CN112225786A - 对松墨天牛具有高毒力的Bt毒素 - Google Patents

对松墨天牛具有高毒力的Bt毒素 Download PDF

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CN112225786A
CN112225786A CN202011168858.0A CN202011168858A CN112225786A CN 112225786 A CN112225786 A CN 112225786A CN 202011168858 A CN202011168858 A CN 202011168858A CN 112225786 A CN112225786 A CN 112225786A
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吴松青
张飞萍
郭雅洁
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Fujian Agriculture and Forestry University
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Abstract

本发明属于生物防治领域，具体涉及一种对松墨天牛具有高毒力的Bt毒素，所述Bt毒素为毒素BRC2007，其氨基酸序列如SEQ ID NO.2所示，该毒素对松墨天牛具有良好的杀虫活性，本发明为松墨天牛的生物防控提供了有效途径。

Description

对松墨天牛具有高毒力的Bt毒素
技术领域
本发明属于林业害虫的生物防治领域，具体涉及一种对松墨天牛具有高毒力的Bt毒素。
背景技术
松材线虫病是由松材线虫（Bursaphelenchus xylophilus）寄生于松树并引发松树萎焉而死亡的一种毁灭性病害。松材线虫病起源于北美洲，主要为美国和加拿大，在20世纪松材线虫病传入日本，接着在1970-1980年间传播到中国，朝鲜等亚洲国家，随后传入葡萄牙和西班牙等欧洲国家甚至尼日利亚和墨西哥等国家。松材线虫病是松属植物中最具破坏性的病害之一，在亚洲，其主要为害树种包括中国马尾松（Pinus massoniana）、日本黑松（P. thunbergii）以及日本红松（P. densiflora），而在欧洲主要危害海岸松（P. pinaster），且该病在世界范围内造成环境和经济损失达数千万美元。由于松材线虫病的为害，每年日本至少损失700,000 m3的松树。而该病自1982年传入我国江苏省南京市以来，在我国呈现快速扩散的态势，已扩散到18个省（区、市）、666个县级行政区，疫情发生面积达974万亩，直接威胁着我国近9亿亩松树的安全，且已经造成了上千亿元的经济损失和生态服务价值损失。
天牛科（Cerambycidae）墨天牛属（Monochamus）的昆虫是松材线虫的主要传播媒介，将松材线虫从枯死的松树上传播到活着的易感松树上。在我国，主要的传播媒介为松墨天牛，其也是主要的松树蛀干害虫，又被称为次期性害虫，生活在韧皮部或者木质部之中，具有很强的隐蔽性。目前，针对松墨天牛的防治手段主要有四种，即物理防治、化学防治、营林防治和生物防治。然而，这些防治方法各有优劣，对松墨天牛的防治未能达到最佳效果。其中生物防治具有绿色环保，持效期长的优点，进一步寻找有效的微生物制剂对松墨天牛进行生物防治，对于防控松材线虫病的扩散蔓延具有重要的意义。
苏云金芽胞杆菌（Bacillus thuringiensis，简称Bt）是目前应用最为广泛的生物农药，对16个目3000多种害虫有活性。苏云金芽胞杆菌的主要特征为在产生孢子期间能够合成晶体蛋白，这些晶体蛋白中的毒力因子为δ-内毒素，且这些毒素主要被分为两个家族包括Cry和Cyt。目前已发现的Cry毒素有759种，根据氨基酸序列的同源性高低，可以将其分为75类，不同类别的Cry毒素的杀虫谱表现出显著的特异性，分别对鳞翅目，鞘翅目，双翅目的昆虫表现出杀虫活性。在Cry毒素家族中，已有研究发现Cry3、Cry7、Cry8和Cry34/35类毒素对鞘翅目的幼虫有活性。其中，Cry3A是第一个分离到的对鞘翅目昆虫有杀虫活性的毒素，且已被应用于一些鞘翅目昆虫的防治。然而，由于Cry3A毒素对天牛科幼虫的低毒力使得防治效果并不理想。因此，为有效防治天牛科墨天牛属昆虫，迫切需要解决Cry3A毒素对其弱毒的问题。
在Cry3Aa毒素作用过程中，当其被昆虫摄取后，原毒素会被昆虫肠道蛋白酶酶解活化出55 kDa大小的活性片段，该片段对鞘翅目昆虫表现出杀虫活性（Walter et al.,2008; Li et al., 2011）。然后，单体的活性毒素寡聚化形成的低聚物与中肠上皮细胞刷状缘膜囊泡（BBNV）上的受体结合，从而引起膜穿孔并导致细胞死亡（Pardo-López et al.,2013）。而已有研究表明，Cry3Aa毒素在松墨天牛幼虫中肠的过度酶解是导致其弱毒的主要原因（Guo et al., 2020）。因此，为解决昆虫肠道蛋白酶对毒素的过度酶解现象，本发明以cry3Aa2基因为对象，通过分子改造的手段对毒素中的胰蛋白酶识别位点进行突变，开发能够产生对墨天牛属昆虫具有高毒力的新型Cry毒素，是一项具有重大意义和应用价值的研究。
发明内容
本发明的目的在于提供了一种对松墨天牛具有高毒力的Bt毒素，以SEQ ID NO.4（Cry3Aa2）为支架毒素蛋白，通过分子改造的手段对毒素中的胰蛋白酶识别位点进行突变，开发能够产生对墨天牛属昆虫具有高毒力的新型Cry毒素。
为实现上述目的，采用以下技术方案：
对松墨天牛具有高毒力的Bt毒素为毒素BRC2007蛋白，其氨基酸序列如SEQ ID NO.2所示。编码如所述Bt毒素的基因，所述基因其核苷酸序列如SEQ ID NO.1所示。
与在SEQ ID NO.4所列出的毒素Cry3Aa2蛋白的初始氨基酸序列相比，为克服昆虫肠道蛋白酶对毒素的过度酶解，本发明的BRC2007蛋白中包括了五个位点的氨基酸突变，将五个胰蛋白酶的识别位点突变为不被识别的丙氨酸，分别为：结构域I中在位置65处由赖氨酸置换成丙氨酸，在位置70处由赖氨酸置换成丙氨酸，在位置231处由赖氨酸置换成丙氨酸，结构域II中在位置468处由赖氨酸置换成丙氨酸，以及结构域III中在位置596处由赖氨酸置换成丙氨酸（图1）。
结构域I的基因序列和氨基酸序列分别如SEQ ID NO.5和SEQ ID NO.6所示；
结构域II基因序列和氨基酸序列分别如SEQ ID NO.7和SEQ ID NO.8所示；
