CN111196844B - 一种杀虫蛋白及其核酸、制备方法和应用 - Google Patents
一种杀虫蛋白及其核酸、制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种杀虫蛋白,其氨基酸序列如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5和SEQ ID No.6所示的至少一种。这些蛋白质具有一致的杀虫谱,均对大猿叶甲、小猿叶甲、黄曲条跳甲、榆蓝叶甲和马铃薯甲虫等鞘翅目害虫具有良好的杀虫活性,特别是氨基酸序列如SEQ ID No.4所示的蛋白的杀虫效果尤为突出。
Description
本发明为于2016年9月21日提交的发明名称为一种杀虫蛋白及其核酸、制备方法和应用,申请号为201610836625.0的专利申请的分案。
技术领域
本发明涉及杀虫蛋白领域。
背景技术
鞘翅目害虫在我国农业生产中的危害逐年加重,且造成了严重的经济损失,因此,急需发掘更多的对鞘翅目害虫具有毒杀作用的菌株及基因。目前,从苏云金芽胞杆菌(Bacillus thuringiensis)中发现了一些的Cry蛋白对鞘翅目害虫具有杀虫活性。然而,为了丰富生物防治的种类,特别是蛋白对鞘翅目害虫防治的种类,以期获得具有不同杀虫机理的生物防治材料,来应对因长期使用某单一药剂可能产生的抗药性。因此,仍然有必要寻找新的具有优良活性的新型杀虫蛋白。
发明内容
本发明之一提供了一种不同于现有技术的新型的杀虫蛋白,其氨基酸序列为如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5和SEQ ID No.6所示的至少一种。根据本发明的发现,这些蛋白质具有一致的杀虫谱,例如它们均对大猿叶甲、小猿叶甲、黄曲条跳甲、榆蓝叶甲和马铃薯甲虫等鞘翅目害虫具有良好的杀虫活性,特别是氨基酸序列如SEQ ID No.4所示的蛋白的杀虫效果尤为突出。
在一个具体实施方式中,所述杀虫蛋白为氨基酸序列如SEQ ID No.1、SEQ IDNo.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5和SEQ ID No.6所示的至少一种的氨基酸序列经过取代和/或缺失和/或添加一个或多个氨基酸,且与氨基酸序列如SEQ ID No.1、SEQID No.2、SEQ ID No.3、SEQ ID No.4、SEQ ID No.5和SEQ ID No.6所示的蛋白中的至少一种具有相同功能的蛋白。
发明人在研究过程中,对其中对鞘翅目害虫杀虫活性突出的蛋白(如SEQ ID No.4所示)进行了一系列的截短,以期提高该序列的活性。最后,经实验证实,氨基酸序列如SEQID No.7所示的截短蛋白获得了较野生型序列(如SEQ ID No.4所示)更优的杀虫效果。因此,在一个优选的具体实施方式中,所述杀虫蛋白的氨基酸序列如SEQ ID No.7所示。
本发明之二提供了一种能够翻译为本发明之一的杀虫蛋白的核酸。
在一个具体实施方式中,能够翻译为如SEQ ID No.1所示的氨基酸序列的核酸的序列如SEQ ID No.8所示,能够翻译为如SEQ ID No.2所示的氨基酸序列的核酸的序列如SEQ ID No.9所示,能够翻译为如SEQ ID No.3所示的氨基酸序列的核酸的序列如SEQ IDNo.10所示,能够翻译为如SEQ ID No.4所示的氨基酸序列的核酸的序列如SEQ ID No.11所示,能够翻译为如SEQ ID No.5所示的氨基酸序列的核酸的序列如SEQ ID No.12所示,能够翻译为如SEQ ID No.6所示的氨基酸序列的核酸的序列如SEQ ID No.13所示,能够翻译为如SEQ ID No.7所示的氨基酸序列的核酸的序列如SEQ ID No.14所示。
本发明之三提供了一种制备杀虫晶体蛋白的方法,其包括如下步骤:在活体生物中表达本发明之一的杀虫蛋白;特别是在含有本发明之二的核酸的活体生物中表达本发明之一的杀虫蛋白。本领域的技术人员熟知每一个三联体密码相对应于一种氨基酸编码,而一种氨基酸则有可能相对应于多个三联体密码,因此,在氨基酸序列一定的情况下,可以存在与其对应的多种DNA序列。
在一个具体实施方式中,所述活体生物在16-20℃下表达所述杀虫蛋白。
在一个具体实施方式中,所述活体生物为微生物,进一步地,所述微生物为野生微生物和/或遗传工程微生物。
在一个具体实施方式中,所述活体生物为芽胞杆菌(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方式中,所述芽胞杆菌为苏云金芽胞杆菌(Bacillusthuringiensis)、枯草芽胞杆菌(Bacilllus subtilis)、萎缩芽胞杆菌(Bacillusatrophaeus)和蜡样芽胞杆菌(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(Escherichia coli)。
在一个具体实施方式中,在所述活体生物中表达所述杀虫蛋白后,还包括从所述活体生物中纯化所述杀虫蛋白的步骤。其中纯化的方式可通过本领域技术人员所知晓的常规方法实现。
本发明之四提供了本发明之一的杀虫蛋白在防治有害生物中的应用。
在一个具体实施方式中,本发明之四中所述有害生物为昆虫中的至少一种。
在一个具体实施方式中,本发明之四中所述有害生物为鞘翅目昆虫中的至少一种。
在一个具体实施方式中,本发明之四中所述有害生物为大猿叶甲(Colaphellusbowringii Baly)、小猿叶甲(Phaedon brassicae Baly)、黄曲条跳甲(Phyllotretastriolata)、榆蓝叶甲(Pyrrhalta aenescens)和马铃薯甲虫(Leplinotarsadecernlineata)中的至少一种。
本发明之五提供了本发明之二的核酸在构建防治有害生物的转基因植物中的应用。通过构建所述转基因植物,本发明之一的杀虫蛋白能够在所述转基因植物中表达,或者进一步地在所述转基因植物中的特定组织中表达,在有害生物为害该转基因植物时,由于食用表达所述杀虫蛋白的植株,而被毒杀或者被抑制生长,从而达到防治有害生物的目的。
在一个具体实施方式中,为核酸在构建防治鞘翅目昆虫中的至少一种的转基因植物中的应用。
在一个具体实施方式中,为核酸在构建防治大猿叶甲(Colaphellus bowringiiBaly)、小猿叶甲(Phaedon brassicae Baly)、黄曲条跳甲(Phyllotreta striolata)、榆蓝叶甲(Pyrrhalta aenescens)和马铃薯甲虫(Leplinotarsa decernlineata)中的至少一种的转基因植物中的应用。
附图说明
图1为在可溶组分(上清)中目标基因表达产物的SDS-PAGE分析。其中,LM:低分子量蛋白参照;1:Rosetta-pET21b(阴性对照);2:Rosettaip1;3:Rosetta-ip1;4:Rosetta-ip1;5:Rosetta-ip1;6:Rosetta-ip1;7:Rosetta-ip1。
图2为不可溶组分(沉淀)中目标基因表达产物的SDS-PAGE分析。其中,LM:低分子量蛋白参照;1:Rosetta-pET21b(阴性对照);2:Rosettaip1;3:Rosetta-ip1;4:Rosetta-ip1;5:Rosetta-ip1;6:Rosetta-ip1;7:Rosetta-ip1。
