CN108546694B - 亚洲小车蝗蛋白酪氨酸磷酸酶ptpn4及其编码基因与应用 - Google Patents
亚洲小车蝗蛋白酪氨酸磷酸酶ptpn4及其编码基因与应用 Download PDFInfo
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- CN108546694B CN108546694B CN201810455252.1A CN201810455252A CN108546694B CN 108546694 B CN108546694 B CN 108546694B CN 201810455252 A CN201810455252 A CN 201810455252A CN 108546694 B CN108546694 B CN 108546694B
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Abstract
本发明公开了亚洲小车蝗蛋白酪氨酸磷酸酶PTPN4及其编码基因与应用。本发明首先从亚洲小车蝗中克隆了亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4，并对亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4设计引物，合成用于干扰亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4的dsRNA，且运用注射法将dsRNA导入亚洲小车蝗对亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4进行RNAi。结果表明：dsRNA注射亚洲小车蝗后，生长速率和体重增加量均显著提高，说明亚洲小车蝗蛋白酪氨酸磷酸酶PTPN4在昆虫生长发育过程中起着重要作用。本发明为害虫的分子调控提供了新的方法，也为深入理解昆虫生长发育机制、创制新的生物农药制剂提供理论基础。
Description
技术领域
本发明属于生物技术领域，具体涉及亚洲小车蝗蛋白酪氨酸磷酸酶PTPN4及其编码基因与应用。
背景技术
农作物病虫害是我国的主要农业灾害之一，它具有种类多、影响大、并时常暴发成灾的特点，其发生范围和严重程度对我国国民经济、特别是农业生产常造成重大损失。传统病虫害防治多以应急防治和化学防治为主，对病虫害防治过分依赖化学农药。这都表现了现阶段农作物病虫害防治存在相对滞后的弊端。病虫害的防治是一个漫长的过程，需要在不断的探索、改进和总结中进行，引进新的病虫害防治技术并不代表完全抛弃传统防治方式，只有做到新技术与传统防治方式相结合，才能在病虫害防治上取得新的突破。
蛋白酪氨酸磷酸酶(protein tyrosine phosphatase,non-receptor type 4，PTPN4)属于蛋白质酪氨酸磷酸酶家族，与蛋白酪氨酸激酶(proteintyrosinekinase，PTK)共同维持着酪氨酸蛋白磷酸化的平衡。PTPN4通过对胰岛素受体或其底物上的酪氨酸残基去磷酸化作用，对胰岛素信号转导进行负调节，组织细胞中PTPN4过表达都会降低PTK的活性，使胰岛素受体无法与胰岛素结合，进而引起胰岛素抵抗。
发明内容
本发明要解决的技术问题是如何调控昆虫生长发育。
为了解决上述技术问题，本发明首先提供了一种蛋白酪氨酸磷酸酶。
本发明提供的蛋白酪氨酸磷酸酶来源于亚洲小车蝗，将其命名为PTPN4，是如下a)或b)或c)或d)的蛋白质：
a)氨基酸序列是序列2所示的蛋白质；
b)在序列2所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质；
c)将序列2所示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/或添加得到的具有相同功能的蛋白质；
d)与序列2所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的蛋白质。
为了使a)中的蛋白质便于纯化，可在序列表中序列2所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1、标签的序列
| 标签 | 残基 | 序列 |
| Poly-Arg | 5-6(通常为5个) | RRRRR |
| Poly-His | 2-10(通常为6个) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
上述c)中的蛋白质PTPN4，所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10个氨基酸残基的取代和/或缺失和/或添加。
上述c)中的蛋白质PTPN4可人工合成，也可先合成其编码基因，再进行生物表达得到。
