CN105925592B - 一种几丁质代谢调控基因NlTPS、编码蛋白、载体、工程菌及其应用 - Google Patents
一种几丁质代谢调控基因NlTPS、编码蛋白、载体、工程菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种几丁质代谢调控基因NlTPS、编码蛋白、载体、工程菌及其应用,所述基因NlTPS核苷酸序列为下列之一:a、SEQ.ID.NO.1或SEQ.ID.NO.2;b、与SEQ.ID.NO.1或SEQ.ID.NO.2所示的核苷酸序列互补的序列;本发明NlTPS能够调控昆虫几丁质酶及几丁质合成酶在mRNA水平上的表达,从而调控昆虫几丁质代谢,导致昆虫畸形甚至死亡。
Description
(一)技术领域
本发明涉及一种昆虫几丁质代谢调控基因及其应用,特别涉及一种昆虫几丁质代谢调控基因NlTPS(褐飞虱海藻糖合成酶:Trehalose-6-phosphate synthase,包括NlTPS1和NlTPS2)及其应用。
(二)背景技术
我国是世界上农作物有害生物发生最为严重的国家之一。农作物生物灾害发生频繁、为害严重、损失巨大。害虫一直严重威胁全球农作物的安全生产(植物保护工程建设规划(2006-2010年),据报道全球约有植物重要害虫6000多种,其中,在我国有发生的比例达到30%,为1800多种。据估计,全球每年仅害虫危害造成的产量损失已达总产量的18%,经济损失达1000亿美元,其中仅每年用于植食性害虫防治的费用就达75.6亿美元。且目前至少有30种农作物害虫对40种杀虫剂产生了不同程度的抗药性,采用传统的单一农药控制害虫的为害将会越来越困难。由此,害虫防治的指导思想也已从“杀死害虫”转变为“调控害虫”。通过调控害虫的生长发育将成为害虫控制的重要手段,找到能够起调控作用的靶标基因对农业生产中害虫的防治具有重大意义。
海藻糖为一种非还原性双糖,其不但是昆虫血淋巴中的重要能量物质,而且由于其在昆虫的生长、发育、蜕皮变态过程中具有重要的作用,被成为昆虫的“血糖”,海藻糖合成是昆虫体内合成海藻糖的酶,其表达被抑制后会直接影响昆虫的生长和变态发育,因此TPS发现和研究对害虫的防治具有重要意义。
(三)发明内容
本发明目的是提供一种能够调控几丁质代谢表达的NlTPS基因,包 括NlTPS1和NlTPS2,其表达被抑制后,能够调控几丁质合成酶及几丁质酶在mRNA水平上的表达,该基因还能应用于农业生产中害虫的防治。
本发明采用的技术方案是:
本发明提供一种几丁质代谢调控基因NlTPS(褐飞虱海藻糖合成酶:Trehalose-6-phosphate synthase),所述基因核苷酸序列为下列之一:a、SEQ.ID.NO.1或SEQ.ID.NO.2所示的核苷酸序列;b、与SEQ.ID.NO.1或SEQ.ID.NO.2所示的核苷酸序列互补的序列;本发明也不排除对上述a或b所述序列进行一个或多个碱基的取代、缺失或修饰的核苷酸序列;或与上述a或b所述序列具有至少90%同源性的核苷酸序列。
进一步,所述的几丁质代谢调控基因NlTPS核苷酸序列优选为SEQ.ID.NO.1(NlTPS1)或SEQ.ID.NO.2(NlTPS2)所示。
本发明还提供一种几丁质代谢调控基因NlTPS编码蛋白,所述的蛋白的氨基酸序列为SEQ.ID.NO.3或SEQ.ID.NO.4所示。
本发明涉及一种由所述几丁质代谢调控基因NlTPS构建的重组载体,及所述重组载体构建的重组基因工程菌,优选大肠杆菌DH5α。
本发明还涉及一种所述几丁质代谢调控基因NlTPS在制备几丁质代谢酶活性抑制剂中的应用,所述几丁质代谢酶为几丁质酶或几丁质合成酶。
本发明还涉及一种所述几丁质代谢调控基因NlTPS在制备防治害虫药物中的应用,所述药物为防治褐飞虱(Nilaparvata lugens)药物,具体所述的应用为:利用RNAi的方法抑制NlTPS的表达(以SEQ.ID.NO.1或SEQ.ID.NO.2所示序列为模板,合成dsRNA,显微注射褐飞虱),导致昆虫出现一定的死亡率和畸形率,畸形形态主要包括蜕皮困难、蜕皮困难且翅型异常以及翅型异常三种。此外NlTPS的表达被抑制后还会导致褐飞虱体内几丁质合成酶以及几丁质酶在mRNA水平上的表达下降。
本发明利用同源克隆和RACE技术获得褐飞虱NlTPS中间序列及全长cDNA序列,具体方法如下:
本发明提取褐飞虱RNA,反转录合成cDNA,设计引物,引物序列如下:
NlTPS1-F:5’-ATGATTGATAATCCTGACAC-3’
NlTPS1-R:5’-TTATCGTTTGGTATCTGACGG-3’
NlTPS2-F:5’-ATGGATTCAATAAAGGATATG-3’
NlTPS2-R:5’-CTAATATCTAGCCATAACTGG-3’
利用上述引物进行常规聚合酶链式反应(PCR)。将PCR产物与克隆载体(PMD18-T)连接转化大肠杆菌DH5α感受态细胞,然后筛选阳性重组子,进行测序,得到长度为2421bp和2460bp的NLTPS序列,将该序列分别记为NlTPS1和NlTPS2。
该NLTPS1序列SEQ.ID.NO.1信息如下:
长度:2421bp
类型:核酸
链型:双链
来源:褐飞虱
该NlTPS2序列SEQ.ID.NO.2信息如下:
长度:2460bp
类型:核酸
链型:双链
来源:褐飞虱
本发明所述几丁质代谢调控基因NlTPS1编码蛋白质的氨基酸序列SEQ.ID.NO.3信息如下:
长度:807
类型:氨基酸
来源:褐飞虱
本发明所述几丁质代谢调控基因NlTPS2编码蛋白质的氨基酸序列SEQ.ID.NO.4信息如下:
长度:820
类型:氨基酸
来源:褐飞虱
NlTPS1序列SEQ.ID.NO.1和NlTPS2序列SEQ.ID.NO.2起始密码子和终止密码子用下划线标记。
与现有技术相比,本发明的有益效果主要体现在:本发明提供了一种几丁质代谢调控基因NlTPS(包括NlTPS1和NlTPS2),其表达被抑制后,能够调控几丁质合成酶及几丁质酶在mRNA水平上的表达;对褐飞虱进行NlTPS干扰后,NlTPS1和NlTPS2的表达被抑制,褐飞虱表现出一定的畸形率(蜕皮困难、蜕皮失败且翅型异常、翅型异常)和死亡率,该基因还能应用于农业生产中害虫的防治。
(四)附图说明
图1dsTPS注射后被干扰基因相对表达量,图中数据为平均数±标准差,对照组为dsGFP注射组;*代表P<0.05,差异显著,**代表P<0.01,差异极显著,下同;A为NlTPS1相对表达量,B为NlTPS2相对表达量。
图2dsTPS注射后褐飞虱的变态发育情况:A表示dsTPS注射后飞虱的畸形形态,B表示dsTPS注射后褐飞虱的死亡率和畸形率,C表示dsTPS注射后褐飞虱三种畸形百分比;
图3为dsTPS注射后几丁质合成酶的相对表达情况,A为CHS1相对表达量,B为可变剪接体CHS1a相对表达量;C为可变剪接体CHS1b的相对表达量;
图4dsTPS注射后几丁质酶及几丁质酶复合体的相对表达情况:10个几丁质酶及成虫盘生长因子(Imaginal Disc Growth Factor,IDGF)基因和β-N-乙酰葡糖胺内切酶(endo-β-N-acetylglucosaminidase,ENGase)基因 的相对表达量(A-L)。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
褐飞虱是水稻上的重要害虫,全世界各地都有分布,该虫具有繁殖速度快、生命周期短、内禀增长率高、环境适应性强等特点,在外界环境适宜时极易暴发成灾,给水稻生产造成巨大的损失。在下述实施例中以褐飞虱为例对本发明进行了详细说明。
氨苄LB固体培养基、LB固体培养基均为公知常识。
实施例1:昆虫几丁质代谢相关基因NlTPS的获取
1.RNA提取:
利用Trizol法提取室内饲养的褐飞虱(饲养条件:温度25±1℃,湿度70%,光照比16L:8D)的RNA,作为模板RNA。
cDNA单链合成:取1μg模板RNA,利用AMV反转录试剂盒(Takara,大连宝生)合成单链cDNA,作为扩增中间片段序列的模板。
2.NlTPS基因中间片段序列扩增
根据褐飞虱cDNA序列设计引物,引物序列如下:
NlTPS1-F:5’-ACCAGGAGTTGAAGGAGGAG-3’
NlTPS1-R:5’-GATCAGGGTGCCCATAGC-3’
NlTPS2-F:5’-CACCAAAGGTCTAAGGCACA-3’
NlTPS2-R:5’-CATCGTTGATCTCGTAGGGA-3’