图3为截短基因表达情况的SDS-PAGE分析。其中,LM:低分子量蛋白参照;1:Rosetta-pET21b可溶组分(阴性对照);2:Rosetta-ip4S的可溶组分;6:Rosetta-ip4S的不可溶组分。忽略其他泳道的蛋白条带。
具体实施方式
以下通过优选的实施例的形式对本发明的上述内容再作进一步的详细说明,但不构成对本发明的限制。
1)液体LB:胰蛋白胨1%,酵母粉0.5%,NaCl 1%,pH 7.0,15磅灭菌15min。用于培养大肠杆菌。
2)固体LB:在液体LB培养基中加1.3%琼脂,15磅灭菌15min。用于培养大肠杆菌。
3)抗生素:氨苄青霉素水溶液100mg/ml,用时稀释500倍-20℃保存。其中pEB质粒载体具有氨苄青霉素抗性。
蛋白电泳检测及定量分析
将诱导表达的大肠杆菌菌体经过常规的超声波处理之后分离为上清和沉淀两部分,并根据实际需要,分别对上清样品和沉淀样品或者单独的上清样品或沉淀样品进行蛋白电泳。其中目标蛋白可能含有以可溶形式(存在于超声破碎后的样品的上清中)和/或不溶形式(存在于超声破碎后的样品的沉淀中)被表达。因此,需要指出的是上清样品指的是经过超声波处理后离心得到的液体;而沉淀样品指的是经过超声波处理后离心的沉淀部分用缓冲液(例如Tris缓冲液)悬浮后的样品,该沉淀部分中含有的蛋白在经过重悬、煮沸等一系列处理后,也可以成为可溶样品,从而可以将其用于蛋白电泳分析。
蛋白检测分析:
120V预电泳约10-20min,取样品40μL,加入10μL 5×加样缓冲液,混匀,煮沸5-10min,12000rpm离心5min以去除不溶成分,取10μL样品上样。80V恒压电泳至样品浓缩呈直线,150V恒压电泳至指示的溴酚蓝到达凝胶底部。脱色:电泳后取出凝胶,电泳后取出凝胶并用蒸馏水冲洗,加入50mL溶液I(50%乙醇,10%乙酸,40%超纯水),微波炉中加热30s,60rpm振荡10min。
染色:倒掉溶液I后加入含有溶液III(0.25%R250的95%乙醇溶液)的溶液II(7.5%乙酸,5%乙醇,87.5%超纯水)(每50mL溶液II加200μL溶液III)微波炉加热30s,60rpm振荡15min以上。倒掉溶液II,加入无菌水,凝胶成像系统照相保存。
观察结果,根据蛋白条带着色程度比较目的蛋白表达情况,用凝胶成像系统照相保存。
蛋白定量分析:
制备以下5种不同浓度梯度的BSA:0.8μg/μL、0.4μg/μL、0.2μg/μL、0.1μg/μL、0.05μg/μL,同时分别将目标蛋白进行梯度稀释,使其终浓度介于0.8-0.05μg/μL,目标蛋白和5种不同浓度的BSA上样量均为10μL,进行蛋白电泳定量分析。其中SDS聚丙烯酰氨凝胶为8%的分离胶和4%的浓缩胶。
1.ip1(SEQ ID No.8)、ip2(SEQ ID No.9)、ip3(SEQ ID No.10)、ip4(SEQ IDNo.11)、ip5(SEQ ID No.12)和ip6(SEQ ID No.13)基因的克隆
1.1引物:
设计了三对引物,分别为:ip-AF(SEQ ID No.15):GATGGATGGTTTTTAGCTAGATTCTGGAT,ip-AR(SEQ ID No.16):GCTCTATAATATGGAT TAGCTCCAT;ip-BF(SEQ ID No.17):ACGCGTCGACATGAAATACAAGT TTTCAAAAGTC,ip-BR(SEQ ID No.18):CCGCTCGAGGATTTTATTTTAT TAATTTCCACT;ip-CF(SEQ ID No.19):ACGCGTCGACATGAAAAATAGATTTTCAAAAGTG,ip-CR(SEQ ID No.20):CCGCTCGAGCTACAATTTAAAGTTTTCACTTAAT。其中引物序列中加横线的部分为引入的酶切位点,即Sal I酶切位点:GTCGAC,以及Xho I酶切位点:CTCGAG。
1.2对实验室已保存的255株标准菌株进行基因组提取:
(1)将新鲜的单株菌种样品接种到LB固体培养基上,在30℃恒温箱中培养至菌体裂解80%以上;
(2)刮取固体培养基上的菌体至1.5mL EP管中,加入SI溶液(1mmol/L EDTA,10mmol/L Tris-HCl,1mol/L蔗糖,1μL RNase溶液,pH 7.0)150μL和100mg石英砂,组织破碎仪上震荡1min;
(3)加入SII溶液(4%SDS)200μL,充分混匀;
(4)加入SIII溶液(1mol/L NaAc,5mol/L异硫氰酸胍,pH 4.8)400μL,充分混匀,12000g,离心10min;
(5)取上清液加入等体积异丙醇,-20℃静置20min;
(6)12000g,离心10min,弃上清,70%乙醇清洗沉淀,晾干后加入100μL超纯水或TE缓冲液溶解;
(7)取少量(例如5-20μL或100-500ng)利用0.7%琼脂糖凝胶电泳检测提取的Bt基因组的质量和浓度,其余部分-20℃保存备用。
1.3基因检测、克隆和表达
利用引物对ip-AF和ip-AR,分别以根据1.1.2的方法提取的本实验室保存的255株标准菌株(部分菌株来源于美国的芽胞杆菌遗传资源中心,Bacillus Genetic StockCenter,简称BGSC)基因组为模板进行常规的PCR检测。
将检测到的PCR产物进行基因序列测定。利用Vector NTI软件对测序结果进行聚类比对分析;然后根据聚类分析结果,选取标准菌株BGSC No.4A4、BGSC No.4A5、BGSCNo.6A25、BGSC No.4K9、Bt851的基因组分别作为模板,利用引物对ip-BF和ip-BR,以及引物对ip-CF和ip-CR对该五株菌进行扩增。并将扩增的产物经过双酶切和DNA琼脂糖凝胶回收连接到利用与PCR产物相同的酶酶切过的pET-21b质粒片段上,然后将连接产物转化到大肠杆菌感受态细胞中,进行重组质粒的提取和测序鉴定,获得了重组质粒pET-ip1(含有SEQID No.8所示的序列)、pET-ip2(含有SEQ ID No.9所示的序列)、pET-ip3(含有SEQ IDNo.10所示的序列)、pET-ip4(含有SEQ ID No.11所示的序列)、pET-ip5(含有SEQ ID No.12所示的序列)和pET-ip6(含有SEQ ID No.13所示的序列)。并进一步将这6种重组质粒分别转入到大肠杆菌表达菌株Rosetta(DE3)感受态细胞中,对鉴定正确的阳性转化子在16℃-20℃的低温下利用常规用量的IPTG进行蛋白诱导表达。
利用聚丙烯酰胺凝胶电泳分别对目标蛋白的可溶形式和不可溶形式进行检测。结果见图1和图2。根据表达结果发现,Ip6主要表达可溶组分,而其他五种蛋白,即Ip1、Ip2、Ip3、Ip4和Ip5,主要表达不可溶组分。
其中,Ip1蛋白的氨基酸序列如SEQ ID No.1所示,Ip2蛋白的氨基酸序列如SEQ IDNo.2所示,Ip3蛋白的氨基酸序列如SEQ ID No.3所示,Ip4蛋白的氨基酸序列如SEQ IDNo.4所示,Ip5蛋白的氨基酸序列如SEQ ID No.5所示,Ip6蛋白的氨基酸序列如SEQ IDNo.6所示。
1.4ip4的截短与表达
设计了引物对:ip4S-F(SEQ ID No.21):ACGCGTCGACGCAGAAGCCAAATCACC AGATTT;并引入了Sal I酶切位点:GTCGAC;ip4S-R(SEQ ID No.22):CCGCTCGAGCAATTTAAAGTTTTCACTTAAT,并引入了Xho I酶切位点:CTCGAG。