结构域III基因序列和氨基酸序列分别如SEQ ID NO.9和SEQ ID NO.10所示。
所述的毒素BRC2007的三个结构域，这三个结构域中的任意一个结构域均可与Bt毒素的其他家族毒素的结构域任意组合获得对鞘翅目具有高毒力的毒素。
所述的Bt毒素的其他家族毒素，这些毒素均具有三级结构域，包括但不限于Cry3、Cry7、Cry8类毒素。
所述对墨天牛属昆虫具有高毒力的Bt毒素在防治天牛科墨天牛属昆虫中的应用。
所述墨天牛属昆虫包括松墨天牛（Monochamus alternatus enda、Monochamus alternatus alternatus）、云杉花墨天牛（Monochamus saltuarius）、云杉小墨天牛（Monochamus sutor）、卡来罗纳黑天牛（Monochamuscarolinensis）、密毛白点墨天牛（Monochamus scutellatus）和松墨斑墨天牛（Monochamus mutator）和樟子松墨天牛（Monochamus galloprovincialis）。
本发明的BRC2007晶体蛋白用于墨天牛属昆虫的生物测定。在具体实施例中，优选地用于防治松墨天牛（Monochamus alternatusalternatus）。
本发明的优点在于：
本发明的BRC2007毒素与SEQ ID NO. 4所示的毒素Cry3Aa2蛋白在体外酶解活化效率、杀虫活性和目标昆虫种类谱相比，BRC2007毒素能够克服被昆虫肠道蛋白酶过度酶解的现象，即酶解活化出更多的55 KDa大小的活性片段，从而提高了对目标昆虫的杀虫活性。
附图说明
图1为BRC2007与Cry3Aa2蛋白氨基酸序列的比对。
图2为BRC2007基因克隆到pHT304-G载体上。
图3为pHT304-G穿梭载体图谱。
图4为BRC2007晶体蛋白的SDS-PAGE的结果图。其中泳道1为蛋白Marker，泳道2为BRC2007晶体蛋白。
具体实施方式
以下叙述本发明的实施例。应说明的是，本发明的实施例对于本发明只有说明作用，并不对本发明的范围构成任何限制。任何熟悉本领域的技术人员，在不脱离本发明技术方案的情况下，对本发明做出改进，变动或修饰均等同于等效实施案例，均属于本发明所附权力要求保护范围内。
实施例1 BRC2007基因的克隆
对cry3Aa2基因上五位点进行氨基酸的突变获得BRC2007基因序列，分别为：在位置65处由赖氨酸（K）置换成丙氨酸（A），在位置70处由赖氨酸（K）置换成丙氨酸（A），在位置231处由赖氨酸（K）置换成丙氨酸（A），在位置468处由赖氨酸（K）置换成丙氨酸（A），以及在位置596处由赖氨酸（K）置换成丙氨酸（A）（图1）。生物合成的BRC2007基因（序列如SEQ ID NO.1所示）直接通过XhoI和BamHI酶切位点利用T4连接酶克隆到穿梭载体pHT304-G中。具体步骤如下：
目的片段双酶切体系 pHT304-G表达载体双酶切体系
10×Cutsmart buffer 2 μL 10×Cutsmart buffer 2 μL
BamHI 1 μL BamHI 1 μL
XhoI 1 μL XhoI 1 μL
目的质粒 6 μL pHT304-G质粒 6 μL
ddw 10 μL ddw 10 μL
Total 20 μL Total 20 μL
黏性末端双酶切处理：将上述所加试剂于冰上溶解后按照顺序依次加入到200 μL的PCR管中，最后加入Phusion DNA聚合酶，然后混匀，放入PCR仪，37℃反应1 h。
酶切产物切胶回收纯化：酶切后的产物包括线性DNA片段以及线性化pHT304-G载体片段利用OMEGA的Gel Extraction Kit试剂盒进行产物纯化。
T4连接体系：将上一步纯化回收的酶切的线性DNA片段以及线性化的pHT304-G载体利用T4连接酶进行连接，然后反应体系所加试剂按照如下顺序依次加入到200 μL的PCR管中，然后混匀，放入PCR仪，25℃，30min，65℃，30min，最后4℃保存。连接体系如下：
10×T4 Ligase buffer 1 μL
目的基因全长（双酶切处理后） 5 μL
pHT304-G载体（双酶切处理后） 1 μL
T4 DNA Ligase 1 μL
ddw 2 μL
Total 10 μL
通过以上步骤最终获得含有BRC2007基因的pHT304-G载体（图2）。
实施例2穿梭载体pHT304-G的构建
以苏云金杆菌/大肠杆菌的pHT304穿梭载体为基础，设计合成cry3Aa2基因的启动子，包括启动cyt1A基因的BtI和BtII启动序列，STAB-SD序列以及多克隆位点序列（XhoI，SphI，XbaI，BamHI，KpnI和SacI）。该合成片段通过HindIII和EcoRI酶切位点连入pHT304质粒中（图3），最终获得穿梭载体pHT304-G。
实施例3 BRC2007毒素在苏云金芽胞杆菌中的表达及纯化
将毒素BRC2007利用电转化方法转入无晶体突变株HD73Cry-中，通过挑取单菌落在LB培养基中30℃培养过夜，然后保存甘油菌，即为BRC2007菌液。
其中Bt电击转化具体方法为：
1）2 mm电击杯使用前置于4℃冰箱中。
2）10 μL质粒加入到200 μL感受态细菌中，混合均匀，冰浴10 min。
3）冰浴后进行电击转化，条件为：电压2.5 kv，电阻200 Ω，电容25 μF。
4）电击结束后加1800 μL的LB培养液，混匀后转入1.5 mL离心管，置于30℃摇床中慢速培养3 h。
5）取适量菌液于含有10 μg/mL红霉素（Em）的LB固体培养基上，30℃培养至长出单菌落。
6）挑斑并在1/2 LB平板上划线30℃培养。
7）三天半后镜检挑出晶体较多的保存甘油菌。
将BRC2007菌液接入PM培养基（酵母粉2 g/L，胰蛋白胨10 g/L，KH2PO4 1 g/L，FeSO4•7H2O 0.02 g/L，MgSO4•7H2O 0.3 g/L，MnSO4•7H2O 0.02 g/L，ZnSO4•7H2O 0.02 g/L，pH 7.0）中30℃培养至晶体释放期（60 h），经过受体菌体，晶体蛋白的溶解后，利用盐酸将pH调至4.5，离心收集沉淀，获得BRC2007晶体蛋白（图4），其序列如SEQ ID NO.2所示。
实施例4 BRC2007毒素对松墨天牛（Monochamus alternatusalternatus）的生物测定
天牛人工饲料的配方：麸皮60 g，虾粉10 g，山梨酸2 g，苯钾酸钠4 g，酵母粉25 g，琼脂30 g，松树韧皮部100 g，松树木质部50 g，蔗糖40 g，水300 mL。