利用上述引物进行PCR扩增,体系如下:10×Buffer 5μL,10mmoL/L dNTP 2μL,PCR引物各25pmol,cDNA模板1μL(约200ng),Taq酶2U,加灭菌双蒸水至50μL。PCR反应步骤为:94℃预变性5min;93℃变性30sec,50℃退火30sec,72℃延伸90sec,循环30次;最后72℃延伸10min。1%琼脂糖凝胶电泳,利用胶回收试剂盒(TransGen,Beijing)回收片段,将所得片段连入PMD18-T载体(Takara),转化大肠杆菌DH5α感受态细胞(TransGen,Beijing),然后利用氨苄LB固体培养基筛选阳性重组子,并进行PCR鉴定,然后进行测序,分别获得2421bp和2460bp的NlTPS1(核苷酸序列为SEQ ID NO.1,氨基酸序列为SEQ ID NO.3)和NlTPS2序列(核苷酸序列为SEQ ID NO.2,氨基酸序列为SEQ ID NO.4)。实施例2:NlTPS在害虫防治中的应用
1.dsRNA的合成:以实施例1方法进行TPS1、TPS2和GFP(绿色荧光蛋白:GreenFluorescent Protein)中间片段的扩增、胶回收、连接、转化,并进行PCR鉴定,然后进行测序,将测序正确转化子转到含Amp+(100μg/ml)的LB液体培养基中,250rpm、37℃震荡培养过夜,利用质粒抽屉试剂盒进行质粒的抽提。然后利用T7 Express RNAiSystem试剂盒(Promega)合成dsTPS1、dsTPS2以及dsGFP,具体操作参照T7RiboMAX ExpressRNAi System试剂盒说明书,所用的引物序列如下:
DSNLTPS1-F:5-ACCAGGAGTTGAAGGAGGAG-3
DSNLTPS1-R:5-GATCAGGGTGCCCATAGC-3
DSNLTPS2-F:5-CACCAAAGGTCTAAGGCACA-3
DSNLTPS2-R:5-CATCGTTGATCTCGTAGGGA-3
DSNLGFP-F:5-AAGGGCGAGGAGCTGTTCACCG-3
DSNLGFP-R:5-CAGCAGGACCATGTGATCGCGC-3
dsTPS1合成的模板为SEQ ID NO.1所示的序列,dsTPS2合成的模板为SEQ IDNO.2,dsGFP合成的模板为公知序列。
2.dsRNA的显微注射:将步骤1合成的dsRNA用适量的DEPC水溶解,注射前首先用标准毛细管定量注射器每次泵出的dsRNA的体积,并 通过调整氮气压来调整dsRNA的体积使其符合注射量。将5龄第一天活体褐飞虱用CO2麻醉后放至注射盘上,使其腹部朝上,注射腹面口器,注射量为200ng/头,注射后将其放至装有新鲜水稻的试管中,分别于注射后48h,72h取材。
3.dsRNA注射后被干扰基因的相对定量表达情况
对步骤2取材的褐飞虱进行液氮研磨,抽提RNA,取总量1μg的RNA进行cDNA单链合成,取反转录产物1μl用于qRT-PCR的模板。qRT-PCR反应体系为20μl,具体如下:正、反向引物各1μl,cDNA 1μl,SYBR Premix ExTaq(Takara)10μl,灭菌超纯水补至20μl,引物序列如下:
RTNLTPS1-F:5’-AAGACTGAGGCGAATGGT-3’
RTNLTPS1-R:5’-AAGGTGGAAATGGAATGTG-3’
RTNLTPS2-F:5-AGAGTGGACCGCAACAACA-3
RTNLTPS2-R:5-TCAACGCCGAGAATGACTT-3
采用方法和STATISTICA 6.0中的ANOVA进行数据分析,结果如图1所示。
4.dsRNA注射后褐飞虱的变态发育情况:
利用超微量显微注射技术分别对5龄褐飞虱若虫进行dsTPS1和dsTPS2注射,dsGFP注射作为对照,注射方法同步骤2,于注射后72h后观察褐飞虱的畸形形态,并统计畸形率和死亡率,结果如图2所示。
5.几丁质合成酶、几丁质酶以及几丁质酶复合体的相对定量表达
利用步骤3方法进行对褐飞虱几丁质合成酶、几丁质酶以及几丁质酶复合体进行qRT-PCR检测,引物序列如下:
18SF:5’-CGCTACTACCGATTGAA-3’
18SR:5’-GGAAACCTTGTTACGACTT-3’
CHS1F:5’-CCGCAAACGATTCCTACAGA-3’
CHS1R:5’-AGGTCCTTGACGCTCATTCC-3’
CHS1aF:5’-TGTTCTTGCTACAACTCAATAAA-3’
CHS1aR:5’-ACACCAATCCGATAGGCTC-3’
CHS1bF:5’-GCTGTCTTTGCTTTCTTCAT-3’
