飞蝗漆酶基因mRNA表达及功能研究
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摘要
漆酶(Laccase)属于酚氧化酶,是一种结合多个铜离子的蛋白质,可将酚类物质(如多巴胺)氧化成相应的醌,在昆虫的生长发育和变态过程中发挥着重要作用。漆酶主要参与昆虫表皮鞣化,促进表皮的硬化和暗化,这个过程对于昆虫的生长发育至关重要。直翅目昆虫飞蝗是重要的农业害虫,常暴发成灾,对农业生产构成严重威胁。目前,漆酶在分子水平上的研究主要集中在完全变态昆虫,而在不完全变态昆虫中尚未见报道。本文以飞蝗Locusta migratoria为研究对象,对漆酶基因(Laccase)进行了分子特性及功能研究,这对深入了解漆酶在飞蝗表皮形成过程中的作用提供了基础资料,丰富了目前昆虫漆酶的研究内容,同时为研发以该酶为靶标的新型飞蝗绿色防控新技术提供科学依据。本文主要研究内容有如下三个方面：
一、飞蝗漆酶基因的克隆及序列分析
基于飞蝗表达序列标签(Expressing sequence tag, ESTs)数据库,采用生物信息学方法获得2条漆酶ESTs,经比对确定其分别为Laccasel和Laccase2cDNA序列片段,分别将其命名为LmLacl和LmLac2。其中LmLacl的部分核苷酸序列长为757bp,编码252个氨基酸；LmLac2的部分核苷酸序列长为509bp,采用RACE技术克隆获得2026bp LmLac2cDNA序列,其中开放式阅读框(ORF)为1866bp,编码621个氨基酸,3’非编码区(3’-UTR)的长度为160bp。
二、飞蝗漆酶基因在不同发育阶段及不同组织部位的mRNA表达分析
采用qPCR技术对漆酶基因在不同龄期和不同组织部位的mRNA表达水平进行了分析。结果显示：LmLacl主要在中肠表达,而LmLac2在体表特异性表达；LmLacl在各个龄期都有表达,其中在卵期表达量最低,成虫期第5天表达量达到最高；LmLac2在5龄第一天不同时间段的表皮中(0h,2h,4h,8h,24h)都有表达,且随着体表暗化程度的加深,表达量呈上升趋势,之后,LmLac2在5龄每一天均有表达,虽然表达量最高的第1天是表达量最低的第5天的3.9倍,但统计学分析无显著性差异。
三、飞蝗漆酶基因的功能研究
采用RNAi技术研究飞蝗漆酶基因在生长发育过程中的功能。飞蝗4龄若虫注射dsRNA(dsLmLacl/dsLmLac2)后,检测到目标基因的表达均被有效沉默。dsLmLacl注射组与dsGFP注射组相比,体壁鞣化程度相同,且无致死现象；dsLmLac2注射组中,若虫蜕至下一龄期均出现体色变淡,虫体变软,活动能力下降等现象。为了进一步研究LmLac2基因沉默后是否对之后各发育龄期若虫的表皮鞣化都有影响,选取2龄第1天若虫注射dsRNA,之后取每一龄期的表皮做石蜡切片观察分析,发现dsLmLac2注射组的若虫在蜕至3龄和4龄后与对照组相比,其表皮明显变薄,但蜕至5龄的若虫表皮厚度与对照组无差异。这些结果表明,漆酶LmLacl不是表皮鞣化所必需的,而LmLac2对飞蝗表皮的硬化和暗化起着重要的作用。
四、中华稻蝗几丁质合成酶1基因的mRNA表达及功能
根据已知中华稻蝗几丁质合成酶1基因(OcCHSl)保守区域部分cDNA序列(GenBank登录号：HM214491),运用RT-PCR和qPCR技术分析mRNA表达特性,采用RNA干扰技术研究其生物学功能。结果表明,OcCHSl在中华稻蝗各龄期都有表达,其体表为高表达,其次是气管,其它组织部位表达量低。RNA干扰实验发现,4龄第4天若虫注射OcCHSl的双链RNA(dsRNA)后,与对照组相比, OcCHSl mRNA表达量被沉默了70.8%,稻蝗出现蜕皮延迟、不能完成蜕皮或腹部皱缩死亡等现象,注射dsOcCHSl组死亡率为85.2%,与对照组(7.4%)相比差异显著。这些结果表明：几丁质合成酶1主要参与体表和气管几丁质的合成,对中华稻蝗的生长发育具有重要作用,RNA干扰使该基因沉默后可导致虫体死亡。
Laccases, a member of phenoloxidase, are copper-containing oxidase enzymes. Laccases oxidize catechols (eg.dopamine) to corresponding quinines, which plays an important role in insect growing development and metamorphosis. In insects, most researches on laccases have focused on the main role in cuticle tanning (sclerotization and pigmentation). Locusta migratoria, a member of orthopterans, is the important agriculture pest, the outbreak of locusts has made a serious effect on agricultural production. So far, the research concerning insect laccase genes mainly focuses on the holometabolous insects, while there is no information of laccase genes from hemimetabolous insects. In this thesis, we focused on molecular characterization and functions of laccase genes from L. migratoria, a typical hemimetabolous insects. The results will provide the theoretical basis for gaining further insight into the biological function of LmLacs. These findings supported our notion that LmLac could potentially serve as an excellent target for developing novel strategies for controlling this important pest. The contents are as follows:
1. cDNA cloning and sequences analysis of LmLacs
Two expressing sequence tags (ESTs) of Laccase were obtained by bioinformatics methods from the database of ESTs in L. migratoria (LocustDB). Blast analysis showed that they were the partial cDNA sequences of Laccase1and Laccase2. The length of cDNA fragment of LmLacl is757bp, encoding252amino acid residues; the length of cDNA fragment of LmLac2is507bp. The full length cDNA of LmLac2was amplified by RACE-PCR, obtained cDNA consists of2026nucleotides that include a1866-bp open reading frame (ORF) encoding621amino acid residues and a160-bp non-coding region at the3'-end of the cDNA.
2. Tissue distribution and developmental expression patterns of LmLacs
The5th-instar nymphs of L. migratoria were dissected for tissue distribution analysis, qPCR revealed that LmLacl was highly expressed in the midgut, while LmLac2was mainly expressed in the integument. Developmental expression patterns by qPCR analyses showed LmLacl was expressed at all life stages of the locusts. The expression of LmLac2was detected during tanning time (from0h to24h after newly molt to5th-instar nymphs), the results indicated mRNA expression of LmLac2was gradually increased when nymphs body colour became dark brown, the expression reached highest level at24h. After that, LmLac2was expressed in all5th-instar developmental stages, and there was no significant difference.
3. Functional analysis of two Laccases of L. migratoria
We performed RNA interference experiments to investigate biological function of LmLacl and LmLac2in insect development. dsRNA was synthesized and injected into the newly molt4th-instar nymphs, the expression level of both genes were significantly decreased after dsRNA injection. The dsLm/ac2-injected locusts failed to tan, and showed soft-body, while no observable phynotypes were found in dsLmLacl injection locusts; Paraffin section was performed to study the histological structures of integuments, dsLmLac2was injected into the2nd-instar (day1) nymphs and the dsLm/ac2-injected locusts appeared thinner procuticle compared to dsGFP-injected locusts when dsRNA-injected nymphs molted to3rd-,4th-instar nymphs, however, the thickness of cuticle in5th-instar nymphs is similar to the control locusts. The results presented here indicated laccase2could be involved in cuticle tanning, laccase1appeared no contribution for cuticle tanning.