生物测定方法：松墨天牛幼虫分别培养在含有0.1g人工饲料的500 µL离心管中。每一支离心管对应一只三龄初期幼虫，分别加入150 µL不同浓度梯度的Cry3Aa2原毒素和BRC2007毒素（200、100、50、25、10和5 µg/mL)。各浓度梯度三组重复，每组含有10只幼虫作为重复（各浓度梯度测试30只）。以单蒸水作为空白对照。所有离心管放置在25℃、75%湿度的环境下培养，并对其进行光周期16:8处理（光照培养16小时，黑暗培养8小时）。观察各组幼虫的生长状况并在两周后计算各组幼虫死亡率，运用概率单位分析计算出致死中浓度（LC50）。BRC2007毒素对松墨天牛三龄初期幼虫两周的LC50值为10.2 μg/mL，与Cry3Aa2原始毒素相比，杀虫活性提高了11.5倍（详见表1）。
表1 Cry3Aa2和BRC2007毒素对松墨天牛幼虫的杀虫活性
Figure 692756DEST_PATH_IMAGE001
SEQUENCE LISTING
序 列 表
<110> 福建农林大学
<120> 对松墨天牛具有高毒力的Bt毒素
<130>
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 1945
<212> DNA
<213> 苏云金杆菌（Bacillus thuringiensis）人工合成
<400> 1
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ccaactaacc atgttcaata tcctttagcg gaaactccaa atccaacact agaagattta 120
aattataaag agtttttaag aatgactgca gataataata cggaagcact agatagctct 180
acaacagcag atgtcattca agcaggcatt tccgtagtag gtgatctcct aggcgtagta 240
ggtttcccgt ttggtggagc gcttgtttcg ttttatacaa actttttaaa tactatttgg 300
ccaagtgaag acccgtggaa ggcttttatg gaacaagtag aagcattgat ggatcagaaa 360
atagctgatt atgcaaaaaa taaagctctt gcagagttac agggccttca aaataatgtc 420
gaagattatg tgagtgcatt gagttcatgg caaaaaaatc ctgtgagttc acgaaatcca 480
catagccagg ggcggataag agagctgttt tctcaagcag aaagtcattt tcgtaattca 540
atgccttcgt ttgcaatttc tggatacgag gttctatttc taacaacata tgcacaagct 600
gccaacacac atttattttt actaaaagac gctcaaattt atggagaaga atggggatac 660
gaaaaagaag atattgctga attttatgca agacaactaa aacttacgca agaatatact 720
gaccattgtg tcaaatggta taatgttgga ttagataaat taagaggttc atcttatgaa 780
tcttgggtaa actttaaccg ttatcgcaga gagatgacat taacagtatt agatttaatt 840
gcactatttc cattgtatga tgttcggcta tacccaaaag aagttaaaac cgaattaaca 900
agagacgttt taacagatcc aattgtcgga gtcaacaacc ttaggggcta tggaacaacc 960
ttctctaata tagaaaatta tattcgaaaa ccacatctat ttgactatct gcatagaatt 1020
caatttcaca cgcggttcca accaggatat tatggaaatg actctttcaa ttattggtcc 1080
ggtaattatg tttcaactag accaagcata ggatcaaatg atataatcac atctccattc 1140
tatggaaata aatccagtga acctgtacaa aatttagaat ttaatggaga aaaagtctat 1200
agagccgtag caaatacaaa tcttgcggtc tggccgtccg ctgtatattc aggtgttaca 1260
aaagtggaat ttagccaata taatgatcaa acagatgaag caagtacaca aacgtacgac 1320
tcaaaaagaa atgttggcgc ggtcagctgg gattctatcg atcaattgcc tccagaaaca 1380
acagatgaac ctctagaagc gggatatagc catcaactca attatgtaat gtgcttttta 1440
atgcagggta gtagaggaac aatcccagtg ttaacttgga cacataaaag tgtagacttt 1500
tttaacatga ttgattcgaa aaaaattaca caacttccgt tagtaaaggc atataagtta 1560
caatctggtg cttccgttgt cgcaggtcct aggtttacag gaggagatat cattcaatgc 1620
acagaaaatg gaagtgcggc aactatttac gttacaccgg atgtgtcgta ctctcaaaaa 1680
tatcgagcta gaattcatta tgcttctaca tctcagataa catttacact cagtttagac 1740
ggggcaccat ttaatcaata ctatttcgat aaaacgataa atgcaggaga cacattaacg 1800
tataattcat ttaatttagc aagtttcagc acaccattcg aattatcagg gaataactta 1860
caaataggcg tcacaggatt aagtgctgga gataaagttt atatagacaa aattgaattt 1920