以提纯的pET-ip1质粒为模板,利用如上引物对进行PCR扩增。然后以1.1.3相同的程序和载体进行截短序列克隆的表达与蛋白检测。
其中获得的含有截短序列ip4S(序列如SEQ ID No.14所示,其对应的氨基酸序列如SEQ ID No.7所示)的重组质粒命名为pET-ip4S。
并进一步将该重组质粒转入到大肠杆菌表达菌株Rosetta(DE3)感受态细胞中,对鉴定正确的阳性转化子在16℃-20℃的低温下利用常规用量的IPTG进行蛋白诱导表达。
利用聚丙烯酰胺凝胶电泳分别对目标蛋白的可溶形式和不可溶形式进行检测。结果见图3。根据表达结果发现,Ip4S可表达可溶组分和不可溶组分。
2.蛋白的纯化
(1)镍亲和层析柱的制备
1)使用20%乙醇清洗空柱子,将柱料Chelating Sepharose Fast Flow(GE)摇匀后,取2mL加入亲和层析柱子(20mL);
2)向柱子内加入5倍柱体积的超纯水进行冲洗;
3)向柱子内加入2倍柱体积的1mol/L NiSO4,放置20min使Ni离子与柱料充分结合;
4)向柱子内加入10倍柱体积的超纯水进行冲洗,洗掉未与柱料结合的Ni离子,防止其与蛋白结合;
5)向柱子内加入5倍柱体积的结合缓冲液(Binding Buffer),平衡柱料。
(2)蛋白样品的上柱
1)对超声波处理后获得的蛋白样品进行预处理:4℃,10000g离心20min,并将上清转入新的50mL离心管;
2)将处理好的蛋白样品(步骤1)中的上清)分多次加入柱子即反复上样3-4次,使蛋白样品充分挂柱;
3)向柱子内加入5倍柱体积的结合缓冲液,洗去杂蛋白;
4)向柱子内加入3倍柱体积的洗脱缓冲液,洗脱目的蛋白,可通过SDS-PAGE分析洗脱下来的蛋白;
5)向柱子内加入超纯水清洗柱子,之后用20%乙醇清洗,并于20%乙醇中4℃长期保存。
(3)透析袋的处理
剪透析袋约10cm,放入到含有2%(w/v)NaHCO3的500mL的1mmol/L EDTA·2Na液中,煮沸10min;用超纯水将透析袋彻底清洗干净,再用500mL的1mmol/L EDTA·2Na煮沸10min;冷却后,置于50%乙醇中,放于4℃保存;在使用前,用超纯水将透析袋里外进行彻底清洗。
(4)纯化蛋白的透析
将镍柱纯化后的蛋白装入处理好的透析袋,用夹子封紧,放入2L预冷透析液20mmol/L Tris-HCl(pH 8.0),置于磁力搅拌机上使之旋转透析,每3h更换一次透析液,透析三次后换新的透析液,4℃过夜透析,获得纯化后的蛋白。
可以利用SDS-PAGE电泳对纯化的蛋白进行分析或者定量。
3.生物活性测定
将经过1.1.5纯化的7种蛋白分别定量后,针对大猿叶甲(Colaphellus bowringiiBaly)、小猿叶甲(Phaedon brassicae Baly)、黄曲条跳甲(Phyllotreta striolata)、榆蓝叶甲(Pyrrhalta aenescens)和马铃薯甲虫(Leplinotarsa decernlineata)的初孵幼虫分别进行生物活性测定,以清水作为空白对照。其中大猿叶甲、小猿叶甲用小油菜叶片进行生物活性测定,黄曲条跳甲用白萝卜进行生物活性测定,榆蓝叶甲用榆树叶片进行生物活性测定,马铃薯甲虫用马铃薯叶片进行生物活性测定。
操作步骤如下:
(1)选取鲜嫩一致的叶片或白萝卜片(根据试虫选择合适的食物),用清水洗净,晾干,剪成一次性平皿(直径=6cm)大小;
(2)将叶片或白萝卜片在配制好的一定量蛋白样品(加入洗涤剂,终浓度0.1%)中浸泡30s;
(3)在实验台上铺一层保鲜膜,蘸有样品的叶片或白萝卜片置于保鲜膜上,室温晾干;
(4)将叶片或白萝卜片分别放于培养皿中,培养皿(直径=9cm)中垫入无菌水喷湿的滤纸(需用锡箔纸包装灭菌);
(5)用毛笔轻轻接入初孵幼虫,每个叶片或白萝卜片接幼虫30头,每个处理做三次重复,接完虫后以2层吸水纸严格密封,并用橡皮筋固定,防止幼虫爬出;
(6)放置25℃生化培养箱中,光周期为16h:8h,湿度50%左右,每天观察叶片或白萝卜片是否腐烂或干枯,是否有水蒸气凝结,并在生化培养箱内适时适量增加水分,保持箱内湿度;
(7)48h后分别调查死、活虫数,计算死亡率、存活率、校正死亡率和LC50。
其中,对试虫进行LC50测定之前,首先经过初筛初步确定杀虫的浓度范围,然后再测定一系列的浓度梯度下的杀虫活性,进而计算出LC50。在某一特定浓度下的死亡率和校正死亡率计算公式如下:
根据生测数据采用SPSS(V13.0)软件计算致死中浓度(LC50)。结果见表1。
表1
从表1可以看出,本发明的杀虫蛋白对大猿叶甲、小猿叶甲、黄曲条跳甲、榆蓝叶甲和马铃薯甲虫均表现出了杀虫活性,特别是Ip4和其截短蛋白Ip4S对这几种鞘翅目的害虫表现尤为突出。
序列表
<110> 中国农业科学院植物保护研究所
<120> 一种杀虫蛋白及其核酸、制备方法和应用
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Gly His Lys Ala Ile Val Lys His Asp Leu Arg Lys Met Val Tyr Ser
245 250 255
Gly Thr His Asp Leu Lys Gly Asp Leu Ile Val Ser Phe Asn Asp Lys
260 265 270
Glu Ile Val Gln Lys Phe Ile Tyr Pro Asn Tyr Arg Glu Ile Asn Leu
275 280 285
Ser Asp Ile Arg Glu Thr Met Ile Glu Ile Asp Glu Trp Asn His Val
290 295 300
Asn Pro Val Asn Phe Tyr Glu Leu Val Gly Val Lys Asn His Ile Lys
305 310 315 320
Asn Gly Glu Thr Leu Tyr Ile Asp Thr Pro Ala Lys Phe Met Phe Asn
325 330 335
Gly Ala Asn Pro Tyr Tyr Arg Ala Thr Phe Thr Glu Tyr Asp Gly Asn
340 345 350
Asn Asn Pro Val Gln Thr Lys Val Leu Ser Glu Asn Phe Lys Leu
355 360 365
<210> 5
<211> 364
<212> PRT
<213> 苏云金芽胞杆菌(Bacillus thuringiensis)
<400> 5
Met Lys Tyr Lys Phe Ser Lys Val Val Lys Cys Thr Leu Pro Ala Leu
1 5 10 15
Met Ile Thr Thr Phe Val Thr Pro Ser Met Ala Val Phe Ala Ala Glu
20 25 30
Thr Lys Ser Pro Asn Leu Asn Ala Ser Gln Gln Ala Ile Thr Pro Tyr
35 40 45
Ala Glu Ser Tyr Ile Asp Thr Val Gln Asp Arg Met Lys Gln Arg Asp
50 55 60
Arg Glu Ser Lys Leu Thr Gly Lys Pro Ile Asn Met Gln Glu Gln Ile
