CN107759702A - 一种抗稻褐飞虱的杀虫蛋白HY131c及其编码基因与应用 - Google Patents
一种抗稻褐飞虱的杀虫蛋白HY131c及其编码基因与应用 Download PDFInfo
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- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
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- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
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- C—CHEMISTRY; METALLURGY
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Landscapes
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种抗稻褐飞虱的杀虫蛋白及其编码基因与应用。本发明提供的蛋白质,包括Cry1Ac的结构域I、Cry30Fa1结构域II及Cry1Ac结构域III;为将所述Cry1Ac蛋白中的结构域II替换为所述Cry30Fa1蛋白的结构域II得到的蛋白质。本发明的实验证明,本发明提供的新型杀虫蛋白对稻褐飞虱有高毒力,为水稻抗稻褐飞虱提供了新的策略,具有广阔的应用前景。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种抗稻褐飞虱的杀虫蛋白及其编码基因与应用。
背景技术
水稻是世界范围内重要的粮食作物,然而水稻的病虫害严重影响了水稻的生产。随着转Cry1Ab及Cry1Ac等Bt蛋白水稻株系的推广种植预期可有效的防治鳞翅目和鞘翅目害虫的危害(Shu,Ye et al.2000,Breitler,Vassal et al.2004,Cohen,Chen etal.2008)。但是,也不可避免的促进了非靶标害虫昆虫如褐飞虱的种群数量和危害程度(Chen et al.2011)。因此,创制发展对稻褐飞虱等害虫有高毒力的新型杀虫基因迫在眉睫。
苏云金芽孢杆菌(Bacillus thuringiensis简称Bt)是一种土壤杆菌,它在逆境条件下会形成芽孢并产生伴胞晶体,伴孢晶体由不同种Cry蛋白组成(亦被称为δ内毒素;δ-Endotoxins),其对鳞翅目、鞘翅目、双翅目等多种害虫具有杀虫活性(Bravo,Likitvivatanavong et al.2011)。Cry蛋白的杀虫活性区通常由三个结构域组成,其中结构域I主要负责在昆虫中肠膜上形成孔洞,结构域II主要负责与昆虫中肠膜上受体结合,由此确定毒素特异性,结构域III的功能比较复杂,包括维持蛋白质的稳定性、协助结构域I在膜上穿孔及协助结构域II与膜上受体结合等等。通过对不同Cry蛋白的结构域II互换,会产生对某种害虫毒性增强的新的杀虫蛋白,因此通过结构域互换产生新的杀虫蛋白的方法被广泛的应用于Cry蛋白的改造(Saraswathy and Kumar 2004,Florez,Osorio etal.2012)。
Cry蛋白对害虫的毒力非常高,且杀虫谱很窄,某一特定的Cry蛋白通常只对一种或几种昆虫起作用而对非靶标害虫及人畜完全无害。鉴于Cry蛋白的高效性和安全性,Cry基因也成为目前应用最广的用于抗虫转基因作物的杀虫基因(Romeis,Meissle etal.2006,Sanahuja,Banakar et al.2011)。然而,目前所发现的Cry蛋白主要是针对鳞翅目、鞘翅目和双翅目等害虫,对具有刺吸式口器的半翅目昆虫没有明显毒性(Chougule andBonning 2012)。鉴于稻褐飞虱对水稻生产的重大危害,迫切的需要研发针对此类害虫的新型抗性基因。
发明内容
本发明的一个目的是提供抗稻褐飞虱的杀虫蛋白及其编码基因。
本发明提供的蛋白质,为HY131c,包括Cry1Ac的结构域I、Cry30Fa1结构域II及Cry1Ac结构域III。
该蛋白为将所述Cry1Ac蛋白中的结构域II替换为所述Cry30Fa1蛋白的结构域II得到的蛋白质。
根据本发明,如在此定义的通过交换Cry1Ac及Cry30Fa1的结构域II,设计了相比于Cry1Ac及Cry30Fa1具有实质上改变的生化特性及杀虫活性的HY131c蛋白。
本发明所创制HY131c蛋白全长为619位氨基酸,其序列1到254位氨基酸来源于Cry1Ac,具有强的在昆虫中肠膜上形成空洞的能力;255到467位氨基酸来源于Cry30Fa1,具有与稻褐飞虱中肠膜上受体特异结合的能力;468到619位氨基酸来源于Cry1Ac,具有稳定蛋白空间结构等功能。其序列经NCBI Blastp分析第48-251位氨基酸具有δ内毒素结构域I特征,其序列第261-465位氨基酸具有δ内毒素结构域II特征;其序列475-613位氨基酸具有δ内毒素结构域III特征。本发明所创制蛋白分子量为69 353.4Da,等电点为4.98。本发明所创制HY131c蛋白序列与NCBI数据库中已知序列比对,同源性最高为73%。
上述蛋白质为如下a)-e)中任一种蛋白质:
a)氨基酸序列包括序列表中序列2所示的氨基酸序列的蛋白质;
b)氨基酸序列由序列表中序列2所示的氨基酸残基组成;
c)将a)或b)或c)所限定的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有抗虫功能的蛋白质;
d)与a)或b)或c)所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有抗虫功能的蛋白质;
e)a)-d)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
上述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过一个或几个氨基酸残基的取代和/或缺失和/或添加。
编码上述蛋白的核酸分子也是本发明保护的范围。
上述核酸分子为如下1)-6)中任一种所示的核酸分子:
1)其编码序列包括序列表中序列1;
2)其编码序列包括序列表中序列3;
3)其编码序列为序列表中序列1;
4)其编码序列为序列表中序列3;
5)在严格条件下与1)-4)中任一限定的DNA分子杂交且编码权利要求1所述蛋白的DNA分子;
6)与1)-4)中任一限定的DNA分子具有80%以上或90%以上的同源性且编码权利要求1所述蛋白的DNA分子。
HY131c蛋白编码基因的核酸序列全长为1860bp。本发明所创制HY131c基因序列与NCBI数据库中已知序列比对,仅有65%的部分与已提交序列有同源性,这一部分来源于Cry1Ac基因。与蛋白质序列相对应的,HY131c基因序列第1-762位核苷酸来自于Cry1Ac基因,第763-1401位核苷酸来自于Cry30Fa1基因,第1402-1860位核苷酸来自于Cry1Ac基因。本序列所用密码子为水稻优势密码子。本序列亦去除了影响mRNA稳定的一些基序,包括可能的mRNA剪切位点、可能的mRNA加PolyA位点、重复的PolyA及PolyT序列以及一些正向或反向的重复序列。用此序列产生的转基因水稻株系可以高效的表达本基因所编码蛋白,并提供高水平的防治害虫的能力。
序列3为序列1的优化前序列。本优化方案适用于但不限于在水稻中高水平表达HY131c蛋白。首先本序列所用密码子为水稻最优势密码子。序列优化时同时考虑到5’非编码区及3’非编码区对基因所转录之mRNA整体二级结构的影响,以确保mRNA具有更稳定,更利于高效翻译其所编码的蛋白的能力。本序列亦去除了影响mRNA稳定的一些基序,包括可能的mRNA剪切位点、可能的mRNA加PolyA位点、重复的PolyA及PolyT序列以及一些正向或反向的重复序列。另外,为了有效的翻译起始,可能需要修饰与起始甲硫氨酸相邻的序列,Lütcke(Lütcke,Chow et al.1987)提出针对植物的一种合适的共有序列为:AACAAUGGC,Joshi(Joshi,Zhou et al.1997)等提出适合单子叶植物的序列C(A/C)(A/G)CAAUGGCG,Furukawa(Furukawa,Maekawa et al.2006)等发现了一个高效的适用于水稻的序列GCCGCCATGG。另外,为了有效的提高翻译效率同时引入了Ω序列(Sleat,Gallic etal.1987,Gallie,Sleat et al.1988)。应用本优化方案所得到的序列所产生的转基因水稻株系可以高水平的表达本基因所编码蛋白,并提供高水平的防治害虫的能力。
下述1)-4)中的任一种生物材料也是本发明保护的范围:1)含有上述核酸分子的表达盒;2)含有上述核酸分子的重组载体;3)含有上述核酸分子的重组菌;4)含有上述核酸分子的转基因细胞系。
上述含有核酸分子的重组载体,在本发明的实施例中为将序列表中序列1所示的核苷酸插入原核表达载体pET28a的BamHI和XhoI酶切位点间得到的载体。
上述蛋白质或上述核酸分子或上述表达盒、重组载体、重组菌或转基因细胞系在作为或制备Cry类杀虫剂的应用也是本发明保护的范围。
上述蛋白质或上述核酸分子或上述表达盒、重组载体、重组菌或转基因细胞系在杀虫中的应用也是本发明保护的范围。
上述蛋白质或上述核酸分子或上述表达盒、重组载体、重组菌或转基因细胞系在防治虫害和/或制备防治虫害产品中的应用也是本发明保护的范围。
上述应用中,所述虫为刺吸式口器昆虫;所述刺吸式口器昆虫为稻褐飞虱。
本发明提供新颖的工程化杀虫蛋白HY131c,这一蛋白比Cry1Ac及Cry30Fa1有实质上的改变,包括靶标害虫品种及杀虫活力,这一蛋白的靶标害虫是且不仅限于稻褐飞虱(BPH;Brown plant hopper;Nilapavata lugens)。
本发明近一步涉及编码此HY131c蛋白的核苷酸序列,并且涉及含有这些HY131c蛋白的组合物以及配制品,它们能够抑制害虫的生存、生长以及繁殖的能力或者能够限制昆虫相关的对作物植物的损害或损失。
上述蛋白质或上述核酸分子或上述的表达盒、重组载体、重组菌或转基因细胞系在防治稻褐飞虱和/或制备防治稻褐飞虱产品中的应用也是本发明保护的范围。