上述c)中的蛋白质PTPN4的编码基因可通过将序列1所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
为了解决上述技术问题，本发明还提供了与PTPN4蛋白质相关的生物材料。
本发明提供的与PTPN4蛋白质相关的生物材料为下述A1)至A8)中的任一种：
A1)编码PTPN4蛋白质的核酸分子；
A2)含有A1)所述核酸分子的表达盒；
A3)含有A1)所述核酸分子的重组载体；
A4)含有A2)所述表达盒的重组载体；
A5)含有A1)所述核酸分子的重组微生物；
A6)含有A2)所述表达盒的重组微生物；
A7)含有A3)所述重组载体的重组微生物；
A8)含有A4)所述重组载体的重组微生物。
上述生物材料中，A1)所述核酸分子为如下1)或2)或3)所示的基因：
1)其编码序列是序列1所示的cDNA分子或基因组DNA分子；
2)与1)限定的核苷酸序列具有75％或75％以上同一性，且编码PTPN4蛋白质的cDNA分子或基因组DNA分子；
3)在严格条件下与1)或2)限定的核苷酸序列杂交，且编码PTPN4蛋白质的cDNA分子或基因组DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
其中，序列1由2670个核苷酸组成，编码序列2所示的氨基酸序列。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码PTPN4蛋白质的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的PTPN4的核苷酸序列75％或者更高同一性的核苷酸，只要编码PTPN4且具有相同功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码序列2所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。
为了解决上述技术问题，本发明还提供了PTPN4蛋白质或上述生物材料的新用途。
本发明提供了PTPN4蛋白质或上述生物材料在如下a1)-a6)中任一种中的应用：
a1)调控昆虫生长发育；
a2)制备调控昆虫生长发育的产品；
a3)提高昆虫体重增加量；
a4)制备提高昆虫体重增加量的产品；
a5)提高昆虫生长速率；
a6)制备提高昆虫生长速率的产品。
上述应用中，所述调控为促进；所述昆虫为亚洲小车蝗。
为了解决上述技术问题，本发明还提供了一种促进昆虫生长发育的方法。
本发明提供的促进昆虫生长发育的方法包括降低昆虫中蛋白酪氨酸磷酸酶的表达量和/或活性的步骤，从而实现促进昆虫生长发育；
所述降低昆虫中蛋白酪氨酸磷酸酶的表达量和/或活性的方法是将抑制昆虫中蛋白酪氨酸磷酸酶的编码基因表达的物质导入昆虫；
所述抑制昆虫中蛋白酪氨酸磷酸酶的编码基因表达的物质为抑制昆虫中蛋白酪氨酸磷酸酶的编码基因表达的dsRNA。
上述方法中，所述蛋白酪氨酸磷酸酶为PTPN4蛋白质；抑制昆虫中PTPN4蛋白质的编码基因表达的dsRNA为由序列表中序列3所示的核苷酸和其反向互补序列所示的核苷酸组成的双链RNA。
上述方法中，所述导入的方式为注射。
上述方法中，所述昆虫为亚洲小车蝗。
为了解决上述问题，本发明最后还提供了抑制PTPN4蛋白质的编码基因表达的物质。
本发明提供的抑制PTPN4蛋白质的编码基因表达的物质为由序列表中序列3所示的核苷酸和其反向互补序列所示的核苷酸组成的双链RNA。
上述抑制PTPN4蛋白质的编码基因表达的物质在促进昆虫生长发育中的应用也属于本发明的保护范围。
上述应用中，所述昆虫为亚洲小车蝗。
本发明首先从亚洲小车蝗中克隆了亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4，并对亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4设计引物，合成了用于干扰亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4的dsRNA，且运用注射法将dsRNA导入亚洲小车蝗对亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4进行RNAi。结果表明：dsRNA注射亚洲小车蝗后，生长速率和体重增加量均显著提高，说明亚洲小车蝗蛋白酪氨酸磷酸酶PTPN4在昆虫生长发育过程中起着重要作用。本发明为害虫的分子调控提供了新的方法，也为深入理解昆虫生长发育机制、创制新的生物农药制剂提供理论基础。
附图说明
图1为实验组和对照组亚洲小车蝗体重增加量、生长速率的统计结果。
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。
下述实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中的供试虫源：锡林浩特市采集5龄亚洲小车蝗蝗蝻(发育一致)，在智能人工气候箱中进行饲养，饲养条件如下：温度26℃，湿度70％，光照黑暗比为16h：8h，统一以克氏针茅饲喂。