4. mRNA expression and function of chitin synthasel from Oxya chiensis (Thunberg)
Based on the known partial conservative cDNA fragment of OcCHSl (GenBank accession number:HM214491), primers were designed for mRNA expression analysis. The developmental and tissue-specific expression patterns of OcCHS1were determined by RT-PCR and qPCR. RNAi experiment was performed to explore the biological function. OcCHSl was expressed at all the developmental stages. Tissue-specific analysis showed that OcCHSl was highly expressed in the integument, followed in the trachea, and lowest in other tissues. RNAi-based gene silencing experiment showed that the fourth day of the4th instar nymphs injected by dsOcCHSJ could repress the transcript levels of OcCHSl by70.8%compared to those in the control nymphs. Moreover, dsRNA injections significantly increased abnormalities, such as stunted development, uncompleted molting, crimpy abdomen and eventually died. Consequentially,85.2%of mortalities were observed, while the mortality in the control nymphs was only7.4%. OcCHSl is essential for development and mainly responsible for chitin synthesis in cuticle and trachea of O. chinensis. Moreover, silencing of chitin synthase1gene by RNAi is lethal to O. chinensis.
一、飞蝗漆酶基因的克隆及序列分析
基于飞蝗表达序列标签(Expressing sequence tag, ESTs)数据库,采用生物信息学方法获得2条漆酶ESTs,经比对确定其分别为Laccasel和Laccase2cDNA序列片段,分别将其命名为LmLacl和LmLac2。其中LmLacl的部分核苷酸序列长为757bp,编码252个氨基酸；LmLac2的部分核苷酸序列长为509bp,采用RACE技术克隆获得2026bp LmLac2cDNA序列,其中开放式阅读框(ORF)为1866bp,编码621个氨基酸,3’非编码区(3’-UTR)的长度为160bp。
二、飞蝗漆酶基因在不同发育阶段及不同组织部位的mRNA表达分析
采用qPCR技术对漆酶基因在不同龄期和不同组织部位的mRNA表达水平进行了分析。结果显示：LmLacl主要在中肠表达,而LmLac2在体表特异性表达；LmLacl在各个龄期都有表达,其中在卵期表达量最低,成虫期第5天表达量达到最高；LmLac2在5龄第一天不同时间段的表皮中(0h,2h,4h,8h,24h)都有表达,且随着体表暗化程度的加深,表达量呈上升趋势,之后,LmLac2在5龄每一天均有表达,虽然表达量最高的第1天是表达量最低的第5天的3.9倍,但统计学分析无显著性差异。
三、飞蝗漆酶基因的功能研究
采用RNAi技术研究飞蝗漆酶基因在生长发育过程中的功能。飞蝗4龄若虫注射dsRNA(dsLmLacl/dsLmLac2)后,检测到目标基因的表达均被有效沉默。dsLmLacl注射组与dsGFP注射组相比,体壁鞣化程度相同,且无致死现象；dsLmLac2注射组中,若虫蜕至下一龄期均出现体色变淡,虫体变软,活动能力下降等现象。为了进一步研究LmLac2基因沉默后是否对之后各发育龄期若虫的表皮鞣化都有影响,选取2龄第1天若虫注射dsRNA,之后取每一龄期的表皮做石蜡切片观察分析,发现dsLmLac2注射组的若虫在蜕至3龄和4龄后与对照组相比,其表皮明显变薄,但蜕至5龄的若虫表皮厚度与对照组无差异。这些结果表明,漆酶LmLacl不是表皮鞣化所必需的,而LmLac2对飞蝗表皮的硬化和暗化起着重要的作用。
四、中华稻蝗几丁质合成酶1基因的mRNA表达及功能
根据已知中华稻蝗几丁质合成酶1基因(OcCHSl)保守区域部分cDNA序列(GenBank登录号：HM214491),运用RT-PCR和qPCR技术分析mRNA表达特性,采用RNA干扰技术研究其生物学功能。结果表明,OcCHSl在中华稻蝗各龄期都有表达,其体表为高表达,其次是气管,其它组织部位表达量低。RNA干扰实验发现,4龄第4天若虫注射OcCHSl的双链RNA(dsRNA)后,与对照组相比, OcCHSl mRNA表达量被沉默了70.8%,稻蝗出现蜕皮延迟、不能完成蜕皮或腹部皱缩死亡等现象,注射dsOcCHSl组死亡率为85.2%,与对照组(7.4%)相比差异显著。这些结果表明：几丁质合成酶1主要参与体表和气管几丁质的合成,对中华稻蝗的生长发育具有重要作用,RNA干扰使该基因沉默后可导致虫体死亡。
Laccases, a member of phenoloxidase, are copper-containing oxidase enzymes. Laccases oxidize catechols (eg.dopamine) to corresponding quinines, which plays an important role in insect growing development and metamorphosis. In insects, most researches on laccases have focused on the main role in cuticle tanning (sclerotization and pigmentation). Locusta migratoria, a member of orthopterans, is the important agriculture pest, the outbreak of locusts has made a serious effect on agricultural production. So far, the research concerning insect laccase genes mainly focuses on the holometabolous insects, while there is no information