本发明所选用改造方法为交换两个不同杀虫蛋白的某一序列区段,使其同时具有两个杀虫蛋白的特征和优点。本发明所选用的两个杀虫蛋白其中Cry1Ac具有对鳞翅目昆虫强的杀虫毒性,Cry30Fa1具有对半翅目稻褐飞虱较微弱的毒性。本发明所用两个杀虫蛋白的编码基因序列都经过序列优化以增强在水稻等植物体中高水平表达。本发明所采用的策略为:用Cry30Fa1能与褐飞虱中肠受体结合的结构域II替换对稻褐飞虱没有毒性的Cry1Ac的结构域II,利用Cry1Ac强的、能在昆虫中肠膜表面形成孔洞的能力,创制形成对稻褐飞虱毒性显著增强的新型杀虫蛋白HY131c。结构域II的替换所采用的方法为重叠PCR(OverlapPCR)法。
定义
“杀虫蛋白”是指能够控制昆虫的的生存、生长以及繁殖的蛋白,并且主要是通过杀死昆虫的方式实现其生物活性。
“δ-内毒素”是指由苏云金芽孢杆菌在产生的,具有对不同昆虫具有毒性的蛋白质。
“靶标害虫”是指对某一特定杀虫蛋白敏感的昆虫称为此杀虫蛋白的靶标害虫,与此对应的对此蛋白不敏感的害虫称为非靶标害虫。
“结构域”:是蛋白质中的一类结构单元,是构成蛋白质三级结构的基本单元。它由不同的α螺旋、β折叠或者其它蛋白质二级结构组成,具有相对独立的空间结构和功能。
“工程化杀虫蛋白HY131c”:是指包括HY131c蛋白编码序列的一种核酸,这种工程化HY131c基因可以衍生于一种天然的Cry类基因或者一种合成的Cry类基因。
“基因”是指编码一种特定蛋白的全部或部分的核酸片段,并包括该编码区前面(5’非编码区)和后面(3’非编码区)的调控序列。
“植物”是指处于任何发育阶段的任何植物,特别是种子植物。
“转化”是指将外源核酸引入至一种宿主细胞或生物中的一个过程。
“外源基因”是指正常情况下宿主生物中没有的、通过基因转移导入的基因;也可指正常存在于宿主生物中的,但又被重新导入其基因组中其天然基因座之外的另一基因座的基因,这种变化会导致一种内源基因的编码序列的一个或几个额外拷贝的出现。
“基序”是指DNA,蛋白质等生物大分子中的保守序列,在反式作用因子的结构中,基序一般指构成任何一种特征序列的基本结构(既指此具功能的基本结构,也指编码此结构的蛋白质/DNA序列),作为结构域中的亚单元,其功能是体现结构域的多种生物学作用。
“mRNA剪切”是指从DNA模板链转录出的最初转录产物中除去内含子,并将外显子连接起来形成一个连续的RNA分子的过程。
“密码子”是指信使RNA链上决定一个氨基酸的相邻的三个碱基亦称三联体密码。同一个氨基酸可由不同的密码子编码称为“密码子的简并性”。
“密码子偏爱性”是指由于密码子的简并性,每个氨基酸至少对应1种密码子,最多有6种对应的密码子。不同物种、不同生物体的基因密码子使用存在着很大的差异。各种生物体似乎更偏爱使用某些同义三联密码子。其中在某一物种中使用频率高的密码子称为该物种的“优势密码子”。
“重叠PCR”:是指采用具有互补末端的引物,使PCR产物形成重叠链,从而在随后的扩增反应中通过重叠链的延伸,将不同来源的扩增片段重叠拼接起来形成一条杂合DNA链。
“保护碱基”是指限制性内切酶识别特定的DNA序列,除此之外,酶蛋白还要占据识别位点两边的若干个碱基,这些碱基对内切酶稳定的结合到DNA双链并发挥切割DNA作用是有很大影响的,被称为保护碱基。
“半致死浓度”是指在动物急性毒性试验中,使受试动物半数死亡的毒物浓度,用LC50表示。
本发明的实验证明,本发明在前期工作的基础上,创造性的改造了Cry1Ac及Cry30Fa1杀虫蛋白,并得到了对褐飞虱等刺吸式口器昆虫具有强杀虫活性的新型杀虫蛋白HY131c,从而为防治稻褐飞虱等水稻害虫提供了新的策略,具有广阔的应用前景;以Cry1Ac和Cry30Fa1基因为基础,通过交换Cry1Ac和Cry30Fa1的结构域II,开发对稻褐飞虱具有高毒力的新型杀虫蛋白,是一项具有重大理论和应用价值的研究。
附图说明
图1为Cry1Ac NCBI Blastp CDD分析结果。
图2为Cry30Fa1NCBI Blastp CDD分析结果。
图3为HY131c NCBI Blastp CDD分析结果。
图4为HY131c重组示意图。
图5为HY131c编码序列获得过程overlap PCR结果。
图6为pET28a-HY131c质粒BamHI、XhoI双酶切鉴定结果。
图7为pET28a-HY131c的质粒图谱。
图8为Cry30Fa1、HY131c及Cry1Ac氨基酸序列比对结果。
图9为pET28a-HY131c原核表达结果。
图10为pET28a-HY131c纯化结果。
图11为HY131c编码序列优化后密码子使用情况与水稻密码子比较结果。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例构建过程中质粒提取、酶切、DNA的回收、纯化均按分子克隆中的方法进行(Sambrook and Russell 2001)。
实施例1、新型杀虫蛋白基因HY131c的获得
一、Cry1Ac及Cry30Fa1蛋白及基因序列分析
将Cry1Ac及Cry30Fa1氨基酸序列应用NCBI网站Blastp功能分析其保守结构域Conserved domains,并对应的找出其编码核苷酸序列。得出Cry1Ac结构域I为Cry1Ac蛋白第48-251位氨基酸,对应的Cry1Ac核酸编码序列为第142-753位核苷酸;结构域II为Cry1Ac蛋白第259-461位氨基酸,对应的Cry1Ac核酸编码序列为第775-1383位核苷酸;结构域III为Cry1Ac蛋白第471-609位氨基酸,对应Cry1Ac的核酸编码序列为第1411-1827位核苷酸,其余序列为结构域间连接序列,如图1所示。Cry30Fa1结构域I为Cry30Fa1蛋白第201-302位氨基酸,对应的Cry30Fa1核酸编码序列为第601-906位核苷酸;结构域II为Cry30Fa1蛋白第310-514位氨基酸,对应的Cry30Fa1核酸编码序列为第928-1542位核苷酸;结构域III为Cry30Fa1蛋白第524-678位氨基酸,对应的Cry30Fa1核酸编码序列为第1570-2034位核苷酸,其余序列为结构域间连接序列,如图2所示。
根据以上信息设计引物,用Cry30Fa1的结构域II替换掉Cry1Ac的结构域II,结构域I与结构域II、结构域II与结构域III的连接部分也来自于Cry30Fa1,应用NCBI网站Blastp功能分析结果如图3所示,构建模式如图4所示,与Cry1Ac及Cry30Fa1的氨基酸序列比对结果见图8。具体引物序列如下:
表1为用来制造编码HY131c蛋白的序列的引物
引物名称 | 引物序列 |
Cry1AcL | 5’-TTTGTGGATCCATGGACAACAACCCA-3’ |
Cry1AcR | 5’-GGGCACTCGAGTTCAGCCTCGAGTGT-3’ |
Cry30R | 5’-GGGCAAAGCTTTTAGTTCACTGGACAAGCAAATGC-3’ |
DS1Ⅰ3ⅡL | 5’-CGAACTATGACTCCAGAACCTGCAAGTACCCCATTTCCAC-3’ |
DS1Ⅰ3ⅡR | 5’-GTGGAAATGGGGTACTTGCAGGTTCTGGAGTCATAGTTCG-3’ |
DS3Ⅱ1ⅢL | 5’-ATACTAGTGTCGACCCTGATAACATCATCGCATCCGATAG-3’ |
DS3Ⅱ1ⅢR | 5’-CTATCGGATGCGATGATGTTATCAGGGTCGACACTAGTAT-3’ |
二、HY131c基因PCR扩增
全部构建过程PCR扩增选用高保真Fast Pfu DNA Polymerase购自全式金生物技术公司,货号为:AP221。扩增体系采用50μl体系,具体如下:
表2为扩增体系
PCR扩增程序为:
表3为扩增程序
具体流程为:用引物cry1AcL和引物DS1Ⅰ3ⅡR以Cry1Ac基因为模板扩增得到产物P100(788bp);用引物DS1Ⅰ3ⅡL和引物Cry30R以Cry30Fa1基因为模板扩增得到产物P033(1175bp)。接下来,用引物cry1AcL和引物Cry30R以P100和P033的混合物为模板扩增,得到产物P133(1943bp)。再然后,用引物cry1AcL和引物DS3Ⅱ1ⅢR以P133为模板扩增得到产物P130(1427bp);用引物DS3Ⅱ1ⅢL和引物Cry1AcR以Cry1Ac基因为模板扩增得到产物P001(532bp)。
最后,用引物cry1AcL和引物Cry1AcR以P130和P001的混合物为模板扩增,得到1860bp的PCR产物,其核苷酸序列为序列表中序列3,为优化前HY131c基因。
全部构建过程的PCR结果见图5。
三、HY131c基因的优化
序列优化采用金斯瑞公司密码子优化软件OptimumGeneTM,采用水稻(Oryzasativa subsp.indica)最优势密码子,参考密码子表:(http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=311553&aa=1&style=GCG),去除XmaI、KpnI、NotI和XhoI等酶切位点。序列优化时同时考虑到5’非编码区及3’非编码区对基因所转录之mRNA整体二级结构的影响,去除影响mRNA稳定的一些基序,包括可能的mRNA剪切位点、可能的mRNA加PolyA位点、重复的PolyA及PolyT序列以及一些正向或反向的重复序列。优化后序列与优化前序列比较参数如下表4和表5。
表4.HY131c序列优化前后碱基组成比较
表5.HY131c序列优化前后密码子适应指数(CAI)及自由能比较
图11为HY131c编码序列优化后密码子使用情况与水稻密码子比较结果。
按照上述方法,优化后的PCR产物核苷酸序列为序列表中序列1,将该PCR产物的基因命名为HY131c,该基因编码的蛋白命名为HY131c,其氨基酸序列为序列表中序列2。
四、杀虫蛋白HY131c的原核表达及纯化
1、重组载体的构建
将上述三获得的优化后的PCR产物经1%琼脂糖凝胶6v/cm,20min分离后从琼脂糖中切出含目标条带并用SV Gel and PCR Clean-Up System试剂盒纯化回收。回收产物HY131c基因用BamHI及XhoI双酶切(全部酶切),并连接入同是BamHI及XhoI双酶切的原核表达载体pET28a(100ng/μl×45μl)。具体酶切体系为:
50μl酶切体系:
表6为酶切体系
组分 | 体积 |
PCR回收产物 | 45μl |
10×buffer | 5μl |
Enzyme | 1μl each |
37℃,1h。
酶切产物再次经过1%琼脂糖凝胶6v/cm,20min分离并用SV Gel andPCRClean-Up System试剂盒纯化回收,回收产物按以下条件连接,其中连接酶购自NEB公司T4DNALigase(货号为M0202L):
