CN101210252B - 鳞翅目害虫基质金属蛋白酶基因mmp及其应用 - Google Patents
鳞翅目害虫基质金属蛋白酶基因mmp及其应用 Download PDFInfo
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- CN101210252B CN101210252B CN2007101648100A CN200710164810A CN101210252B CN 101210252 B CN101210252 B CN 101210252B CN 2007101648100 A CN2007101648100 A CN 2007101648100A CN 200710164810 A CN200710164810 A CN 200710164810A CN 101210252 B CN101210252 B CN 101210252B
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Abstract
本发明公开了一种鳞翅目害虫基质金属蛋白酶基因mmp,其具有SEQ ID No:1所示的核苷酸序列。本发明还公开了上述基因mmp编码的蛋白质,其具有SEQ ID NO:2所示的氨基酸序列。本发明还公开了上述基因mmp的用途:利用该基因物调节鳞翅目害虫的***反应。
Description
技术领域
本发明属于昆虫生物技术领域,具体的说涉及茶尺蠖基质金属蛋白酶基因的分离,以及利用该基因物调节鳞翅目害虫的***反应。
背景技术
茶尺蠖等鳞翅目昆虫是重要的茶树害虫种类之一,对这些害虫的有效控制是茶叶高产优质的根本保证。但是随着现有农药使用频度和剂量的增加,鳞翅目害虫的抗药性逐渐增强,使防治效果下降。因此需要开发一些针对性强、作用位点明确、防效好的新农药。
基质金属蛋白酶(matrix metalloproteinase,mmp)是生物降解细胞外基质成分的最主要蛋白水解***,在组织重构中起重要作用。mmp是一类锌、钙依赖性的蛋白水解酶家族,是细胞外基质降解的主要酶类,MMPs在体内的表达、激活及对底物的分解都受到严格的调控。已有的研究显示,mmp的表达与活性与脊椎动物的免疫行为息息相关,它们在肿瘤细胞侵袭转移中起关键性作用,还与细胞的凋亡有关。但是现有对茶尺蠖等鳞翅目害虫的该基因及其产物的研究很少。
发明内容
本发明要解决的技术问题是提供一种鳞翅目害虫基质金属蛋白酶基因mmp及其编码的蛋白质,该基因物能用于调节鳞翅目害虫的***反应。
为了解决上述技术问题,本发明提供一种鳞翅目害虫基质金属蛋白酶基因mmp,其具有SEQ ID No:1所示的核苷酸序列。
本发明还提供了上述基因mmp编码的蛋白质,其具有SEQ ID NO:2所示的氨基酸序列。
本发明还提供了上述基因mmp的用途,利用该基因物调节鳞翅目害虫的***反应。
作为本发明的基因mmp的用途的改进:将根据SEQ ID No:1所示的核苷酸序列设计的mmp siRNA注射入鳞翅目害虫体内,使内源mmp表达下调,达到鳞翅目害虫***过程出现严重障碍。
作为本发明的基因mmp的用途的进一步改进:鳞翅目害虫为茶尺蠖。
本发明通过分子生物学技术手段分离和分析与鳞翅目害虫***反应密切相关的基因,并明确这些基因的生理功能,提出鳞翅目害虫农药设计的新作用位点。本发明是采用cDNA-AFLP技术分离与鳞翅目害虫与***反应有密切关系的基因mmp;所得的基质金属蛋白酶与鳞翅目害虫***过程中基质水解的再利用相关。
实现本发明的具体技术步骤如下:
一、茶树鳞翅目害虫mmp基因片段分离和分析
取进食期(feeding stage)和休眠期(wandering stage)的茶尺蠖幼虫若干头。以TRIZOL(Invitrogen公司)试剂提取总RNA,并以UNIQ-10柱式mRNA抽提纯化试剂盒(上海生工生物工程有限公司)分离mRNA。分别取1-5μg mRNA进行cDNA合成,双链cDNA合成采用TakaRa MMLV RTase cDNA Synthesis Kit(宝生物工程[大连]有限公司)进行。双链cDNA经异丙醇沉淀纯化后,以TakaRa Afl II/Nsp I(宝生物工程[大连]有限公司)组合酶切消化。消化后的片段与事先设计的2个接头SEQ ID No:3和SEQ ID No:4,以T4 DNA连接酶(上海生工生物工程有限公司)连接。以连接完成的cDNA片段为模板,以预扩增引物SEQID No:5和SEQ ID No:6进行PCR预扩增,具体扩增条件见表1第1列,将预扩增产物适当稀释后,以选择性扩增引物组合SEQ ID No:7和SEQ ID No:8,以及SEQ ID No:9和SEQ IDNo:10,分别进行PCR选择性扩增,具体扩增条件见表1第2列。扩增产物以6%变性聚丙烯酰胺凝胶进行电泳分析,以核酸银染试剂盒(上海生工生物工程有限公司)进行显带。