65 70 75 80
Ile Asp Gly Trp Phe Leu Ala Arg Phe Trp Ile Phe Lys Asp Gln Asn
85 90 95
Asn Asn His Gln Thr Asn Arg Phe Ile Ser Trp Phe Lys Asp Asn Leu
100 105 110
Ala Ser Ser Lys Gly Tyr Asp Ser Ile Ala Glu Gln Met Gly Leu Lys
115 120 125
Ile Glu Ala Leu Asn Asp Met Asp Val Thr Asn Ile Asp Tyr Thr Ser
130 135 140
Lys Thr Gly Asp Thr Ile Tyr Asn Gly Ile Ser Glu Leu Thr Asn Tyr
145 150 155 160
Thr Gly Thr Thr Gln Lys Met Lys Thr Asp Ser Phe Gln Arg Asp Tyr
165 170 175
Thr Lys Ser Glu Ser Thr Ser Val Thr Asn Gly Leu Gln Leu Gly Phe
180 185 190
Lys Val Ala Ala Lys Gly Val Val Ala Leu Ala Gly Ala Asp Phe Glu
195 200 205
Thr Ser Val Thr Tyr Asn Leu Ser Ser Thr Thr Thr Glu Thr Asn Thr
210 215 220
Ile Ser Asp Lys Phe Thr Val Pro Ser Gln Glu Val Thr Leu Ser Pro
225 230 235 240
Gly His Lys Ala Val Val Lys His Asp Leu Arg Lys Met Val Tyr Phe
245 250 255
Gly Thr Gln Asp Leu Lys Gly Asp Leu Lys Val Ser Phe Asn Asp Lys
260 265 270
Glu Ile Val Gln Lys Phe Ile Tyr Pro Asn Tyr Arg Ser Ile Asp Leu
275 280 285
Ser Asp Ile Arg Lys Thr Met Ile Glu Ile Asp Lys Trp Asn His Val
290 295 300
Asn Thr Ile Asp Phe Tyr Gln Leu Val Gly Val Lys Asn His Ile Lys
305 310 315 320
Asn Gly Asp Thr Leu Tyr Ile Asp Thr Pro Ala Glu Phe Thr Phe Asn
325 330 335
Gly Ala Asn Pro Tyr Tyr Arg Ala Thr Phe Thr Glu Tyr Asp Glu Asn
340 345 350
Gly Asn Pro Val Gln Thr Lys Ile Leu Ser Gly Asn
355 360
<210> 6
<211> 328
<212> PRT
<213> 苏云金芽胞杆菌(Bacillus thuringiensis)
<400> 6
Met Lys Asn Ser Lys Lys Leu Lys Arg Lys Ile Leu Ala Cys Gly Ala
1 5 10 15
Ile Ala Ser Ile Ser Thr Thr Leu Val Thr Pro Leu Pro Thr Leu Ala
20 25 30
Ser Ala Asp Gln Ile Asn Thr Ser Glu Leu Glu Lys Asp Ala Lys Val
35 40 45
Asp Thr Ala Ile Leu Glu Trp Lys Val Pro Leu Phe Lys Ala Thr Glu
50 55 60
Ile Tyr Gly Lys Asn Ile Val Val Pro Ser Gly Tyr Glu Phe Arg Ser
65 70 75 80
Leu Thr Ser His Gly Phe Asn Thr Ser Tyr Lys Asn Leu Glu Tyr His
85 90 95
Gln Phe Ser Val Glu Ala Asp Gly Ser Pro Ile Ile Thr Asn Ser Asn
100 105 110
Asn Ile Phe Val Gly Lys Thr Thr Leu Thr Asn Asn Thr Asp Gln Glu
115 120 125
Gln Thr Leu Ser Thr Asn Ser Phe Ser Lys Met Ile Ser Asn Ser Val
130 135 140
Thr His Ser Thr Thr His Gly Phe Lys Phe Gly Thr Lys Ala Ser Ala
145 150 155 160
Lys Phe Asn Ile Pro Phe Val Gly Glu Thr Gly Ile Glu Leu Ser Ala
165 170 175
Glu Tyr Asn Phe Ser Asp Thr Ser Ser Glu Thr Ser Ser Glu Ser Phe
180 185 190
Thr Tyr Ile Ala Thr Pro Gln Asn Ile Lys Val Pro Ala His Ser Ser
195 200 205
Val Glu Val Val Val Ser Leu Asp Thr Val Lys Ala Asn Gly Asn Val
210 215 220
Lys Leu Leu Ala Lys Met Ser Gly Glu Asp Met Gly Ser Phe Asn Tyr
225 230 235 240
Lys Ser Thr Thr Gly Gly Ile Gly Lys Ser Tyr Val Tyr Asn Lys Ser
245 250 255
Phe Asn Ser Leu Val Thr Tyr Ala Ser Lys Ile Glu Lys Leu Gln Asn
260 265 270
Ile Ser Ala Asn Pro Asp Gly Lys Thr Ile Asn Ile Ile Gly Ser Gly
275 280 285
Lys Tyr Glu Ala Glu Tyr Gly Thr Glu Phe Asn Val Thr Val Thr Pro
290 295 300
Ile Asp Lys Asn Gly Lys Ser Val Asp Glu Gly Tyr Thr Tyr Lys Val
305 310 315 320
Lys Pro Glu Ile Thr Lys Glu Lys
325
<210> 7
<211> 334
<212> PRT
<213> 苏云金芽胞杆菌(Bacillus thuringiensis)
<400> 7
Ala Glu Ala Lys Ser Pro Asp Leu Asn Val Ser Gln Gln Val Ile Gly
1 5 10 15
Pro Tyr Ala Glu Ser Tyr Ile Asp Ile Val Gln Asp Arg Met Lys Gln