10μl连接体系:
表7为连接体系
Components | Volume |
HY131c(BamHI+XhoI) | 4.5μl |
pET28a(BamHI+XhoI) | 4μl |
10×buffer | 1μl |
Enzyme | 0.5μl |
16℃,1h。
连接产物全部转化Trans1-T1Phage Resistant化学感受态细胞(CD501),并涂布含50ng/μl kanamycin LB平板。1L LB平板配方为:胰蛋白胨10g,酵母提取物5g,NaCl 10g,琼脂粉15g,121°高压灭菌20min。
第二天挑单克隆于10ml含50ng/μl kanamycin不含琼脂的LB液体培养基中,37℃,200rpm摇菌12h,用Plus SV Minipreps DNA Purification System提取质粒酶切鉴定,结果见图6,并送睿博兴科公司进行测序,该质粒为将序列表中序列1所示的HY131c基因插入pET28a载体得到的重组载体,命名为pET28a-HY131c,其物理图谱见图7。
1L LB液体培养基配方为:胰蛋白胨10g,酵母提取物5g,NaCl 10g,121°高压灭菌20min。
2、重组菌的构建及目的蛋白的表达和纯化
将上述重组载体pET28a-HY131c转化大肠杆菌表达菌株Trans BL21(DE3)(货号:CD601)转化操作按照产品说明进行操作,得到重组菌BL21(DE3)/pET28a-HY131c。
采用同样的方法将pET28a空载体转化大肠杆菌表达菌株Trans BL21(DE3)中,得到重组菌BL21(DE3)/pET28a。
第二天挑选重组菌BL21(DE3)/pET28a-HY131c单克隆于10ml含50mg/μlkanamycin不含琼脂的LB液体培养基中,37℃,200rpm摇菌12h。然后按照1:100接入新鲜的含50ng/μl kanamycin的100ml LB液体培养基中,37℃,200rpm摇菌,2h左右检测OD600,当OD600=0.6时取500ul未诱导样品作为阴性对照。加入终浓度为1mM IPTG,并每2h取500ul诱导样品,重组菌BL21(DE3)/pET28a的样品只取第6个小时的样品。诱导6个小时后将各样品12000rpm离心1min加50μl 1×PBS(137mM NaCl,2.7mM KCl,10mM Na2HPO4,2mM KH2PO4,PH 7.4)涡旋混匀,加50μl 2×SDS-PAGE loading buffer(120mM Tris-HCl 6.8,20%Glycerol,4%SDS,3%β-mercaptoethanol,0.02%bromophenol blue)混匀,沸水浴5min,跑1.2%SDS-PAGE胶检测。
结果如图9所示,其中,M:分子量marker;28a-:BL21(DE3)/pET28a未诱导对照;28a+:BL21(DE3)/pET28a诱导对照;131c-:BL21(DE3)/pET28a-HY131c未诱导对照;131c+:BL21(DE3)/pET28a-HY131c诱导结果,可以看出,BL21(DE3)/pET28a-HY131c诱导条带与理论分子量69.35kDa相符,说明蛋白HY131c在大肠杆菌中得到诱导表达。
纯化过程为:挑选单克隆于10ml含50ng/μl kanamycin不含琼脂的LB液体培养基中,37℃,200rpm摇菌12h。然后按照1:100接入新鲜的含50mg/μl kanamycin的200ml LB液体培养基中,37℃,200rpm摇菌,2h左右检测OD600,当OD600=0.6时加入终浓度为1mMIPTG,16℃,200rpm诱导过夜。收集过夜诱导表达菌体,10,000g离心10分钟收集菌体。弃上清,用20ml 1×Ni-NTA结合缓冲液(50mM NaH2PO4,pH 8.0,300mM Nacl,10mM咪唑)重悬。并将重悬液置于大小合适的容器中,200W,10s,10s,超声至澄清。裂解物14,000g离心20min去除细胞碎片。离心后上清经0.45μm滤膜过滤(MiniNML Syringe
Filters-17598),与Ni-NTA His·Bind树脂混合,旋转混合器200rpm,4℃结合1h。用4ml 1×Ni-NTA漂洗缓冲液(50mM NaH2PO4,pH8.0,300mM Nacl,20mM咪唑)漂洗2次,用1ml1×Ni-NTA洗脱缓冲液(50mM NaH2PO4,pH8.0,300mM Nacl,2500mM咪唑)洗脱,收集洗脱液。
将洗脱液用SDS-PAGE检测,结果如图10,可以看出,得到分子量大小为69.35kDa的目的蛋白HY131c。
洗脱后的蛋白用1×PBS透析缓冲液(137mM NaCl,2.7mM KCl,10mM Na2HPO4,2mMKH2PO4,10%v/v甘油,pH 10.0),4℃透析12h,中间换两次缓冲液。
纯化后的蛋白经BCA法(康为世纪试剂盒CW0014)测定浓度。
实施例2、新型杀虫蛋白基因HY131c抗虫生物活性测定
供试虫源:稻褐飞虱(Nilapavata lugens)为中国水稻所,赖凤香老师实验室提供。
供试样品:
实施例1制备的抗稻褐飞虱蛋白HY131c,为原核表达纯化可溶蛋白,浓度及产量表明在对应蛋白的保存管上,干冰寄送,-80℃保存;
Cry30Fa1蛋白:采用实施例1的原核表达的方法,制备Cry30Fa1蛋白,其氨基酸序列为序列表中序列4。由于Cry30Fa1蛋白表达量太低,不足以进行生测,对Cry30Fa1蛋白进行了截短,以提高其表达量。在不影响三个功能结构域的前提下,去掉了Cry30Fa1蛋白第1到49位氨基酸,新的蛋白命名为30T蛋白,其氨基酸序列为序列表中序列5。采用实施例1的原核表达的方法,制备30T蛋白。
Cry1Ac蛋白:采用实施例1的原核表达的方法,制备Cry1Ac蛋白,其氨基酸序列为序列表中序列6;
HY113蛋白:采用实施例1的原核表达的方法,制备HY113蛋白,其氨基酸序列为序列表中序列7,HY113蛋白的结构域Ⅰ和结构域Ⅱ来自Cry1Ac、结构域Ⅲ来自Cry30Fa1。
HY133蛋白:采用实施例1的原核表达的方法,制备HY133蛋白,其氨基酸序列为序列表中序列8;HY133蛋白的结构域Ⅰ来自Cry1Ac、结构域Ⅱ和结构域Ⅲ来自Cry30Fa1。
实验地点:委托中国水稻所赖凤香老师实验室进行。
实验方法:实验于中国水稻所人工气候培养室进行,环境温度为:27±1℃。飞虱饲喂采用全人工饲料,饲育器为长15cm,直径2.5cm的无底的圆筒形玻璃器皿,饲料室由双层拉薄的Parafilm膜夹饲料液滴组成。
生测时将各供试样品进行浓度梯度(具体浓度梯度见表8)稀释混入人工饲料饲喂飞虱,并设定无蛋白添加的纯阴性对照(CK)。
实验具体方案为:每管接初孵(1龄)若虫25头后用纱布封口,平放,用黑布遮光保湿,只露出Parafilm膜封口端。每处理设3次重复,每2天查虫,记录活虫数。以第6天计算LC50,(因第6天虫龄为3-4龄,如果试虫还未死亡,为害苗已经很严重了)。具体结果见表8,半致死浓度LC50结果见表9。
表8为杀虫蛋白HY131c对稻褐飞虱毒性测定第6天结果
表9为杀虫蛋白HY131c对稻褐飞虱第8天半致死浓度
蛋白名称 | LC50 |
Cry30Fa1(30T) | 96.58 |
HY131c | 59.54 |
HY113 | 103.29 |
HY133 | 269.76 |
Cry1Ac | Not available |
可以看出,杀虫蛋白HY131c比Cry1Ac和Cry30Fa1对稻褐飞虱的杀虫率更高。
序列表
<110>中国科学院遗传与发育生物学研究所
<120>一种抗稻褐飞虱的杀虫蛋白HY131c及其编码基因与应用
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 1860
<212> DNA
<213> 人工序列
<220>
<223>
<400> 1
atggacaaca acccgaacat caacgagtgc atcccgtaca actgcctcag caacccagag 60
gtggaggtgc tcggaggaga gaggatcgag accggctaca ccccgatcga catcagcctc 120
tccctcaccc agttcctcct cagcgagttc gtgccgggcg caggattcgt gctcggactc 180
gtggacatca tctggggaat cttcggacca tcccagtggg acgccttcct cgtgcagatc 240
gagcagctca tcaaccagcg catcgaggag ttcgccagga accaggcaat cagccgcctg 300
gagggactct ccaacctcta ccagatctac gcagagtcct tccgcgagtg ggaggcagac 360
ccaaccaacc cagcactccg cgaggagatg aggatccagt tcaacgacat gaacagcgca 420
ctcaccaccg caatcccact cttcgccgtg cagaactacc aggtgccgct cctctccgtc 480
tacgtgcagg cagccaacct ccacctcagc gtgctcaggg acgtgtccgt gttcggacag 540
cgctggggat tcgacgcagc aaccatcaac agcaggtaca acgacctcac ccgcctcatc 600
ggcaactaca ccgaccacgc cgtgaggtgg tacaacaccg gactggagag ggtgtgggga 660
ccagactccc gcgactggat caggtacaac cagttccgca gggagctcac cctcaccgtg 720
ctcgacatcg tgagcctctt cccgaactac gactccagga cctgcaagta cccgatcagc 780
accaagaccg agctcacccg caaggtgtac atgccgtcct tctacctcca ggccctccag 840
cacagcaaca tcgagaccct ggagaaccag ctcacccacc cgccgtccct cttcacctgg 900
ctcaacgagc tcaacctcta caccatcagg gagaacttca acccagcact ccaggtgtcc 960