从凝胶上割取在进食期无表达、而在休眠区有强表达的对应条带。割取目标带,以纯水浸提过夜后,取适量浸提液作为模板,以选择性引物组合进行PCR,产物进行电泳。电泳结束后割取目标条带,以UNIQ-10柱式DNA胶回收试剂盒(上海生工生物工程有限公司)进行片段回收,并***pUCm-T载体(上海生工生物工程有限公司),阳性克隆委托Invitrogen公司进行序列分析,确定了其中一个726bp的显著差异序列与茶尺蠖***密切相关的片断,其核苷酸顺序见SEQ ID No:1,经与美国生物信息数据库http://www. ncbi.nlm.nih.gov比对,证实该序列与果蝇、蜜蜂等基质金属蛋白酶具有很高同源性。因此定名为茶尺蠖mmp。并采用Editseq(DNAStar公司)软件进行推演,获得该核苷酸序列的氨基酸顺序,见SEQ ID No:2。
表1
PCR预扩增 | PCR选择性扩增 | PCR常规扩增 |
94℃变性30秒;56℃退火60秒;72℃延伸60秒;共30个循环。 | 94℃变性30秒;65℃退火30秒;72℃延伸60秒;1个循环。94℃变性30秒;65℃退火30秒,并以每循环下降0.7℃;72℃延伸60秒;12个循环。94℃变性30秒;56℃退火30秒;72℃延伸60秒;23个循环。 | 94℃变性4分钟;一个循环。94℃变性30秒;55℃退火30秒;72℃延伸60秒;35个循环。72℃延伸5分钟。 |
注:本发明中未做特别说明的PCR或PCR扩增均指PCR常规扩增
二、茶树鳞翅目害虫mmp基因表达***行为的关系
以茶尺蠖mmp(SEQ ID No:1)为材料,采用TaKaRa siRNA Cocktail Kit(si-RNAaseIII TM)(宝生物工程[大连]有限公司)制备可与茶尺蠖内源mmp发生双链干涉作用的mmp-siRNAs。将刚刚进入前蛹阶段的茶尺蠖分成2组,一组用于mmp-siRNAs处理,另一组对照。将制备的mmp-siRNAs用水溶解后,在前蛹胸腹部注射mmp-siRNAs,注射剂量为每头lng-1000ng,同时以注射等量纯水为对照。注射处理24-48小时后,分别在处理组、对照组中选取2-3头,以TRIZOL(Invitrogen公司)进行总RNA提取。以SEQ ID No:11和SEQ ID No:12为引物,以MMLV一步法RT-PCR扩增试剂盒进行RT-PCR,具体操作参照试剂盒使用说明。结果表明,注射mmp基因双链干涉siRNAs的个体,其内源的mmp基因表达显著受到抑制。形态观察分析显示,注射过mmp-siRNAs的前蛹化蛹的比例显著低于对照。本发明证明茶尺蠖内源mp基因的正常表达对茶尺蠖虫态变化极为重要,该mmp基因及其产物是茶尺蠖等害虫新型农药研发和设计中潜在作用位点。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1是实施例1中的扩增产物电泳分析后,以核酸银染试剂盒进行显带所得的图;
注:M为分子量标记,1为进食期,2为进食间隔休眠期。
图2是实施例2中注射mmp基因双链干涉siRNAs后,个体内源的mmp基因表达图;
注:本图示mmp-siRNA干涉处理后茶尺蠖前蛹内源性mmp的表达下降;目标条带分子量为725bp;M分子量标记,1为mmp-siRNA处理的前蛹,2为对照。
图3是实施例2中由两种不同的前蛹***反应成蛹粒的图;
注:图中左边蛹粒由注射纯水的前蛹***发育而成;右边为注射mmp-siRNA的前蛹发育形成非正常蛹粒。
具体实施方式
实施例1
取3龄进食期和3龄期末的休眠期的茶尺蠖幼虫各2-3头。以TRIZOL(Invitrogen公司)试剂提取总RNA,并以UNIQ-10柱式mRNA抽提纯化试剂盒(上海生工生物工程有限公司)分离mRNA。分别取1μg mRNA进行cDNA合成,双链cDNA合成采用TakaRa MMLV RTase cDNASynthesis Kit(宝生物工程[大连]有限公司)进行。双链cDNA经异丙醇沉淀纯化后,以TakaRa Afl II/Nsp I(宝生物工程[大连]有限公司)组合酶切消化。消化后的片段与事先设计的2个接头SEQ ID No:3和SEQ ID No:4,以T4 DNA连接酶(上海生工生物工程有限公司)连接。以连接完成的cDNA片段为模板,以预扩增引物SEQ ID No:5和SEQ ID No:6进行PCR预扩增,将预扩增产物20倍稀释后,以选择性扩增引物组合SEQ ID No:7和SEQ IDNo:8,以及SEQ ID No:9和SEQ ID No:10,分别进行PCR选择性扩增。扩增产物以6%变性聚丙烯酰胺凝胶进行电泳分析,以核酸银染试剂盒(上海生工生物工程有限公司)进行显带,见图1。
从凝胶上割取在进食期无表达、而在休眠区有强表达的对应条带。割取目标带,以纯水浸提过夜后,取适量浸提液作为模板,以选择性引物组合SEQ ID No:7和SEQ ID No:8进行PCR,产物进行电泳。电泳结束后割取目标条带,以UNIQ-10柱式DNA胶回收试剂盒(上海生工生物工程有限公司)进行片段回收,并***pUCm-T载体(上海生工生物工程有限公司),阳性克隆委托Invitrogen公司进行序列分析,确定了其中一个726bp的显著差异序列与茶尺蠖虫态变换密切相关的片断,其核苷酸顺序见SEQ ID No:1,经与美国生物信息数据库http://www.ncbi.nlm.nih.gov比对,证实该序列与果蝇、蜜蜂等基质金属蛋白酶具有70以上同源性。并采用Editseq(DNAStar公司)软件进行推演,获得该核苷酸序列的氨基酸顺序,见SEQ ID No:2,该序列与蜜蜂等的基质金属蛋白酶具有75%左右的同源性。因此定名为茶尺蠖mmp。
实施例2