下述实施例中的主要试剂及试剂：
分离试剂(invitrogen原装)，RNA spin column(全式金)，胶回收试剂盒(Axygen)，EX Taq DNA聚合酶(Takara)，T4DNA连接酶(Takara)，pGEM-T Easy Vector Systems(Promega)，无水乙醇、异丙醇、丙三醇等试剂均为国产分析醇。
下述实施例中的主要仪器：超净工作台(上海博讯实业有限公司)、东胜龙ETC-811PCR仪(北京东胜创新生物科技有限公司)、德国Sigma 3K15冷冻离心机(德国希格玛离心机有限公司)、NanoPhotometer微量分光光度计(德国IMPLEN公司)、HPX-9052MBE数显电热培养箱(上海博讯实业有限公司)、THZ-D台式恒温振荡器(华美生化仪器厂)、漩涡振荡器QL-901(海门市其林贝尔仪器制造)、高压灭菌锅YXQ-LS-50SII(上海博讯实业有限公司医疗设备厂)。
实施例1、亚洲小车蝗蛋白酪氨酸磷酸酶PTPN4及其编码基因的获得
1、亚洲小车蝗总RNA的提取
用
分离试剂提取亚洲小车蝗组织样品的RNA。具体步骤如下：
1)2mL匀浆器放于烘箱中，160℃，3个小时，冷却至室温备用。
2)将匀浆器置于冰上，加入1mL
分离试剂和100-200mg亚洲小车蝗组织，研磨。
3)将匀浆液转移至1.5mL离心管中，室温静置5min。4℃，13000r离心5min。
4)将上清转移至干净的1.5mL离心管中，加入200μL氯仿，漩涡震荡15s。室温放置5min。4℃，13000r离心10min。
5)吸取400μL上清转移至新的1.5mL离心管中，加入200μL氯仿，漩涡震荡30s。室温放置5min。4℃，13000r离心10min。
6)吸取300μL上清，加入300μL异丙醇，漩涡震荡30s，转移至RNA spin column中，冰上静置10min。
7)4℃，13000r离心2min，弃滤液。
8)加入600μL RNA washsolution，吸打使沉降物洗涤，4℃，13000r离心2min，弃滤液。再次加入600μL RNA washsolution，吸打使沉降物洗涤，4℃，13000r离心2min，弃滤液。4℃，13000r空离3min，除去多余乙醇，空气吹干3min。
9)更换一个新的收集管，向RNA spin column中加入50μL 65℃预热的RNase-free-water，余热下热置5min，4℃，13000r离心3min。
10)收集滤出液，用NanoPhotometer微量分光光度计检测RNA浓度和OD260/280值，确认RNA质量。同时，取2μL提取的RNA，琼脂糖凝胶电泳检测，剩余RNA储存于-20℃备用。
2、反转录
用PrimeScriptTM 1st strand cDNASynthesis Kit反转录试剂盒反转录获得cDNA。具体步骤如下：
1)配置10μL体系：Oligo dT Primer(50μM)1μL、dNTP Mixture(10mM each)1μL、total RNA<5μL、RNase Free dH2O补足体系至10μL。
2)65℃保温5min后，冰上迅速冷却。
3)配置20μL反应液：5×PrimeScript Buffer 4μL、RNase Inhibitor(400U/μL)0.5μL(20units)、PrimeScript RTase(200U/μL)1μL(200units)、RNase Free dH2O补足体系至20μL。
4)缓慢混匀。
5)42℃保温30-60min。
6)95℃保温5min，使酶失活，冰上放置，-20℃保存cDNA。
3、亚洲小车蝗蛋白酪氨酸磷酸酶及其编码基因的获得
1)引物设计
根据前期获得的亚洲小车蝗转录组得到PTPN4基因序列，利用DNAMAN8设计PTPN4基因全长引物或片段引物。设计的引物如下：
PTPN4-F：5'-GATTGAAAGTGTTTCTCGACGTGC-3'；
PTPN4-R：5'-TCTTCTAAGGGTTTCACAAGGCC-3'。
2)PCR反应
以亚洲小车蝗cDNA为模板，以引物PTPN4-F/PTPN4-R进行PCR扩增，得到PCR产物。
PCR反应体系如下(总体积50μL)：cDNA模板1μL、dNTP 4μL、10×Buffer 5μL、前引物1μL、后引物1μL、Taq酶0.25μL、ddH2O 38μL。
PCR反应条件如下：95℃3min；95℃30s，55℃30s，72℃1min30s，35个循环；72℃10min；4℃保存。
3)PCR产物回收、克隆、测序
3-1)将PCR产物在TAE配制的1％的琼脂糖凝胶上进行电泳，当目标带分离较好时，用刀片切下目标带所在的胶块放入无菌的离心管中。再用胶回收试剂盒(Axygen)回收纯化目标带，回收纯化过程参照试剂盒说明书进行。