agcctcagcg gactccaggc caagtaccgc tacacccaga actccaccat cctcccaaac 1020
ccaccagcac agggcatcac caacggcacc ccgatcccga tcatcggcct caacaacctc 1080
ttcatctaca agctcagcat gtcccagtac aggcacccaa acgactgcgt gccaatcgca 1140
ggcatcagcg acatgacctt ctacaagtcc gactacaacg gcaacgcaag cgcaacccag 1200
acctaccagg caggccgcaa cagcaacaac gtgatcgaca ccttcatgaa cggcccgcag 1260
aacgcctcca gctccaacaa catctccatc aaccagacca accacatcct cagcgacatc 1320
aagatgaact acgcaaggtc cggaggagtg tacgacttcg gctactcctt cgcatggacc 1380
cacaccagcg tggacccaga caacatcatc gccagcgact ccatcaccca gatcccggcc 1440
gtgaagggca acttcctctt caacggcagc gtgatctccg gcccaggctt caccggaggc 1500
gacctcgtga ggctcaacag ctccggcaac aacatccaga accgcggcta catcgaggtg 1560
ccgatccact tcccaagcac ctccacccgc tacagggtgc gcgtgaggta cgcatccgtg 1620
accccgatcc acctcaacgt gaactggggc aacagctcca tcttcagcaa caccgtgccg 1680
gcaaccgcaa cctccctcga caacctccag agctccgact tcggctactt cgagagcgcc 1740
aacgccttca ccagctccct cggcaacatc gtgggcgtga ggaacttctc cggcaccgca 1800
ggcgtgatca tcgaccgctt cgagttcatc ccggtgaccg ccaccctgga ggccgagtga 1860
<210> 2
<211> 619
<212> PRT
<213> 人工序列
<220>
<223>
<400> 2
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
20 25 30
Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45
Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile
50 55 60
Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
65 70 75 80
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95
Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu
100 105 110
Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125
Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala
130 135 140
Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
145 150 155 160
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175
Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190
Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val
195 200 205
Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg
210 215 220
Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
225 230 235 240
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Cys Lys
245 250 255
Tyr Pro Ile Ser Thr Lys Thr Glu Leu Thr Arg Lys Val Tyr Met Pro
260 265 270
Ser Phe Tyr Leu Gln Ala Leu Gln His Ser Asn Ile Glu Thr Leu Glu
275 280 285
Asn Gln Leu Thr His Pro Pro Ser Leu Phe Thr Trp Leu Asn Glu Leu
290 295 300
Asn Leu Tyr Thr Ile Arg Glu Asn Phe Asn Pro Ala Leu Gln Val Ser
305 310 315 320
Ser Leu Ser Gly Leu Gln Ala Lys Tyr Arg Tyr Thr Gln Asn Ser Thr
325 330 335
Ile Leu Pro Asn Pro Pro Ala Gln Gly Ile Thr Asn Gly Thr Pro Ile
340 345 350
Pro Ile Ile Gly Leu Asn Asn Leu Phe Ile Tyr Lys Leu Ser Met Ser
355 360 365
Gln Tyr Arg His Pro Asn Asp Cys Val Pro Ile Ala Gly Ile Ser Asp
370 375 380
Met Thr Phe Tyr Lys Ser Asp Tyr Asn Gly Asn Ala Ser Ala Thr Gln
385 390 395 400
Thr Tyr Gln Ala Gly Arg Asn Ser Asn Asn Val Ile Asp Thr Phe Met
405 410 415
Asn Gly Pro Gln Asn Ala Ser Ser Ser Asn Asn Ile Ser Ile Asn Gln
420 425 430
Thr Asn His Ile Leu Ser Asp Ile Lys Met Asn Tyr Ala Arg Ser Gly
435 440 445
Gly Val Tyr Asp Phe Gly Tyr Ser Phe Ala Trp Thr His Thr Ser Val
450 455 460
Asp Pro Asp Asn Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Ala
465 470 475 480
Val Lys Gly Asn Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly
485 490 495
Phe Thr Gly Gly Asp Leu Val Arg Leu Asn Ser Ser Gly Asn Asn Ile
500 505 510
Gln Asn Arg Gly Tyr Ile Glu Val Pro Ile His Phe Pro Ser Thr Ser
515 520 525
Thr Arg Tyr Arg Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile His
530 535 540
Leu Asn Val Asn Trp Gly Asn Ser Ser Ile Phe Ser Asn Thr Val Pro
545 550 555 560
Ala Thr Ala Thr Ser Leu Asp Asn Leu Gln Ser Ser Asp Phe Gly Tyr
565 570 575
Phe Glu Ser Ala Asn Ala Phe Thr Ser Ser Leu Gly Asn Ile Val Gly
580 585 590
Val Arg Asn Phe Ser Gly Thr Ala Gly Val Ile Ile Asp Arg Phe Glu
595 600 605
Phe Ile Pro Val Thr Ala Thr Leu Glu Ala Glu
610 615
<210> 3
<211> 1860
<212> DNA
<213> 人工序列
<220>
<223>
<400> 3
atggacaaca acccaaacat caacgaatgc attccataca actgcttgag taacccagaa 60
gttgaagtac ttggtggaga acgcattgaa accggttaca ctcccatcga catctccttg 120
tccttgacac agtttctgct cagcgagttc gtgccaggtg ctgggttcgt tctcggacta 180
gttgacatca tctggggtat ctttggtcca tctcaatggg atgcattcct ggtgcaaatt 240
gagcagttga tcaaccagag gatcgaagag ttcgccagga accaggccat ctctaggttg 300
gaaggattga gcaatctcta ccaaatctat gcagagagct tcagagagtg ggaagccgat 360
cctactaacc cagctctccg cgaggaaatg cgtattcaat tcaacgacat gaacagcgcc 420
ttgaccacag ctatcccatt gttcgcagtc cagaactacc aagttcctct cttgtccgtg 480