20 25 30
Arg Asp Lys Gly Ser Lys Leu Thr Gly Lys Pro Ile Asn Met Gln Glu
35 40 45
Gln Ile Ile Asp Gly Trp Phe Leu Ala Arg Phe Trp Ile Phe Lys Asp
50 55 60
Gln Asn Asn Asn His Gln Thr Asn Arg Phe Ile Ser Trp Phe Lys Asp
65 70 75 80
Asn Ile Ala Ser Ser Lys Gly Tyr Asn Ser Ile Ala Glu Gln Met Gly
85 90 95
Leu Lys Ile Glu Ala Glu Asn Asp Met Asp Val Thr Asn Ile Asp Tyr
100 105 110
Thr Ser Lys Thr Gly Asp Thr Ile Tyr Asn Gly Ile Ser Glu Leu Lys
115 120 125
Asn Tyr Thr Gly Ser Thr Gln Lys Met Lys Thr Asp Ser Phe Gln Arg
130 135 140
Asp Tyr Thr Lys Ser Glu Ser Thr Ser Val Thr Asn Gly Leu Gln Leu
145 150 155 160
Gly Phe Lys Val Ala Ala Lys Gly Val Val Ala Leu Ala Gly Ala Asp
165 170 175
Phe Glu Thr Ser Val Thr Tyr Asn Leu Ser Thr Thr Thr Thr Glu Thr
180 185 190
Asn Thr Ile Ser Asp Lys Phe Thr Val Pro Ser Gln Glu Val Thr Leu
195 200 205
Pro Pro Gly His Lys Ala Ile Val Lys His Asp Leu Arg Lys Met Val
210 215 220
Tyr Ser Gly Thr His Asp Leu Lys Gly Asp Leu Ile Val Ser Phe Asn
225 230 235 240
Asp Lys Glu Ile Val Gln Lys Phe Ile Tyr Pro Asn Tyr Arg Glu Ile
245 250 255
Asn Leu Ser Asp Ile Arg Glu Thr Met Ile Glu Ile Asp Glu Trp Asn
260 265 270
His Val Asn Pro Val Asn Phe Tyr Glu Leu Val Gly Val Lys Asn His
275 280 285
Ile Lys Asn Gly Glu Thr Leu Tyr Ile Asp Thr Pro Ala Lys Phe Met
290 295 300
Phe Asn Gly Ala Asn Pro Tyr Tyr Arg Ala Thr Phe Thr Glu Tyr Asp
305 310 315 320
Gly Asn Asn Asn Pro Val Gln Thr Lys Val Leu Ser Glu Asn
325 330
<210> 8
<211> 1095
<212> DNA
<213> 苏云金芽胞杆菌(Bacillus thuringiensis)
<400> 8
atgaaataca agttttcaaa agtcgttaag tgtactttac cagctttaat gattactaca 60
ttcgttactc caagtatggc agtttttgcc gcagaaacca agtcgccaaa tctaaatgca 120
tctcaacaag caataactcc atatgctgaa tcttatattg atactgttca agatagaatg 180
aaacaaagag atagggaatc aaaactaact ggtaaaccaa taaatatgca agaacaaata 240
atagatggat ggtttttagc tagattctgg atatttaaag atcaaaataa caatcatcaa 300
acaaatagat ttatatcctg gtttaaagat aatcttgcta gttcgaaggg gtatgacagt 360
atagcaggac aaatgggctt aaaaatagaa gcattaaatg atatggatgt aacaaatatt 420
gattatacat ctaaaacagg tgataccata tataatggaa tttctgaact aacaaattat 480
acaggaacaa cccaaaaaat gaaaaccgat agttttcaaa gagattatac aaaatctgaa 540
tccacttcag taacaaacgg gttacaatta ggatttaaag ttgctgctaa gggagtagct 600
gcattagcag gtgcagattt tgaaacaagt gttacctata atttatcatc tactacaact 660
gaaacaaata caatatcgga taagtttact gttccatctc aagaagttac attatcccca 720
ggacataaag cagtggtgaa acatgatttg agaaaaatgg tgtattttgg ggctcatgat 780
ttaaagggtg atttaaaagt aggttttaat gataaagaga ttgtacgaaa atttatttat 840
ccaaattata gatcaattga tttatctgat attcgtaaaa caatgattga aattgataaa 900
tggaatcatg taaataccat tgacttttat caattagttg gagttaaaaa tcatataaaa 960
aatggtgata ctttatatat agataccccg gccgaattta catttaatgg agctaatcca 1020
tattatagag caacatttac agaatacgac gagaacggaa atcctgttca aacaaagatt 1080
ttaagtggaa attaa 1095
<210> 9
<211> 1095
<212> DNA
<213> 苏云金芽胞杆菌(Bacillus thuringiensis)
<400> 9
atgaaataca agttttcaaa agtcgttaag tgtactttac cagctttaat gattactaca 60
ttcgctactc caagtatggc agtttttgcc gcagaaacca agtcgccaaa tctaaatgca 120
tctcaacaag caataactcc atatgctgaa tcttatattg atactgttca agatagaatg 180
aaacaaagag atagggaatc aaaactaact ggtaaaccaa taaatatgca agaacaaata 240
