I中制备的上清液配制成系列浓度300mg/L、150mg/L、60mg/L、30mg/L、15mg/L，然后将2ml至5mL稀释为上述系列浓度的上清液分别置于5个离心管中，将韭菜茎剪成2cm小段放入其中震荡浸泡3min，取出放入灭菌的上下均铺有水琼脂和双层滤纸的90mm培养皿中。以与超声破碎后的Rosetta(DE3)上清液(即不含有Cry39A杀虫蛋白)作为阴性对照。每皿接入20头2龄健康的韭菜迟眼蕈蚊(Bradysia odoriphaga)幼虫(俗称韭蛆)，于温度为(24±1)℃、湿度为75-85％、光周期为光照:黑暗＝14:10的光照培养箱中饲养，于接虫后72h调查记录韭蛆的死亡情况，测定不同菌液浓度下的韭蛆死亡率。之后利用POLO Plus软件算出Cry39A对2龄韭蛆的LC50，具体结果见表1。
表1Cry39A蛋白对韭菜迟眼蕈蚊幼虫LC50测定
Claims (10)
1.一种杀虫蛋白，其氨基酸序列为如SEQ ID No.2所示的氨基酸序列。
2.一种杀虫蛋白，其氨基酸序列为与如SEQ ID No.2所示的氨基酸序列的一致性在78％以上，且与如SEQ ID No.2所示的氨基酸序列具有相同功能的氨基酸序列；优选其氨基酸序列为与如SEQ ID No.2所示的氨基酸序列的一致性在95％以上，且与如SEQ ID No.2所示的氨基酸序列具有相同功能的氨基酸序列；特别优选其氨基酸序列为与如SEQ ID No.2所示的氨基酸序列的一致性在99％以上，且与如SEQ ID No.2所示的氨基酸序列具有相同功能的氨基酸序列。
3.一种核苷酸序列，所述核苷酸序列为能够翻译为如权利要求1或2所述的杀虫蛋白的核苷酸序列。
4.根据权利要求3所述的核苷酸序列，其特征在于，所述核苷酸序列为如SEQ IDNo.1所示的核苷酸序列。
5.一种组合物，其包括如权利要求1或2所述的杀虫蛋白；优选能够翻译为所述杀虫蛋白的核苷酸序列如SEQ ID No.1所示。
6.一种含有根据权利要求3或4所述的核苷酸序列，且能产生如权利要求1或2所述的杀虫蛋白的转基因微生物。
7.根据权利要求6所述的转基因微生物，其特征在于，所述转基因微生物包括芽孢杆菌(Bacillus)、假单胞菌(Pseudomonas)、肠杆菌(Escherichia)和酵母(Saccharomyces)中的至少一种；
优选所述芽孢杆菌包括苏云金芽孢杆菌(Bacillus thuringiensis)、枯草芽孢杆菌(Bacillus subtilis)、萎缩芽孢杆菌(Bacillus atrophaeus)和蜡样芽孢杆菌(Bacilluscereus)中的至少一种；所述假单胞菌包括荧光假单胞杆菌(Pseudomonasfluorescens)；所述肠杆菌包括大肠杆菌(Escherichia coli)。
8.根据权利要求3或4所述的核苷酸序列在制备转基因植物中的应用，其中，所述转基因植物能够产生根据权利要求1或2所述的杀虫蛋白；所述转基因植物包括百合科(Liliaceae)、姜科(Zingiberaceae)、菊科(Compositae)、葫芦科(Cucurbitaceae)和伞形科(Umbelliferae)中的至少一种；
优选所述百合科包括葱属(Allium)；更优选包括韭菜(Allium tuberosum)、大葱(Allium fistulosum)、小香葱(Allium ascalonicum)、洋葱(Allium cepa)和大蒜(Allium sativum)中的至少一种；
优选所述姜科包括姜属(Zingiber)、山姜属(Alpinia)、豆蔻属(Amomum)、大豆蔻属(Hornstedtia)、偏穗姜属(Plagiostachys)、直唇姜属(Pommereschea)、喙花姜属(Rhynchanthus)、茴香砂仁属(Achasma)；更优选包括姜(Zingiberofficinale)和/或山姜(Alpinia japonica Miq)；
优选所述菊科包括莴苣属(Lactuca)；更优选包括结球生菜(Lactuca sativaL.var.capitata L)、直立生菜(Lactuca sativa var.c romanaHort)和莴苣(Lactucasativa)中的至少一种；
优选所述葫芦科包括葫芦属(Lagenaria)、黄瓜属(Cucumis)、冬瓜属(Benincasa)、南瓜属(Cucurbita)、丝瓜属(Luffa)、西瓜属(Citrullus)和甜瓜属(Cucumis)中的至少一种；更优选包括葫芦(Lagenaria siceraria)、黄瓜(Cucumissativus L.var.sativus)、冬瓜(Benincasa hispida)、南瓜(Cucurbita moschata)、丝瓜(Luffa cylindrica)、西瓜(Citrullus lanatus)和甜瓜(Cucumis melo)中的至少一种；
优选所述伞形科包括芹属(Apium)，更优选包括欧芹(Apium petroselinum)和/或香芹(Apium graveolens)。
9.一种制备如权利要求1或2所述的杀虫蛋白的方法，其包括利用根据权利要求6或7所述的转基因微生物，和/或利用根据权利要求8所述的应用中的转基因植物来生产所述杀虫蛋白；优选还包括在利用所述转基因微生物和/或转基因植物生产所述杀虫蛋白后，纯化所述杀虫蛋白使所述杀虫蛋白的纯度达到80％以上，更优选使所述杀虫蛋白的纯度达到90％以上，甚至是使所述杀虫蛋白的纯度达到99％以上。
10.根据权利要求1或2所述的杀虫蛋白，根据权利要求5所述的组合物，根据权利要求6或7所述的转基因微生物，以及根据权利要求8所述的应用中的转基因植物中的至少一种在防治双翅目害虫；优选防治蚊类害虫；特别优选防治眼蕈蚊(Sciaridae)中的应用。
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| CN106632624A (zh) * | 2016-09-21 | 2017-05-10 | 中国农业科学院植物保护研究所 | 一种杀虫蛋白及其核酸、制备方法和应用 |
| WO2020047704A1 (en) * | 2018-09-03 | 2020-03-12 | Syngenta Participations Ag | Compositions and methods for controlling plant pests |
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