attccagtga attaatgagg atcca 1945
<210> 2
<211> 646
<212> PRT
<213> 苏云金杆菌（Bacillus thuringiensis）人工合成
<400> 2
Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Thr Thr Glu
1 5 10 15
Asn Asn Glu Val Pro Thr Asn His Val Gln Tyr Pro Leu Ala Glu Thr
20 25 30
Pro Asn Pro Thr Leu Glu Asp Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45
Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Ala Asp
50 55 60
Val Ile Gln Ala Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val
65 70 75 80
Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu
85 90 95
Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu Gln
100 105 110
Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys
115 120 125
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val
130 135 140
Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn Pro
145 150 155 160
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His
165 170 175
Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu
180 185 190
Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu
195 200 205
Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp
210 215 220
Ile Ala Glu Phe Tyr Ala Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr
225 230 235 240
Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly
245 250 255
Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met
260 265 270
Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val
275 280 285
Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu
290 295 300
Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr
305 310 315 320
Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr
325 330 335
Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly
340 345 350
Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro
355 360 365
Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
370 375 380
Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr
385 390 395 400
Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr
405 410 415
Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp
420 425 430
Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val
435 440 445
Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro
450 455 460
Leu Glu Ala Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu
465 470 475 480
Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys
485 490 495
Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu
500 505 510
Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala
515 520 525
Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly
530 535 540
Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys
545 550 555 560
Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr
565 570 575
Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr
580 585 590
Ile Asn Ala Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser
595 600 605
Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly Val
610 615 620
Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe
625 630 635 640
Ile Pro Val Asn Gly Ser
645
<210> 3
<211> 1952
<212> DNA
<213> 苏云金杆菌（Bacillus thuringiensis）
<400> 3
tccatggatg aatccgaaca atcgaagtga acatgataca ataaaaacta ctgaaaataa 60
tgaggtgcca actaaccatg ttcaatatcc tttagcggaa actccaaatc caacactaga 120
agatttaaat tataaagagt ttttaagaat gactgcagat aataatacgg aagcactaga 180
tagctctaca acaaaagatg tcattcaaaa aggcatttcc gtagtaggtg atctcctagg 240
cgtagtaggt ttcccgtttg gtggagcgct tgtttcgttt tatacaaact ttttaaatac 300
tatttggcca agtgaagacc cgtggaaggc ttttatggaa caagtagaag cattgatgga 360
tcagaaaata gctgattatg caaaaaataa agctcttgca gagttacagg gccttcaaaa 420
taatgtcgaa gattatgtga gtgcattgag ttcatggcaa aaaaatcctg tgagttcacg 480
aaatccacat agccaggggc ggataagaga gctgttttct caagcagaaa gtcattttcg 540
taattcaatg ccttcgtttg caatttctgg atacgaggtt ctatttctaa caacatatgc 600
acaagctgcc aacacacatt tatttttact aaaagacgct caaatttatg gagaagaatg 660
gggatacgaa aaagaagata ttgctgaatt ttataaaaga caactaaaac ttacgcaaga 720
atatactgac cattgtgtca aatggtataa tgttggatta gataaattaa gaggttcatc 780
ttatgaatct tgggtaaact ttaaccgtta tcgcagagag atgacattaa cagtattaga 840
tttaattgca ctatttccat tgtatgatgt tcggctatac ccaaaagaag ttaaaaccga 900
attaacaaga gacgttttaa cagatccaat tgtcggagtc aacaacctta ggggctatgg 960
aacaaccttc tctaatatag aaaattatat tcgaaaacca catctatttg actatctgca 1020
tagaattcaa tttcacacgc ggttccaacc aggatattat ggaaatgact ctttcaatta 1080
ttggtccggt aattatgttt caactagacc aagcatagga tcaaatgata taatcacatc 1140
tccattctat ggaaataaat ccagtgaacc tgtacaaaat ttagaattta atggagaaaa 1200
agtctataga gccgtagcaa atacaaatct tgcggtctgg ccgtccgctg tatattcagg 1260
tgttacaaaa gtggaattta gccaatataa tgatcaaaca gatgaagcaa gtacacaaac 1320
gtacgactca aaaagaaatg ttggcgcggt cagctgggat tctatcgatc aattgcctcc 1380
agaaacaaca gatgaacctc tagaaaaggg atatagccat caactcaatt atgtaatgtg 1440
ctttttaatg cagggtagta gaggaacaat cccagtgtta acttggacac ataaaagtgt 1500
agactttttt aacatgattg attcgaaaaa aattacacaa cttccgttag taaaggcata 1560
taagttacaa tctggtgctt ccgttgtcgc aggtcctagg tttacaggag gagatatcat 1620
tcaatgcaca gaaaatggaa gtgcggcaac tatttacgtt acaccggatg tgtcgtactc 1680
tcaaaaatat cgagctagaa ttcattatgc ttctacatct cagataacat ttacactcag 1740
tttagacggg gcaccattta atcaatacta tttcgataaa acgataaata aaggagacac 1800
attaacgtat aattcattta atttagcaag tttcagcaca ccattcgaat tatcagggaa 1860
taacttacaa ataggcgtca caggattaag tgctggagat aaagtttata tagacaaaat 1920
tgaatttatt ccagtgaatt aatgaggatc ca 1952
<210> 4
<211> 644
<212> PRT
<213> 苏云金杆菌（Bacillus thuringiensis）
<400> 4
Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Thr Thr Glu
1 5 10 15
Asn Asn Glu Val Pro Thr Asn His Val Gln Tyr Pro Leu Ala Glu Thr
20 25 30
Pro Asn Pro Thr Leu Glu Asp Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45
Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys Asp
50 55 60
Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val
65 70 75 80
Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu
85 90 95
Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu Gln
100 105 110
Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys
115 120 125
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val
130 135 140
Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn Pro
145 150 155 160
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His
165 170 175
Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu
180 185 190
Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu
195 200 205
Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp
210 215 220
Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr
225 230 235 240
Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly
245 250 255
Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met
260 265 270
Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val
275 280 285
Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu
290 295 300
Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr
305 310 315 320
Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr
325 330 335
Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly
340 345 350
Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro
355 360 365
Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
370 375 380
Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr
385 390 395 400
Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr
405 410 415
Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp
420 425 430
Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val
435 440 445
Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro
450 455 460
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu
465 470 475 480
Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys
485 490 495
Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu
500 505 510
Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala
515 520 525
Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly
530 535 540
Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys
545 550 555 560
Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr
565 570 575
Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr
580 585 590
Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser
595 600 605
Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly Val
610 615 620
Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe
625 630 635 640
Ile Pro Val Asn
<210> 5
<211> 681
<212> DNA
<213> 人工序列
<400> 5
acaacagcag atgtcattca agcaggcatt tccgtagtag gtgatctcct aggcgtagta 60
ggtttcccgt ttggtggagc gcttgtttcg ttttatacaa actttttaaa tactatttgg 120
ccaagtgaag acccgtggaa ggcttttatg gaacaagtag aagcattgat ggatcagaaa 180