以茶尺蠖mmp SEQ ID No:1为材料,采用TaKaRa siRNA Cocktail Kit(si-RNAase IIITM)(宝生物工程[大连]有限公司)制备可与鳞翅目害虫内源mmp发生双链干涉作用的mmp-siRNAs。将刚刚进入前蛹阶段的茶尺蠖分成2组,一组用于mmp-siRNAs处理,另一组对照。将制备的mmp-siRNAs用水溶解后,在前蛹胸腹部注射mmp-siRNAs,注射剂量为150ng(体积为2μL)/头,即每头注射150ng的mmp-siRNAs;同时以注射等体积纯水为对照。注射处理48小时,分别在处理组、对照组中选取2头,以TRIZOL(Invitrogen公司)进行总RNA提取。以SEQ ID No:10和SEQ ID No:11为引物,以MMLV一步法RT-PCR扩增试剂盒进行RT-PCR,具体操作参照试剂盒说明。结果表明,注射mmp基因双链干涉siRNAs的个体,其内源的mmp基因表达显著受到抑制,不足对照的10%,见图2。形态观察分析显示,3-5天后,注射过mmp-siRNAs的前蛹化蛹比例较对照低50%以上,而且主要表现为注射部位不能正常的***反应,见图3。可见利用干涉技术使前蛹mmp表达受到抑制后,前蛹的***反应出现障碍。本发明证明茶尺蠖内源mmp基因的正常表达对茶尺蠖虫态变化极为重要,该mmp基因及其产物是茶尺蠖等害虫新型农药研发和设计中潜在作用位点。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
序列表
SEQ ID No:1
cttaagcggt ggaagagcat cgcatcgcca caaccacaaa cacaactatc ggccaatcag 60
tcctacctat ccacgagaac gagaacccag cagaaatcct acgtattacc ctacaagaca 120
ctacccgaat atcccgaact atccagagcg acctagttat tacccgacac gacatcatta 180
caatgcatcg gaggagtatc cgagaaggac taatccaacc tattatcctc gacctactcc 240
tgagaccacc acgcctccta caacataccg acctcgttat ccagtagatc gttctgatta 300
cccgtataac aagcctaatt atccaactga ctccaggcca agttacccaa gaaaacctta 360
ctatccagag aagacaacca caacaaaacc agctcctaca tcaccagccg atatacctga 420
ggcttgtgac accagttacg acgctgtggc tcttatacga aatgaactgt tcatcttcaa 480
aggcaaatat cattggagaa taggagcgcg cggcaaatat gatgggtacc caatggaaat 540
cagaagaatg tggattgggc ttccgagaga tcttacgcac gtagatgctg tttatgagcg 600
accagatcag aatatagcta tttttgttgg aaaacggctg tacttattca atataagaga 660
gttgttacct ggatacccga aaccgctcac ctccctcgta ccccaaatcg tcgacctgca 720
ggcatg 726
SEQ ID No:2
Leu Ser Gly Gly Arg Ala Ser His Arg His Asn His Lys His Asn
1 5 10 15
Tyr Arg Pro Ile Ser Pro Thr Tyr Pro Arg Glu Arg Glu Pro Ser
20 25 30
Arg Asn Pro Thr Tyr Tyr Pro Thr Arg His Tyr Pro Asn Ile Pro
35 40 45
Asn Tyr Pro Glu Arg Pro Ser Tyr Tyr Pro Thr Arg His His Tyr
50 55 60
Asn Ala Ser Glu Glu Tyr Pro Arg Arg Thr Asn Pro Thr Tyr Tyr
65 70 75
Pro Arg Pro Thr Pro Glu Thr Thr Thr Pro Pro Thr Thr Tyr Arg
80 85 90
Pro Arg Tyr Pro Val Asp Arg Ser Asp Tyr Pro Tyr Asn Lys Pro
95 100 105
Asn Tyr Pro Thr Asp Ser Arg Pro Ser Tyr Pro Arg Lys Pro Tyr
110 115 120
Tyr Pro Glu Lys Thr Thr Thr Thr Lys Pro Ala Pro Thr Ser Pro
125 130 135
Ala Asp Ile Pro Glu Ala Cys Asp Thr Ser Tyr Asp Ala Val Ala
140 145 150
Leu Ile Arg Asn Glu Leu Phe Ile Phe Lys Gly Lys Tyr His Trp