atagatggat ggtttttagc tagattctgg atatttaaag atcaaaataa caatcatcaa 300
acaaatagat ttatatcctg gtttaaagat aatcttgcta gttcgaaggg gtatgacagt 360
atagcagaac aaatgggcct aaaaatagaa gcattaaatg atatggatgt aacaaatatt 420
gattatacat ctaaaacagg tgataccata tataatggaa tttctgaact aacaaattat 480
acaggaacaa cccaaaaaat gaaaaccgat agttttcaaa gagattatac aaaatctgaa 540
tccacttcag taacaaatgg gttacaatta ggatttaaag ttgccgctaa gggagtagtt 600
gcattagcag gtgcagattt tgaaacaagt gttacctata atttatcatc tactacaact 660
gaaacaaata caatatcgga taagtttact gttccatctc aagaagttac attatcccca 720
ggacataaag cagtggtgaa acatgatttg agaaaaatgg tgtattttgg gactcgtgat 780
ttaaagggtg atttaaaagt gggttttaat gataaagaga ttgtacaaaa atttatttat 840
ccaaattata gatcaattga tttatctgat attcgtaaaa caatgattga aattgataaa 900
tggaatcatg taaataccat tgacttttat caattagttg gagttaaaaa tcatataaaa 960
aatggtgata ctttatatat agataccccg gccgaattta catttaatgg agctaatcca 1020
tattatagag caacatttac agaatacgac gagaacggaa atcctgttca aacaaagatt 1080
ttaagtggaa attaa 1095
<210> 10
<211> 1095
<212> DNA
<213> 苏云金芽胞杆菌(Bacillus thuringiensis)
<400> 10
atgaaataca agttttcaaa agtcgttaag tgtactttac cagctttaat gattactaca 60
ttcgttactc caagtatggc agtttttgcc gcagaaacca agtcgccaaa tctaaatgca 120
tctcaacaag caataactcc atatgctgaa tcttatattg atacggttca agatagaatg 180
aaacaaagag atagggaatc aaaactaact ggtaagccaa taaatatgca agaacaaata 240
atagatggat ggtttttagc tagattctgg atatttaaag atcaaaataa caatcatcaa 300
acaaatagat ttatatcctg gtttaaagat aatcttgcta gttcgaaggg gtatgacagt 360
atagcagaac aaatgggctt aaaaatagaa gcattaaatg atatggatgt aacaaatatt 420
gattatacat ctaaaacagg tgataccata tataatggaa tttctgaact aacaaattat 480
acaggaacaa cccaaaaaat gaaaaccgat agttttcaaa gagattatac aaaatctgaa 540
tccacttcag taacaaatgg gttacaatta ggatttaaag ttgctgctaa gggagtagtt 600
gcattagcag gtgcagattt tgaaacaagt gttacctata atttatcatc tactacaact 660
gaaacaaata caatgtcgga taagtttact gttccatctc aagaagttac attatcccca 720
ggtcataaag cagtagtgaa acatgatttg agaaaaatgg tgtattttgg gactcaagat 780
ttaaagggtg atttaaaagt aagttttaat gataaagaga ttgtacaaaa atttatctat 840
ccaaattata gatcaattga tttatctgat attcgtaaaa caatgattga aattgataaa 900
tggaatcatg taaataccat tgacttttat caattagttg gagttaaaaa tcatataaaa 960
aatggtgata ctttatatat agataccccg gccgaattta catttaatgg agctaatcca 1020
tattatagag caacatttac agaatacgac gagaacggaa atcctgttca aacaaagatt 1080
ttaagtggaa attaa 1095
<210> 11
<211> 1104
<212> DNA
<213> 苏云金芽胞杆菌(Bacillus thuringiensis)
<400> 11
atgaaaaata gattttcaaa agtggcatta tgcaccgtac cgattttaat ggtttctaca 60
ttcgccagtt caagcatgtc agcttttgct gcagaagcca aatcaccaga tttaaatgta 120
tctcaacaag taataggtcc ctatgccgaa tcttatattg atattgtgca ggatagaatg 180
aaacaaaggg ataagggatc aaaattaact ggtaaaccaa taaatatgca agaacaaata 240
atagatgggt ggtttctagc tagattttgg atatttaagg atcaaaacaa taaccatcag 300
acaaatagat ttatatcctg gtttaaagat aatattgcta gttcaaaagg gtataatagt 360
attgcggagc aaatgggttt aaaaatagaa gcagaaaacg atatggatgt aacaaatata 420
gattatacat ctaagacagg cgataccatt tataatggta tttcagaatt gaaaaattat 480
acaggatcaa ctcaaaagat gaaaacagat agttttcaaa gagattatac aaaatcagaa 540
tctacttcag taactaatgg attacaatta ggatttaaag ttgctgctaa aggagtagtt 600
gctttggctg gggcagactt tgaaaccagt gttacttata atctatcaac tactacaact 660