3-2)PCR产物回收后连接pGEM-T Easy载体，得到重组载体。连接体系如下：T4DNAligase 1μL、2×buffer 5μL、pGEM-T Easy 1μL、PCR回收产物3μL。连接条件：室温下连接6个小时。
3-3)感受态细胞的制备与转化
在1.5mL离心管中，加入33.3μL Trans1-t1感受态细胞，冰上放置15min，42℃水浴90s，冰上放置10min。在每个1.5mL离心管中加入500μL的液体LB培养液，37℃，200rpm摇菌2h。摇菌后，吸取100μL菌液至1‰AMP LB固体培养基中，37℃培养过夜。挑选单菌落于2mL的离心管中，管中有1mL的1‰AMP LB液体培养基。37℃，200rpm摇菌3-6h，观察生长情况。
3-4)菌液PCR
对3-3)中的菌液进行PCR验证，菌液PCR反应体系(总体积50μL)如下：菌液1μL、dNTP 4μL、10×Buffer 5μL、前引物1μL、后引物1μL、Taq酶0.25μL、ddH2O38μL。
反应条件如下：95℃3min；95℃30s，55℃30s，72℃1min30s，35个循环；72℃10min；4℃保存。
将阳性克隆菌株送到上海生工生物工程技术服务有限公司进行序列测定并对测序结果进行分析。
测序结果表明：PCR扩增得到大小为2670bp的DNA片段，其核苷酸序列如序列1所示，将序列1所示的基因命名为亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4，其编码的蛋白酪氨酸磷酸酶的氨基酸序列如序列2所示，将序列2所示的氨基酸序列命名为亚洲小车蝗蛋白酪氨酸磷酸酶PTPN4。
实施例2、亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4的dsRNA及其在促进昆虫生长发育中的应用
一、dsRNA的合成
采用T7RiboMAX TM Express RNAi System试剂盒合成dsRNA。具体步骤如下：
1)dsRNA引物的合成
根据已克隆出的基因片段设计引物，扩增目的片段为600bp左右，在引物的5'端引入T7启动子。引物序列如下：PTPN4RNAi F：5'-GGATCCTAATACGACTCACTATAGGTCTCGCCAGTTACATTGTTCAG-3'；PTPN4RNAi R：5'-GGATCCTAATACGACTCACTATAGGGGTGTCATAATCCTCCGATGG-3'。
2)DNA模板的制备
用试剂盒提取菌液质粒，以含基因片段的质粒(实施例1中的重组载体)为模板，采用PTPN4RNAi F和PTPN4RNAi R进行PCR扩增，得到含有T7启动子序列的目的片段。
PCR反应体系如下(总体积50μL)：质粒1μL、dNTP 4μL、10×Buffer 5μL、前引物1μL、后引物1μL、Taq酶0.25μL、ddH2O 38μL。
PCR反应条件如下：95℃3min；95℃30s，55℃30s，72℃1min，35个循环；72℃10min；4℃保存。
回收PCR产物，并用NanoPhotometer微量分光光度计检测目标DNA浓度，回收浓度需大于150ng/μL。
3)dsRNA的合成
采用T7RiboMAX TM Express RNAi System试剂盒将回收的DNA体外转录合成亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4的dsRNA，并用NanoPhotometer微量分光光度计检测dsRNA浓度，dsRNA浓度需要大于1000ng/μL。再将dsRNA浓度调整为1ng/μL，得到dsRNA溶液(溶剂为Nucleause free water)。
本发明获得的亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4的dsRNA为双链RNA，由正义链和反义链组成，其正义链的核苷酸序列为序列3，其反义链的核苷酸序列为序列3的反向互补序列。上述亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4的dsRNA也可通过人工合成的方法获得。将亚洲小车蝗蛋白酪氨酸磷酸酶基因PTPN4的dsRNA命名为dsPTPN4。
4)对照GFP的dsRNA
按照上述方法合成对照GFP的dsRNA，将对照GFP的dsRNA命名为dsGFP，得到浓度为1ng/μL的dsGFP溶液。对照GFP的dsRNA为双链RNA，由正义链和反义链组成，其正义链的核苷酸序列为序列4，其反义链的核苷酸序列为序列4的反向互补序列。合成GFP dsRNA的引物如下：
GFP-1F：5’-TAATACGACTCACTATAGGTACGACTCACTATAGGAGTAAAGG-3’；
GFP-1R：5’-TAATACGACTCACTATAGGTAGGTTTGTATAGTTCATCCATACC-3’。
二、dsRNA在促进昆虫生长发育中的应用