tacgttcaag cagctaatct tcacctcagc gtgcttcgag acgttagcgt gtttgggcaa 540
aggtggggat tcgatgctgc aaccatcaat agccgttaca acgaccttac taggctgatt 600
ggaaactaca ccgaccacgc tgttcgttgg tacaacactg gcttggagcg tgtctggggt 660
cctgattcta gagattggat tagatacaac cagttcagga gagaattgac cctcacagtt 720
ttggacattg tgtctctctt cccgaactat gactccagaa cctgcaagta ccccatttcc 780
acgaagactg agctcacacg gaaggtttac atgccgtcct tttatcttca ggcgttgcaa 840
cacagcaaca tcgagactct cgaaaatcag ctgacccatc cgccatccct tttcacgtgg 900
ttgaacgagc tcaatctgta cactatccgg gaaaacttta atccggctct gcaagtgtcg 960
agtctctcag gcctgcaggc aaagtacaga tatacccaaa actcgacaat cctcccaaat 1020
cctcccgctc agggaatcac caatggtaca cctattccca tcattggact taacaatttg 1080
ttcatctaca agctgagtat gtcccaatat cgccacccta acgactgcgt gcccatcgcg 1140
gggattagcg acatgacatt ctacaagtct gattataacg gaaatgcttc agcaacccag 1200
acataccaag ctggtaggaa cagcaacaat gtcatcgata ccttcatgaa cggcccgcag 1260
aatgcatcca gctctaacaa tatctctatt aaccaaacaa accacatcct ctcagacatt 1320
aagatgaact acgccaggtc gggcggggtc tacgatttcg gctattcgtt tgcgtggacg 1380
catactagtg tcgaccctga taacatcatc gcatccgata gtattactca aatccctgca 1440
gtgaagggaa actttctctt caacggttct gtcatttcag gaccaggatt cactggtgga 1500
gacctcgtta gactcaacag cagtggaaat aacattcaga atagagggta tattgaagtt 1560
ccaattcact tcccatccac atctaccaga tatagagttc gtgtgaggta tgcttctgtg 1620
acccctattc acctcaacgt taattggggt aattcatcca tcttctccaa tacagttcca 1680
gctacagcta cctccttgga taatctccaa tccagcgatt tcggttactt tgaaagtgcc 1740
aatgctttta catcttcact cggtaacatc gtgggtgtta gaaactttag tgggactgca 1800
ggagtgatta tcgacagatt cgagttcatt ccagttactg caacactcga ggctgaatga 1860
<210> 4
<211> 687
<212> PRT
<213> 人工序列
<220>
<223>
<400> 4
Met Glu Pro Tyr Gln Ser Glu Asn Glu Tyr Glu Ile Leu Asp Thr Leu
1 5 10 15
Pro Lys Tyr Ser Asn Ile Val Asn Val Tyr Ser Arg Tyr Pro Leu Ala
20 25 30
Asn Asn Pro Gln Val Pro Leu Gln Asn Thr Ser Tyr Lys Asp Trp Leu
35 40 45
Asn Met Cys Gln Thr Ile Thr Pro Leu Cys Thr Pro Ile Asp Ile Asp
50 55 60
Ser Lys Leu Val Ala Thr Ala Ile Gly Ile Leu Gly Ala Ile Phe Lys
65 70 75 80
Ala Met Pro Gly Pro Gly Ser Ala Val Gly Leu Phe Leu Lys Thr Phe
85 90 95
Ser Thr Ile Ile Pro Ile Leu Trp Pro Asn Asp Asn Thr Pro Ile Trp
100 105 110
Lys Glu Phe Thr Lys Gln Gly Leu Gln Leu Phe Arg Pro Glu Leu Gly
115 120 125
Arg Asp Ala Ile Glu Ile Ile Gly Asn Asp Val Gln Ser Gly Phe Asn
130 135 140
Ala Leu Lys Asp His Met Asn Asp Phe Glu Thr Lys Phe Glu Ile Trp
145 150 155 160
Asp Lys Asp Arg Thr Gln Thr Asn Ala Thr Tyr Leu Ile Thr Ala Phe
165 170 175
Gly Val Val Asn Gly Lys Ile Ile Asp Leu Lys Asn Gln Phe Leu Ile
180 185 190
Asn Pro Ala Asn Gln Pro Ala Phe Leu Asn Leu Tyr Ala Gln Thr Ala
195 200 205
Asn Ile Asp Leu Ile Leu Tyr Gln Arg Gly Ala Val Tyr Gly Asp Asp
210 215 220
Trp Ala Lys Ala Ile Asn Asp Ser Ser Ile Ser Pro Phe Asn Ser Ser
225 230 235 240
Gln Ile Phe Tyr Asp Ser Leu Lys Ala Lys Ile Lys Glu Tyr Thr Asn
245 250 255
Tyr Cys Ala Glu Thr Tyr Arg Asn Ser Leu Thr Ile Leu Lys Asn Gln
260 265 270
Pro Asn Ile Gln Trp Asp Ile Tyr Asn Arg Tyr Arg Arg Glu Ala Thr
275 280 285
Leu Gly Ala Leu Asp Leu Val Ala Leu Phe Pro Asn Tyr Asp Ile Cys
290 295 300
Lys Tyr Pro Ile Ser Thr Lys Thr Glu Leu Thr Arg Lys Val Tyr Met
305 310 315 320
Pro Ser Phe Tyr Leu Gln Ala Leu Gln His Ser Asn Ile Glu Thr Leu
325 330 335
Glu Asn Gln Leu Thr His Pro Pro Ser Leu Phe Thr Trp Leu Asn Glu
340 345 350
Leu Asn Leu Tyr Thr Ile Arg Glu Asn Phe Asn Pro Ala Leu Gln Val
355 360 365
Ser Ser Leu Ser Gly Leu Gln Ala Lys Tyr Arg Tyr Thr Gln Asn Ser
370 375 380
Thr Ile Leu Pro Asn Pro Pro Ala Gln Gly Ile Thr Asn Gly Thr Pro
385 390 395 400
Ile Pro Ile Ile Gly Leu Asn Asn Leu Phe Ile Tyr Lys Leu Ser Met
405 410 415
Ser Gln Tyr Arg His Pro Asn Asp Cys Val Pro Ile Ala Gly Ile Ser
420 425 430
Asp Met Thr Phe Tyr Lys Ser Asp Tyr Asn Gly Asn Ala Ser Ala Thr
435 440 445
Gln Thr Tyr Gln Ala Gly Arg Asn Ser Asn Asn Val Ile Asp Thr Phe
450 455 460
Met Asn Gly Pro Gln Asn Ala Ser Ser Ser Asn Asn Ile Ser Ile Asn
465 470 475 480
Gln Thr Asn His Ile Leu Ser Asp Ile Lys Met Asn Tyr Ala Arg Ser
485 490 495
Gly Gly Val Tyr Asp Phe Gly Tyr Ser Phe Ala Trp Thr His Thr Ser
500 505 510
Val Asp Pro Asp Asn Leu Ile Val Pro Asn Arg Ile Thr Gln Ile Pro
515 520 525
Ala Val Lys Ala Asn Tyr Leu Ser Ser Pro Ala Arg Val Ile Ala Gly
530 535 540
Pro Gly His Thr Gly Gly Asp Leu Val Ala Leu Leu Asn Gly Gly Thr
545 550 555 560
Gln Ala Gly Lys Met Gln Ile Gln Cys Lys Thr Gly Ser Phe Thr Gly
565 570 575
Ala Ser Arg Arg Tyr Gly Ile Arg Met Arg Tyr Ala Ala Asn Asn Ala
580 585 590
Phe Thr Val Ser Leu Ser Tyr Thr Leu Gln Gly Gly Asn Thr Ile Gly