gaaacaaata caatatcaga caagtttact gtcccatctc aagaagttac attgcctcca 720
ggacataaag cgatagtgaa acatgattta agaaaaatgg tttattctgg tactcatgat 780
ctaaagggtg atttaattgt gagttttaat gataaagaga ttgtacaaaa atttatttat 840
ccaaattata gagaaattaa tttatctgat atccgtgaaa ctatgattga aattgatgaa 900
tggaatcatg taaaccctgt gaatttttat gaattagttg gggtcaaaaa tcatataaaa 960
aatggtgaaa ctttgtatat agatactcca gctaaattta tgtttaatgg tgctaatcca 1020
tattatagag caacatttac agaatacgac gggaataata atcctgttca aacaaaggta 1080
ttaagtgaaa actttaaatt ctaa 1104
<210> 12
<211> 1095
<212> DNA
<213> 苏云金芽胞杆菌(Bacillus thuringiensis)
<400> 12
atgaaataca agttttcaaa agtcgttaag tgtactttac cagctttaat gattactaca 60
ttcgttactc caagtatggc agtttttgcc gcagaaacca agtcgccaaa tctaaatgca 120
tctcaacaag caataactcc atatgctgaa tcttatattg atacggttca agatagaatg 180
aaacaaagag atagggaatc aaaactaact ggtaagccaa taaatatgca agaacaaata 240
atagatggat ggtttttagc tagattctgg atatttaaag atcaaaataa caatcatcaa 300
acaaatagat ttatatcctg gtttaaagat aatcttgcta gttcgaaggg gtatgacagt 360
atagcagaac aaatgggctt aaaaatagaa gcattaaatg atatggatgt aacaaatatt 420
gattatacat ctaaaacagg tgataccata tataatggaa tttctgaact aacaaattat 480
acaggaacaa cccaaaaaat gaaaaccgat agttttcaaa gagattatac aaaatctgaa 540
tccacttcag taacaaatgg gttacaatta ggatttaaag ttgctgctaa gggagtagtt 600
gcattagcag gtgcagattt tgaaacaagt gttacctata atttatcatc tactacaact 660
gaaacaaata caatatcgga taagtttact gttccatctc aagaagttac attatcccca 720
ggtcataaag cagtagtgaa acatgatttg agaaaaatgg tgtattttgg gactcaagat 780
ttaaagggtg atttaaaagt aagttttaat gataaagaga ttgtacaaaa atttatttat 840
ccaaattata gatcaattga tttatctgat attcgtaaaa caatgattga aattgataaa 900
tggaatcatg taaataccat tgacttttat caattagttg gagttaaaaa tcatataaaa 960
aatggtgata ctttatatat agataccccg gccgaattta catttaatgg agctaatcca 1020
tattatagag caacatttac agaatacgac gagaacggaa atcctgttca aacaaagatt 1080
ttaagtggaa attaa 1095
<210> 13
<211> 987
<212> DNA
<213> 苏云金芽胞杆菌(Bacillus thuringiensis)
<400> 13
atgaaaaata gtaaaaaatt aaagagaaaa atattagctt gtggagcaat cgcatctatt 60
agcacaacac ttgtaacgcc attaccaact ttagctagtg cagatcaaat taatacttca 120
gaattagaaa aagatgcaaa ggttgatact gctatattag aatggaaagt accgcttttt 180
aaggcgacag aaatttatgg gaaaaacata gttgttccaa gtggatatga atttcgctcg 240
cttacttcac atggttttaa tacttcttat aagaatttag aatatcatca attttcagta 300
gaggcagatg gttcgcctat tattactaat tcaaacaata tatttgttgg aaagaccacc 360
ctaactaata atacagacca agaacaaacg ttatctacta atagtttttc aaaaatgatt 420
tcaaattcag ttactcattc aactacacat ggttttaaat ttggaaccaa agcttctgcg 480
aaatttaata ttccttttgt gggagaaaca ggaatagaat tatctgcaga atacaatttt 540
tcggatacat ctagtgaaac aagctctgaa agtttcactt atattgctac tccacagaat 600
ataaaagttc ctgcccattc atctgtagaa gttgttgtaa gtctagatac agttaaagca 660
aatgggaatg taaaacttct tgctaaaatg tcaggtgagg atatgggtag ttttaattat 720
aaatctacaa caggaggtat tggtaaatca tatgtttata ataaatcatt taattcatta 780
gtaacatatg cttcgaagat tgaaaaactt caaaatatct cggctaatcc agatggtaaa 840
acgataaata ttataggttc aggtaaatac gaagctgaat atggaactga gtttaatgta 900
actgtaacac ctattgataa aaatggaaag tcggttgatg aggggtatac atataaggtg 960
aaaccggaga ttacaaaaga aaaataa 987
<210> 14
<211> 1014
<212> DNA
<213> 苏云金芽胞杆菌(Bacillus thuringiensis)