CHS1bR:5’-ACACCAATCCGATAGGCTC-3’
Cht1F:5’-AGGTGGTTAGGGACGAGGAG-3’
Cht1R:5’-TGCGCTTGACATAGTTGGACT-3’
Cht2F:5’-GCAGATTTCTGGACAGGGAA-3’
Cht2R:5’-TGACGCACAAGCGGGAAG-3’
Cht3F:5’-CTACACCTCTGGCTAAACTCGG-3’
Cht3R:5’-AACTTGTCCTTGCGGCTGAT-3’
Cht4F:5’-TTGAGGAGGTTCACGGGTCT-3’
Cht4R:5’-CCTTACTGGAAACGAGGTTGG-3’
Cht5F:5’-AAAGCGTTCGTGATGAAATAGC-3’
Cht5R:5’-GATCCTTTGCCTCAATCCAAT-3’
Cht6F:5’-GCTGGTAAGGAGATGCTATTCG-3’
Cht6R:5’-GTGGTTCTAAGGCTGGCTGTC-3’
Cht7F:5’-CTACTCTGCCATCCCATTCCT-3’
Cht7R:5’-GTCTGGGTTTCTTCACTTCCTG-3’
Cht8F:5’-GAACAAAGTGCAAACTCAGTCC-3’
Cht8R:5’-CACCTTCTGTGGCTTCTGG-3’
Cht9F:5’-GTGCGGTATTGGTTGAAGAGG-3’
Cht9R:5’-GGTATAACGTGATTCCGAGCC-3’
Cht10F:5-CAAGCCAATACCCAACAAAC-3’
Cht10R:5’-ACAGCAAATCCATAGAGCACA-3’
IDGFF:5’-AAAAGAACGAGGAGGAGGG-3’
IDGFR:5’-TTGCTTGAGGATGGGGTAC-3’
ENGaseF:5’-TGTGGCAAGACTTCGTTA-3’
ENGaseR:5’-ATGGGAGGGTTGGGATAG-3’
采用方法和STATISTICA 6.0中的ANOVA进行数据分析,结果如图3和图4所示。
图1的结果表明,TPS1在dsTPS1注射组,TPS2在dsTPS2注射组的表达水平都有明显降低,保证了dsRNA干扰的有效性。图2的结果表明,NlTPS1和NlTPS2的表达被抑制后,褐飞虱表现出一定的畸形率和死 亡率。对褐飞虱进行NlTPS干扰后出现3种主要畸形:蜕皮困难、蜕皮失败且翅型异常、翅型异常。翅型异常在dsTPS1和dsTPS2注射组的百分比分别为50%和70%,蜕皮困难的百分比分别为35.59%和23.09%。TPS1和TPS2表达被抑制后所致的畸形率分别为16.28%和17.7%,死亡率分别为29.63%和28.62%,均显著高于对照组。图3和图4的结果表明,dsTPS注射48后CHS1及其两个可变剪接体CHS1a和CHS1b的表达显均著下降,10个几丁质酶和两个几丁质酶复合体(IDGF和ENGase)的表达也均显著低于对照组。dsTPS注射72h后CHS1b、Cht2、Cht3、Cht6、Cht7、Cht10以及ENGase在mRNA水平的表达均显著下降。NlTPS基因能够调控昆虫几丁质代谢,该基因可作为靶标基因应用于农业生产中的害虫防治。
Claims (10)
1.一种几丁质代谢调控基因NlTPS,其特征在于所述基因核苷酸序列为SEQ ID NO.2所示的核苷酸序列或SEQ ID NO.2所示核苷酸序列互补的序列。
2.如权利要求1所述的几丁质代谢调控基因NlTPS,其特征在于所述基因核苷酸序列为SEQ ID NO.2所示。
3.一种权利要求1所述几丁质代谢调控基因NlTPS编码蛋白。
4.如权利要求3所述几丁质代谢调控基因NlTPS编码蛋白,其特征在于所述蛋白氨基酸序列为SEQ ID NO.4所示。
5.一种由权利要求1所述几丁质代谢调控基因NlTPS构建的重组载体。
6.一种由权利要求5所述重组载体构建的重组基因工程菌。
7.一种权利要求1所述几丁质代谢调控基因NlTPS在制备几丁质代谢酶活性抑制剂中的应用。
8.如权利要求7所述的应用,其特征在于所述几丁质代谢酶为几丁质酶或几丁质合成酶。
9.一种权利要求1所述几丁质代谢调控基因NlTPS在制备防治害虫药物中的应用。
10.如权利要求7所述应用,其特征在于所述药物为防治褐飞虱药物。
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