595 600 605
Thr Thr Phe Ile Thr Glu Arg Thr Phe Ser Arg Pro Asn Asn Ile Ile
610 615 620
Pro Thr Asp Leu Lys Tyr Glu Glu Phe Lys Tyr Lys Glu Tyr Asn Gln
625 630 635 640
Ile Ile Thr Met Thr Ser Pro Gln Asn Thr Ile Val Thr Ile Ala Ile
645 650 655
Gln Gln Leu Asn Pro Ile Pro Asn Asp Gln Leu Ile Ile Asp Arg Ile
660 665 670
Glu Phe Tyr Pro Val Asp Gln Gly Ala Phe Ala Cys Pro Val Asn
675 680 685
<210> 5
<211> 638
<212> PRT
<213> 人工序列
<220>
<223>
<400> 5
Met Cys Gln Thr Ile Thr Pro Leu Cys Thr Pro Ile Asp Ile Asp Ser
1 5 10 15
Lys Leu Val Ala Thr Ala Ile Gly Ile Leu Gly Ala Ile Phe Lys Ala
20 25 30
Met Pro Gly Pro Gly Ser Ala Val Gly Leu Phe Leu Lys Thr Phe Ser
35 40 45
Thr Ile Ile Pro Ile Leu Trp Pro Asn Asp Asn Thr Pro Ile Trp Lys
50 55 60
Glu Phe Thr Lys Gln Gly Leu Gln Leu Phe Arg Pro Glu Leu Gly Arg
65 70 75 80
Asp Ala Ile Glu Ile Ile Gly Asn Asp Val Gln Ser Gly Phe Asn Ala
85 90 95
Leu Lys Asp His Met Asn Asp Phe Glu Thr Lys Phe Glu Ile Trp Asp
100 105 110
Lys Asp Arg Thr Gln Thr Asn Ala Thr Tyr Leu Ile Thr Ala Phe Gly
115 120 125
Val Val Asn Gly Lys Ile Ile Asp Leu Lys Asn Gln Phe Leu Ile Asn
130 135 140
Pro Ala Asn Gln Pro Ala Phe Leu Asn Leu Tyr Ala Gln Thr Ala Asn
145 150 155 160
Ile Asp Leu Ile Leu Tyr Gln Arg Gly Ala Val Tyr Gly Asp Asp Trp
165 170 175
Ala Lys Ala Ile Asn Asp Ser Ser Ile Ser Pro Phe Asn Ser Ser Gln
180 185 190
Ile Phe Tyr Asp Ser Leu Lys Ala Lys Ile Lys Glu Tyr Thr Asn Tyr
195 200 205
Cys Ala Glu Thr Tyr Arg Asn Ser Leu Thr Ile Leu Lys Asn Gln Pro
210 215 220
Asn Ile Gln Trp Asp Ile Tyr Asn Arg Tyr Arg Arg Glu Ala Thr Leu
225 230 235 240
Gly Ala Leu Asp Leu Val Ala Leu Phe Pro Asn Tyr Asp Ile Cys Lys
245 250 255
Tyr Pro Ile Ser Thr Lys Thr Glu Leu Thr Arg Lys Val Tyr Met Pro
260 265 270
Ser Phe Tyr Leu Gln Ala Leu Gln His Ser Asn Ile Glu Thr Leu Glu
275 280 285
Asn Gln Leu Thr His Pro Pro Ser Leu Phe Thr Trp Leu Asn Glu Leu
290 295 300
Asn Leu Tyr Thr Ile Arg Glu Asn Phe Asn Pro Ala Leu Gln Val Ser
305 310 315 320
Ser Leu Ser Gly Leu Gln Ala Lys Tyr Arg Tyr Thr Gln Asn Ser Thr
325 330 335
Ile Leu Pro Asn Pro Pro Ala Gln Gly Ile Thr Asn Gly Thr Pro Ile
340 345 350
Pro Ile Ile Gly Leu Asn Asn Leu Phe Ile Tyr Lys Leu Ser Met Ser
355 360 365
Gln Tyr Arg His Pro Asn Asp Cys Val Pro Ile Ala Gly Ile Ser Asp
370 375 380
Met Thr Phe Tyr Lys Ser Asp Tyr Asn Gly Asn Ala Ser Ala Thr Gln
385 390 395 400
Thr Tyr Gln Ala Gly Arg Asn Ser Asn Asn Val Ile Asp Thr Phe Met
405 410 415
Asn Gly Pro Gln Asn Ala Ser Ser Ser Asn Asn Ile Ser Ile Asn Gln
420 425 430
Thr Asn His Ile Leu Ser Asp Ile Lys Met Asn Tyr Ala Arg Ser Gly
435 440 445
Gly Val Tyr Asp Phe Gly Tyr Ser Phe Ala Trp Thr His Thr Ser Val
450 455 460
Asp Pro Asp Asn Leu Ile Val Pro Asn Arg Ile Thr Gln Ile Pro Ala
465 470 475 480
Val Lys Ala Asn Tyr Leu Ser Ser Pro Ala Arg Val Ile Ala Gly Pro
485 490 495
Gly His Thr Gly Gly Asp Leu Val Ala Leu Leu Asn Gly Gly Thr Gln
500 505 510
Ala Gly Lys Met Gln Ile Gln Cys Lys Thr Gly Ser Phe Thr Gly Ala
515 520 525
Ser Arg Arg Tyr Gly Ile Arg Met Arg Tyr Ala Ala Asn Asn Ala Phe
530 535 540
Thr Val Ser Leu Ser Tyr Thr Leu Gln Gly Gly Asn Thr Ile Gly Thr
545 550 555 560
Thr Phe Ile Thr Glu Arg Thr Phe Ser Arg Pro Asn Asn Ile Ile Pro
565 570 575
Thr Asp Leu Lys Tyr Glu Glu Phe Lys Tyr Lys Glu Tyr Asn Gln Ile
580 585 590
Ile Thr Met Thr Ser Pro Gln Asn Thr Ile Val Thr Ile Ala Ile Gln
595 600 605
Gln Leu Asn Pro Ile Pro Asn Asp Gln Leu Ile Ile Asp Arg Ile Glu
610 615 620
Phe Tyr Pro Val Asp Gln Gly Ala Phe Ala Cys Pro Val Asn
625 630 635
<210> 6
<211> 615
<212> PRT
<213> 人工序列
<220>
<223>
<400> 6
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
20 25 30
Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45
Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile
50 55 60
Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
65 70 75 80
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95
Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu
100 105 110
Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125
Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala
130 135 140
Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
145 150 155 160
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175
Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190
Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val
195 200 205
Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg
210 215 220
Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
225 230 235 240
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro
245 250 255
Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val
260 265 270
Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu
275 280 285
Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr
290 295 300
Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln
305 310 315 320
Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
325 330 335
Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala
340 345 350
Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg
355 360 365
Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp
370 375 380
Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val
385 390 395 400
Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln
405 410 415
Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His
420 425 430
Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile
435 440 445
Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn
450 455 460
Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Ala Val Lys Gly Asn
465 470 475 480
Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly
485 490 495
Asp Leu Val Arg Leu Asn Ser Ser Gly Asn Asn Ile Gln Asn Arg Gly
500 505 510
Tyr Ile Glu Val Pro Ile His Phe Pro Ser Thr Ser Thr Arg Tyr Arg
515 520 525
Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile His Leu Asn Val Asn
530 535 540
Trp Gly Asn Ser Ser Ile Phe Ser Asn Thr Val Pro Ala Thr Ala Thr
545 550 555 560
Ser Leu Asp Asn Leu Gln Ser Ser Asp Phe Gly Tyr Phe Glu Ser Ala
565 570 575
Asn Ala Phe Thr Ser Ser Leu Gly Asn Ile Val Gly Val Arg Asn Phe
580 585 590
Ser Gly Thr Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val
595 600 605
Thr Ala Thr Leu Glu Ala Glu
610 615
<210> 7
<211> 634
<212> PRT
<213> 人工序列
<220>
<223>
<400> 7
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
20 25 30
Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45
Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile
50 55 60
Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
65 70 75 80
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95
Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu
100 105 110
Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125
Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala
130 135 140
Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
145 150 155 160
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175
Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190
Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val
195 200 205
Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg
210 215 220
Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
225 230 235 240
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro
245 250 255
Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val
260 265 270
Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu
275 280 285
Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr
290 295 300
Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln
305 310 315 320
Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
325 330 335
Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala
340 345 350
Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg
355 360 365
Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp
370 375 380
Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val
385 390 395 400
Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln
405 410 415
Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His
420 425 430
Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile
435 440 445
Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Pro Asp Asn
450 455 460
Leu Ile Val Pro Asn Arg Ile Thr Gln Ile Pro Ala Val Lys Ala Asn
465 470 475 480
Tyr Leu Ser Ser Pro Ala Arg Val Ile Ala Gly Pro Gly His Thr Gly
485 490 495
Gly Asp Leu Val Ala Leu Leu Asn Gly Gly Thr Gln Ala Gly Lys Met
500 505 510
Gln Ile Gln Cys Lys Thr Gly Ser Phe Thr Gly Ala Ser Arg Arg Tyr
515 520 525
Gly Ile Arg Met Arg Tyr Ala Ala Asn Asn Ala Phe Thr Val Ser Leu
530 535 540
Ser Tyr Thr Leu Gln Gly Gly Asn Thr Ile Gly Thr Thr Phe Ile Thr
545 550 555 560
Glu Arg Thr Phe Ser Arg Pro Asn Asn Ile Ile Pro Thr Asp Leu Lys
565 570 575
Tyr Glu Glu Phe Lys Tyr Lys Glu Tyr Asn Gln Ile Ile Thr Met Thr
580 585 590
Ser Pro Gln Asn Thr Ile Val Thr Ile Ala Ile Gln Gln Leu Asn Pro
595 600 605
Ile Pro Asn Asp Gln Leu Ile Ile Asp Arg Ile Glu Phe Tyr Pro Val
610 615 620
Asp Gln Gly Ala Phe Ala Cys Pro Val Asn
625 630
<210> 8
<211> 638
<212> PRT
<213> 人工序列
<220>
<223>
<400> 8
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
20 25 30
Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45
Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile
50 55 60
Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
65 70 75 80
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95
Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu
100 105 110
Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125
Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala
130 135 140
Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
145 150 155 160
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175
Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190
Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val
195 200 205
Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg
210 215 220
Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
225 230 235 240
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Cys Lys
245 250 255
Tyr Pro Ile Ser Thr Lys Thr Glu Leu Thr Arg Lys Val Tyr Met Pro
260 265 270
Ser Phe Tyr Leu Gln Ala Leu Gln His Ser Asn Ile Glu Thr Leu Glu
275 280 285
Asn Gln Leu Thr His Pro Pro Ser Leu Phe Thr Trp Leu Asn Glu Leu
290 295 300
Asn Leu Tyr Thr Ile Arg Glu Asn Phe Asn Pro Ala Leu Gln Val Ser
305 310 315 320
Ser Leu Ser Gly Leu Gln Ala Lys Tyr Arg Tyr Thr Gln Asn Ser Thr
325 330 335
Ile Leu Pro Asn Pro Pro Ala Gln Gly Ile Thr Asn Gly Thr Pro Ile
340 345 350
Pro Ile Ile Gly Leu Asn Asn Leu Phe Ile Tyr Lys Leu Ser Met Ser
355 360 365
Gln Tyr Arg His Pro Asn Asp Cys Val Pro Ile Ala Gly Ile Ser Asp
370 375 380
Met Thr Phe Tyr Lys Ser Asp Tyr Asn Gly Asn Ala Ser Ala Thr Gln
385 390 395 400
Thr Tyr Gln Ala Gly Arg Asn Ser Asn Asn Val Ile Asp Thr Phe Met
405 410 415
Asn Gly Pro Gln Asn Ala Ser Ser Ser Asn Asn Ile Ser Ile Asn Gln
420 425 430
Thr Asn His Ile Leu Ser Asp Ile Lys Met Asn Tyr Ala Arg Ser Gly
435 440 445
Gly Val Tyr Asp Phe Gly Tyr Ser Phe Ala Trp Thr His Thr Ser Val
450 455 460
Asp Pro Asp Asn Leu Ile Val Pro Asn Arg Ile Thr Gln Ile Pro Ala
465 470 475 480
Val Lys Ala Asn Tyr Leu Ser Ser Pro Ala Arg Val Ile Ala Gly Pro
485 490 495
Gly His Thr Gly Gly Asp Leu Val Ala Leu Leu Asn Gly Gly Thr Gln
500 505 510
Ala Gly Lys Met Gln Ile Gln Cys Lys Thr Gly Ser Phe Thr Gly Ala
515 520 525
Ser Arg Arg Tyr Gly Ile Arg Met Arg Tyr Ala Ala Asn Asn Ala Phe
530 535 540
Thr Val Ser Leu Ser Tyr Thr Leu Gln Gly Gly Asn Thr Ile Gly Thr
545 550 555 560
Thr Phe Ile Thr Glu Arg Thr Phe Ser Arg Pro Asn Asn Ile Ile Pro
565 570 575
Thr Asp Leu Lys Tyr Glu Glu Phe Lys Tyr Lys Glu Tyr Asn Gln Ile
580 585 590
Ile Thr Met Thr Ser Pro Gln Asn Thr Ile Val Thr Ile Ala Ile Gln
595 600 605
Gln Leu Asn Pro Ile Pro Asn Asp Gln Leu Ile Ile Asp Arg Ile Glu
610 615 620
Phe Tyr Pro Val Asp Gln Gly Ala Phe Ala Cys Pro Val Asn
625 630 635
Claims (9)
1.一种蛋白质,包括Cry1Ac的结构域I、Cry30Fa1结构域II及Cry1Ac结构域III。
2.根据权利要求1所述的蛋白质,其特征在于:所述蛋白质为将所述Cry1Ac蛋白中的结构域II替换为所述Cry30Fa1蛋白的结构域II得到的蛋白质。
3.根据权利要求1或2所述的蛋白质,其特征在于:所述蛋白质为如下a)-e)中任一种蛋白质:
a)氨基酸序列包括序列表中序列2所示的氨基酸序列的蛋白质;
b)氨基酸序列由序列表中序列2所示的氨基酸残基组成;
c)将a)或b)所限定的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有抗虫功能的蛋白质;
d)与a)或b)所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有抗虫功能的蛋白质;
e)a)-d)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
4.编码权利要求1-3中任一所述蛋白的核酸分子。
5.根据权利要求4所述的核酸分子,其特征在于:所述核酸分子为如下1)-6)中任一种所示的核酸分子:
1)其编码序列包括序列表中序列1;
2)其编码序列包括序列表中序列3;
3)其编码序列为序列表中序列1;
4)其编码序列为序列表中序列3;
5)在严格条件下与1)-4)中任一限定的DNA分子杂交且编码权利要求1所述蛋白的DNA分子;
6)与1)-4)中任一限定的DNA分子具有80%以上或90%以上的同源性且编码权利要求1所述蛋白的DNA分子。
6.下述1)-4)中的任一种生物材料:
1)含有权利要求4或5所述核酸分子的表达盒;
2)含有权利要求4或5所述核酸分子的重组载体;
3)含有权利要求4或5所述核酸分子的重组菌;
4)含有权利要求4或5所述核酸分子的转基因细胞系。
7.权利要求1-3中任一所述蛋白质或权利要求4或5所述核酸分子或权利要求6所述的表达盒、重组载体、重组菌或转基因细胞系在作为或制备Cry类杀虫剂中的应用;
或权利要求1-3中任一所述蛋白质或权利要求4或5所述核酸分子或权利要求6所述的表达盒、重组载体、重组菌或转基因细胞系在杀虫中的应用;
或,权利要求1-3中任一所述蛋白质或权利要求4或5所述核酸分子或权利要求6所述的表达盒、重组载体、重组菌或转基因细胞系在防治虫害和/或制备防治虫害产品中的应用。
8.根据权利要求7所述的应用,其特征在于:所述虫为刺吸式口器昆虫。
9.根据权利要求8所述的应用,其特征在于:所述刺吸式口器昆虫为稻褐飞虱。
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CN114573675A (zh) * | 2020-12-01 | 2022-06-03 | 中国科学院微生物研究所 | GrpE蛋白或其编码基因作为特异性抗灰飞虱及水稻条纹病毒的分子靶标的应用 |
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CN110563819A (zh) * | 2019-10-08 | 2019-12-13 | 中国农业科学院植物保护研究所 | 一种杀虫蛋白、其核苷酸序列及其应用 |
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CN111995690A (zh) * | 2020-01-16 | 2020-11-27 | 黑龙江大鹏农业有限公司 | 一种人工合成的抗虫蛋白mCry1Ia2及其制备方法和应用 |
CN111995690B (zh) * | 2020-01-16 | 2022-10-11 | 黑龙江大鹏农业有限公司 | 一种人工合成的抗虫蛋白mCry1Ia2及其制备方法和应用 |
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CN114573675B (zh) * | 2020-12-01 | 2023-06-20 | 中国科学院微生物研究所 | GrpE蛋白或其编码基因作为特异性抗灰飞虱及水稻条纹病毒的分子靶标的应用 |
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