1、实验方法
将dsRNA导入亚洲小车蝗体内。具体步骤如下：用10μL的微量注射器吸取5μL浓度为1ng/μL的dsPTPN4溶液(实验组)和dsGFP溶液(dsGFP对照组)，分别从蝗虫腹部的第二腹节和第三腹节之间的节间膜注射进入，注射器的针头和腹部平行，避免损伤蝗虫的内部器官组织。设置注射5μL Nucleause free water的空白对照组(CK)，每处理重复5次，每重复处理8头5龄亚洲小车蝗(雌雄比1:1)。注射后将其在智能人工气候箱中进行饲养，饲养条件如下：温度26℃，湿度70％，光照黑暗比为16h：8h，每天观察亚洲小车蝗生长发育情况，进入成虫阶段后取样统计体重增加量和生长速率。统计天数D；实验前各处理组平均体重为A1，进入成虫阶段后各处理组平均体重为A2，体重增加量＝A2-A1；生长速率＝(A2-A1)/D。
2、实时荧光定量PCR
注射后72h提取实验组和对照组(dsGFP对照组和空白对照组)的亚洲小车蝗虫体的总RNA，Takara反转录试剂盒合成cDNA，检测PTPN4基因表达量。以actin基因作为内参基因。PTPN4基因引物序列如下：C-PTPN4-149-F：GCCAGTGAAGAAGCAAATAAGAA和C-PTPN4-149-R：GCTGTAACCGTCCCTGAAGAATG；actin基因引物序列为GTTACAAACTGGGACGACAT和AGAAAGCACAGCCTGAATAG。
结果表明：与对照组(dsGFP对照组和空白对照组)相比，dsPTPN4实验组的亚洲小车蝗中的PTPN4基因的表达量显著降低，说明dsPTPN4成功干扰了亚洲小车蝗中PTPN4基因的表达。dsGFP对照组和空白对照组结果无显著性差异。
3、体重增加量、生长速率检测结果
结果如图1所示。PTPN4基因RNAi后的亚洲小车蝗体重增加量、生长速率与对照组(dsGFP对照组和空白对照组)有显著差异(P＜0.05)，干扰PTPN4基因后的亚洲小车蝗各项指标都显著高于对照组，dsGFP对照组和空白对照组结果无显著性差异。说明PTPN4基因RNAi对亚洲小车蝗的生长发育起着促进作用。
序列表
<110>中国农业科学院植物保护研究所
<120>亚洲小车蝗蛋白酪氨酸磷酸酶PTPN4及其编码基因与应用
<160>4
<170>PatentIn version 3.5
<210>1
<211>2670
<212>DNA
<213>人工序列(Artificial Sequence)
<400>1
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gatgatactc aacattgttt ccaaattgag aaacgatcga aaggtcatgt tctacttgat 180
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aatggagcag ccccaacacc cagcaattct gatcttgtgc gttggcttga cccctcaaag 300
ccagtgaaga agcaaataag aaacaatgga aacttctact tccgtgttaa gttctacgtc 360
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tacattgttc agtctgagct gggtgactat caacctgatg aacatctgcc tggatacctt 540
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ggagtgtatg aaatacctgg atatgtagat acacaatcac agatttctga agaagggttg 1440
gttgtcataa gaattacacc tgatgaacaa ggaagatttg gattcaatgt taaaggaggc 1500
gccaatttaa atatgccaat tgttgtatcc agagtagttc caaatacacc agctgaccga 1560
tgctgtccaa aactgaatga gggagatcag gtactcttca ttaatggcca aaacatcagt 1620
ggcatgttac atgaacaagt agttaatttg atccgtgaaa caagagattc ggcttctgga 1680
gagctaatat tgactgtaaa acccagtgcg gtttatgaag ctcaggatat tgaggaacct 1740
ccatatcaat atgttcctga tcgaccacat acaaatggaa tagatcaggc tgatgcattg 1800
gctgaatcca tgctgttact tgctgatgga cttgctagtg gagctctcat tgcacagttc 1860