155 160 165
Arg Ile Gly Ala Arg Gly Lys Tyr Asp Gly Tyr Pro Met Glu Ile
170 175 180
Arg Arg Met Trp Ile Gly Leu Pro Arg Asp Leu Thr His Val Asp
185 190 195
Ala Val Tyr Glu Arg Pro Asp Gln Asn Ile Ala Ile Phe Val Gly
200 205 210
Lys Arg Leu Tyr Leu Phe Asn Ile Arg Glu Leu Leu Pro Gly Tyr
215 220 225
Pro Lys Pro Leu Thr Ser Leu Val Pro Gln Ile Val Asp Leu Gln
230 235 240
Ala
SEQ ID No:3
5’-CTCGTAGACTGCGTACC -3’
3’- CATCTGACGCATGGAATT-5’
SEQ ID No:4
5’- CGATGAGTCCTGAGCATG -3’
3’-TACGCTACTCAGGACTC -5’
SEQ ID No:5
5’-CTCGTAGACTGCGTACCTTAAG
SEQ ID No:6
5’-CGATGAGTCCTGAGCATGC
SEQ ID No:7
5’-CTCGTAGACTGCGTACCTTAAGCG
SEQ ID No:8
5’-CGATGAGTCCTGAGCATGCCT
SEQ ID No:9
5’-CTCGTAGACTGCGTACCTTAAGAG
SEQ ID No:10
5’-CGATGAGTCCTGAGCATGCAT
SEQ ID No:11
5’-TTAAGCGGTGGAAGAGCATCG
SEQ ID No:12
5’-CATGCCTGCAGGTCGACGATTTG
Claims (2)
1.一种鳞翅目害虫基质金属蛋白酶基因mmp,其特征是:其核苷酸序列如SEQ ID No:1所示。
2.如权利要求1所述的基因mmp编码的蛋白质,其特征是:其氨基酸序列如SEQ ID NO:2所示。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1190128A (zh) * | 1997-01-22 | 1998-08-12 | 加拿大自然资源部 | 转基因病毒 |
WO2007074405A2 (en) * | 2005-09-16 | 2007-07-05 | Devgen Nv | Dsrna as insect control agent |
CN101041828A (zh) * | 2007-03-02 | 2007-09-26 | 浙江大学 | 家蚕基因及其在真核细胞中的表达和应用 |
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2007
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1190128A (zh) * | 1997-01-22 | 1998-08-12 | 加拿大自然资源部 | 转基因病毒 |
WO2007074405A2 (en) * | 2005-09-16 | 2007-07-05 | Devgen Nv | Dsrna as insect control agent |
CN101041828A (zh) * | 2007-03-02 | 2007-09-26 | 浙江大学 | 家蚕基因及其在真核细胞中的表达和应用 |
Non-Patent Citations (5)
Title |
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Boran Altincicek,et al.Metamorphosis and collagen-IV-fragments stimulate innateimmune response in the greater wax moth,Galleria mellonella.Developmental & Comparative Immunology 30.2006,(30),1108-1118. |
Boran Altincicek,et al.Metamorphosis and collagen-IV-fragments stimulate innateimmune response in the greater wax moth,Galleria mellonella.Developmental & * |
Comparative Immunology 30.2006,(30),1108-1118. * |
王小纯,等.茶尺蠖小RNA病毒5‘端非编码区的克隆和测序及与哺乳动物小RNA病毒的比较分析.昆虫学报47 5.2004,47(5),573-578. |
王小纯,等.茶尺蠖小RNA病毒5‘端非编码区的克隆和测序及与哺乳动物小RNA病毒的比较分析.昆虫学报47 5.2004,47(5),573-578. * |
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