atagctgatt atgcaaaaaa taaagctctt gcagagttac agggccttca aaataatgtc 240
gaagattatg tgagtgcatt gagttcatgg caaaaaaatc ctgtgagttc acgaaatcca 300
catagccagg ggcggataag agagctgttt tctcaagcag aaagtcattt tcgtaattca 360
atgccttcgt ttgcaatttc tggatacgag gttctatttc taacaacata tgcacaagct 420
gccaacacac atttattttt actaaaagac gctcaaattt atggagaaga atggggatac 480
gaaaaagaag atattgctga attttatgca agacaactaa aacttacgca agaatatact 540
gaccattgtg tcaaatggta taatgttgga ttagataaat taagaggttc atcttatgaa 600
tcttgggtaa actttaaccg ttatcgcaga gagatgacat taacagtatt agatttaatt 660
gcactatttc cattgtatga t 681
<210> 6
<211> 227
<212> PRT
<213> 人工序列
<400> 6
Thr Thr Ala Asp Val Ile Gln Ala Gly Ile Ser Val Val Gly Asp Leu
1 5 10 15
Leu Gly Val Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr
20 25 30
Thr Asn Phe Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala
35 40 45
Phe Met Glu Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr
50 55 60
Ala Lys Asn Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val
65 70 75 80
Glu Asp Tyr Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser
85 90 95
Ser Arg Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln
100 105 110
Ala Glu Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly
115 120 125
Tyr Glu Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His
130 135 140
Leu Phe Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr
145 150 155 160
Glu Lys Glu Asp Ile Ala Glu Phe Tyr Ala Arg Gln Leu Lys Leu Thr
165 170 175
Gln Glu Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp
180 185 190
Lys Leu Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr
195 200 205
Arg Arg Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro
210 215 220
Leu Tyr Asp
225
<210> 7
<211> 615
<212> DNA
<213> 人工序列
<400> 7
gttaaaaccg aattaacaag agacgtttta acagatccaa ttgtcggagt caacaacctt 60
aggggctatg gaacaacctt ctctaatata gaaaattata ttcgaaaacc acatctattt 120
gactatctgc atagaattca atttcacacg cggttccaac caggatatta tggaaatgac 180
tctttcaatt attggtccgg taattatgtt tcaactagac caagcatagg atcaaatgat 240
ataatcacat ctccattcta tggaaataaa tccagtgaac ctgtacaaaa tttagaattt 300
aatggagaaa aagtctatag agccgtagca aatacaaatc ttgcggtctg gccgtccgct 360
gtatattcag gtgttacaaa agtggaattt agccaatata atgatcaaac agatgaagca 420
agtacacaaa cgtacgactc aaaaagaaat gttggcgcgg tcagctggga ttctatcgat 480
caattgcctc cagaaacaac agatgaacct ctagaagcgg gatatagcca tcaactcaat 540
tatgtaatgt gctttttaat gcagggtagt agaggaacaa tcccagtgtt aacttggaca 600
cataaaagtg tagac 615
<210> 8
<211> 205
<212> PRT
<213> 人工序列
<400> 8
Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr Asp Pro Ile Val Gly
1 5 10 15
Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe Ser Asn Ile Glu Asn
20 25 30
Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu His Arg Ile Gln Phe
35 40 45