<400> 14
gcagaagcca aatcaccaga tttaaatgta tctcaacaag taataggtcc ctatgccgaa 60
tcttatattg atattgtgca ggatagaatg aaacaaaggg ataagggatc aaaattaact 120
ggtaaaccaa taaatatgca agaacaaata atagatgggt ggtttctagc tagattttgg 180
atatttaagg atcaaaacaa taaccatcag acaaatagat ttatatcctg gtttaaagat 240
aatattgcta gttcaaaagg gtataatagt attgcggagc aaatgggttt aaaaatagaa 300
gcagaaaacg atatggatgt aacaaatata gattatacat ctaagacagg cgataccatt 360
tataatggta tttcagaatt gaaaaattat acaggatcaa ctcaaaagat gaaaacagat 420
agttttcaaa gagattatac aaaatcagaa tctacttcag taactaatgg attacaatta 480
ggatttaaag ttgctgctaa aggagtagtt gctttggctg gggcagactt tgaaaccagt 540
gttacttata atctatcaac tactacaact gaaacaaata caatatcaga caagtttact 600
gtcccatctc aagaagttac attgcctcca ggacataaag cgatagtgaa acatgattta 660
agaaaaatgg tttattctgg tactcatgat ctaaagggtg atttaattgt gagttttaat 720
gataaagaga ttgtacaaaa atttatttat ccaaattata gagaaattaa tttatctgat 780
atccgtgaaa ctatgattga aattgatgaa tggaatcatg taaaccctgt gaatttttat 840
gaattagttg gggtcaaaaa tcatataaaa aatggtgaaa ctttgtatat agatactcca 900
gctaaattta tgtttaatgg tgctaatcca tattatagag caacatttac agaatacgac 960
gggaataata atcctgttca aacaaaggta ttaagtgaaa actttaaatt ctaa 1014
<210> 15
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gatggatggt ttttagctag attctggat 29
<210> 16
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gctctataat atggattagc tccat 25
<210> 17
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
acgcgtcgac atgaaataca agttttcaaa agtc 34
<210> 18
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ccgctcgagg attttatttt attaatttcc act 33
<210> 19
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
acgcgtcgac atgaaaaata gattttcaaa agtg 34
<210> 20
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ccgctcgagc tacaatttaa agttttcact taat 34
<210> 21
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
acgcgtcgac gcagaagcca aatcaccaga ttt 33
<210> 22
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ccgctcgagc aatttaaagt tttcacttaa t 31
Claims (11)
1.一种杀虫蛋白在防治有害生物中的应用,所述杀虫蛋白的氨基酸序列如SEQ IDNo.6所示;
所述有害生物为大猿叶甲(Colaphellus bowringii Baly)、小猿叶甲(Phaedonbrassicae Baly)、黄曲条跳甲(Phyllotreta striolata)、榆蓝叶甲(Pyrrhaltaaenescens)和马铃薯甲虫(Leplinotarsa decernlineata)中的至少一种。
2.根据权利要求1所述的应用,其特征在于,所述杀虫蛋白在活体生物中在16-20℃下表达。
3.根据权利要求2所述的应用,其特征在于,所述杀虫蛋白在含有能够翻译为如SEQ IDNo.6所示的氨基酸序列的核酸的活体生物中表达。
4.根据权利要求3所述的应用,其特征在于,能够翻译为如SEQ ID No.6所示的氨基酸序列的核酸的序列如SEQ ID No.13所示。
5.根据权利要求2所述的应用,其特征在于,所述活体生物为微生物。
6.根据权利要求5所述的应用,其特征在于,所述活体生物为芽孢杆菌(Bacillus)、假单胞菌(Pseudomonas)、肠杆菌(Escherichia)和酵母(Saccharomyces)中的至少一种。
7.根据权利要求6所述的应用,其特征在于,所述活体生物为苏云金芽孢杆菌(Bacillus thuringiensis)、枯草芽孢杆菌(Bacilllus subtilis)、萎缩芽孢杆菌(Bacillus atrophaeus)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(Escherichia coli)。
8.根据权利要求5所述的应用,其特征在于,所述微生物为野生微生物和/或遗传工程微生物。
9.根据权利要求2至8中任意一项所述的应用,其特征在于,在所述活体生物中表达所述杀虫蛋白后,还包括从所述活体生物中纯化所述杀虫蛋白的步骤。
10.能够翻译为如SEQ ID No.6所示的氨基酸序列的核酸在构建防治有害生物的转基因植物中的应用,其中,
所述应用为在构建防治大猿叶甲(Colaphellus bowringii Baly)、小猿叶甲(Phaedonbrassicae Baly)、黄曲条跳甲(Phyllotreta striolata)、榆蓝叶甲(Pyrrhaltaaenescens)和马铃薯甲虫(Leplinotarsa decernlineata)虫中的至少一种的转基因植物中的应用。
11.根据权利要求10所述的应用,其特征在于,能够翻译为如SEQ ID No.6所示的氨基酸序列的核酸的序列如SEQ ID No.13所示。
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