gagcagttgt atcggaaaaa gccaggcctg acaacaaatg aatccagaaa gcaagaaaat 1920
gtcaataaaa atagatatcg ggatatttct ccatatgatg caacgagggt aattttgcaa 1980
ggtgggacca ctggtgatta cataaatgcc aactatgtta atatggaaat tcctggatct 2040
ggtataatta atagatatat tgcaacacaa ggccctttac caggaacagt agctgatttc 2100
tggcagatgg tagtggaagc tcagagcact ctcattgtca tggtcactcc agttgtggaa 2160
cgtggccgca taaaatgtca caaatactgg cctgccctga cacaaacttt ggaattatcg 2220
cacctccaca tcacatgtac acgagaggaa actgaaccta ctggcagctt tatattcaga 2280
gagtttagac tcacaaatct agagactgag gaggaacgac acatcagcca catgcagtac 2340
ttggcatggc cagaccatgg tgttcctgag gatcccacac aatttctaga gttcacaatg 2400
cgtgtgcgca aagctcggac tggaatggtg gaacccacca ttgtccattg ctctgctggg 2460
ataggtcgga ctggagttct gattctcatg gaaactgcaa tgtgcctcat cgaagctaat 2520
gaaccagttt atcctcttga tattgtacgg gctatgagag atcaacgagg gatgatgata 2580
caaacagcgg cccagtatcg atttgtttgt gagagtgttc acagagctta tagtgaaggc 2640
cttgtgaaac ccttagaaga atttcagagg 2670
<210>2
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<210>3
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Claims (9)
1.蛋白质，是如下a）或b）的蛋白质：
a）氨基酸序列是序列2所示的蛋白质；
b）在序列2所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质。
2.与权利要求1所述的蛋白质相关的生物材料，为下述A1）至A8）中的任一种：
A1）编码权利要求1所述的蛋白质的核酸分子；
A2）含有A1）所述核酸分子的表达盒；
A3）含有A1）所述核酸分子的重组载体；
A4）含有A2）所述表达盒的重组载体；
A5）含有A1）所述核酸分子的重组微生物；
A6）含有A2）所述表达盒的重组微生物；
A7）含有A3）所述重组载体的重组微生物；
A8）含有A4）所述重组载体的重组微生物。
3.根据权利要求2所述的相关生物材料，其特征在于：A1）所述核酸分子为序列1所示的DNA分子。
4.权利要求1所述的蛋白质或权利要求2或3所述的生物材料在如下a1）-a6）中任一种中的应用：
a1）调控亚洲小车蝗生长发育；
a2）制备调控亚洲小车蝗生长发育的产品；
a3）提高亚洲小车蝗体重增加量；
a4）制备提高亚洲小车蝗体重增加量的产品；
a5）提高亚洲小车蝗生长速率；
a6）制备提高亚洲小车蝗生长速率的产品。
5.一种促进亚洲小车蝗生长发育的方法，包括降低亚洲小车蝗中蛋白酪氨酸磷酸酶的表达量和/或活性的步骤，从而实现促进亚洲小车蝗生长发育；所述蛋白酪氨酸磷酸酶为权利要求1所述蛋白质。
6.根据权利要求5所述的方法，其特征在于：所述降低亚洲小车蝗中蛋白酪氨酸磷酸酶的表达量和/或活性的方法是将抑制亚洲小车蝗中蛋白酪氨酸磷酸酶的编码基因表达的物质导入亚洲小车蝗；
或，所述抑制亚洲小车蝗中蛋白酪氨酸磷酸酶的编码基因表达的物质为抑制亚洲小车蝗中蛋白酪氨酸磷酸酶的编码基因表达的dsRNA。
7.根据权利要求5或6所述的方法，其特征在于：
所述抑制亚洲小车蝗中蛋白酪氨酸磷酸酶的编码基因表达的dsRNA为由序列表中序列3所示的核苷酸和其反向互补序列所示的核苷酸组成的双链RNA；
所述导入的方式为注射。
8.抑制权利要求1所述蛋白质的编码基因表达的物质，其为由序列表中序列3所示的核苷酸和其反向互补序列所示的核苷酸组成的双链RNA。
9.抑制权利要求1所述蛋白质的编码基因表达的物质在促进亚洲小车蝗生长发育中的应用。
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