His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn Asp Ser Phe Asn Tyr
50 55 60
Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser Ile Gly Ser Asn Asp
65 70 75 80
Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser Ser Glu Pro Val Gln
85 90 95
Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg Ala Val Ala Asn Thr
100 105 110
Asn Leu Ala Val Trp Pro Ser Ala Val Tyr Ser Gly Val Thr Lys Val
115 120 125
Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu Ala Ser Thr Gln Thr
130 135 140
Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser Trp Asp Ser Ile Asp
145 150 155 160
Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu Ala Gly Tyr Ser
165 170 175
His Gln Leu Asn Tyr Val Met Cys Phe Leu Met Gln Gly Ser Arg Gly
180 185 190
Thr Ile Pro Val Leu Thr Trp Thr His Lys Ser Val Asp
195 200 205
<210> 9
<211> 429
<212> DNA
<213> 人工序列
<400> 9
aacatgattg attcgaaaaa aattacacaa cttccgttag taaaggcata taagttacaa 60
tctggtgctt ccgttgtcgc aggtcctagg tttacaggag gagatatcat tcaatgcaca 120
gaaaatggaa gtgcggcaac tatttacgtt acaccggatg tgtcgtactc tcaaaaatat 180
cgagctagaa ttcattatgc ttctacatct cagataacat ttacactcag tttagacggg 240
gcaccattta atcaatacta tttcgataaa acgataaatg caggagacac attaacgtat 300
aattcattta atttagcaag tttcagcaca ccattcgaat tatcagggaa taacttacaa 360
ataggcgtca caggattaag tgctggagat aaagtttata tagacaaaat tgaatttatt 420
ccagtgaat 429
<210> 10
<211> 143
<212> PRT
<213> 人工序列
<400> 10
Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro Leu Val Lys Ala
1 5 10 15
Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala Gly Pro Arg Phe Thr
20 25 30
Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly Ser Ala Ala Thr Ile
35 40 45
Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys Tyr Arg Ala Arg Ile
50 55 60
His Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr Leu Ser Leu Asp Gly
65 70 75 80
Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr Ile Asn Ala Gly Asp
85 90 95
Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser Phe Ser Thr Pro Phe
100 105 110
Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly Val Thr Gly Leu Ser Ala
115 120 125
Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Asn
130 135 140

Claims (5)

1.对松墨天牛具有高毒力的Bt毒素，其特征在于，所述Bt毒素为毒素BRC2007，其氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的对松墨天牛具有高毒力的Bt毒素，其特征在于，其在选自SEQID NO.4所示的骨架蛋白中的三个结构域中至少包含一个氨基酸的取代。
3.根据权利要求2所述的对松墨天牛具有高毒力的Bt毒素，其特征在于，所述氨基酸的取代具体为：
（1）结构域I中氨基酸取代：在结构域I的位置65处由赖氨酸置换成丙氨酸，在位置70处由赖氨酸置换成丙氨酸，在位置231处由赖氨酸置换成丙氨酸，结构域I的基因序列和氨基酸序列分别如SEQ ID NO.5和SEQ ID NO.6所示；
（2）结构域II中氨基酸取代：在结构域II位置468处由赖氨酸置换成丙氨酸，结构域II基因序列和氨基酸序列分别如SEQ ID NO.7和SEQ ID NO.8所示；
（3）结构域III中氨基酸取代：在结构域III位置596处由赖氨酸置换成丙氨酸，结构域III基因序列和氨基酸序列分别如SEQ ID NO.9和SEQ ID NO.10所示。
4.一种编码如权利要求1所述Bt毒素的基因，其特征在于，所述基因其核苷酸序列如SEQ ID NO.1所示。
5.如权利要求1所述的对松墨天牛具有高毒力的Bt毒素在防治松墨天牛中的应用。
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