of laccase genes from hemimetabolous insects. In this thesis, we focused on molecular characterization and functions of laccase genes from L. migratoria, a typical hemimetabolous insects. The results will provide the theoretical basis for gaining further insight into the biological function of LmLacs. These findings supported our notion that LmLac could potentially serve as an excellent target for developing novel strategies for controlling this important pest. The contents are as follows:
1. cDNA cloning and sequences analysis of LmLacs
Two expressing sequence tags (ESTs) of Laccase were obtained by bioinformatics methods from the database of ESTs in L. migratoria (LocustDB). Blast analysis showed that they were the partial cDNA sequences of Laccase1and Laccase2. The length of cDNA fragment of LmLacl is757bp, encoding252amino acid residues; the length of cDNA fragment of LmLac2is507bp. The full length cDNA of LmLac2was amplified by RACE-PCR, obtained cDNA consists of2026nucleotides that include a1866-bp open reading frame (ORF) encoding621amino acid residues and a160-bp non-coding region at the3'-end of the cDNA.
2. Tissue distribution and developmental expression patterns of LmLacs
The5th-instar nymphs of L. migratoria were dissected for tissue distribution analysis, qPCR revealed that LmLacl was highly expressed in the midgut, while LmLac2was mainly expressed in the integument. Developmental expression patterns by qPCR analyses showed LmLacl was expressed at all life stages of the locusts. The expression of LmLac2was detected during tanning time (from0h to24h after newly molt to5th-instar nymphs), the results indicated mRNA expression of LmLac2was gradually increased when nymphs body colour became dark brown, the expression reached highest level at24h. After that, LmLac2was expressed in all5th-instar developmental stages, and there was no significant difference.
3. Functional analysis of two Laccases of L. migratoria
We performed RNA interference experiments to investigate biological function of LmLacl and LmLac2in insect development. dsRNA was synthesized and injected into the newly molt4th-instar nymphs, the expression level of both genes were significantly decreased after dsRNA injection. The dsLm/ac2-injected locusts failed to tan, and showed soft-body, while no observable phynotypes were found in dsLmLacl injection locusts; Paraffin section was performed to study the histological structures of integuments, dsLmLac2was injected into the2nd-instar (day1) nymphs and the dsLm/ac2-injected locusts appeared thinner procuticle compared to dsGFP-injected locusts when dsRNA-injected nymphs molted to3rd-,4th-instar nymphs, however, the thickness of cuticle in5th-instar nymphs is similar to the control locusts. The results presented here indicated laccase2could be involved in cuticle tanning, laccase1appeared no contribution for cuticle tanning.
4. mRNA expression and function of chitin synthasel from Oxya chiensis (Thunberg)
Based on the known partial conservative cDNA fragment of OcCHSl (GenBank accession number:HM214491), primers were designed for mRNA expression analysis. The developmental and tissue-specific expression patterns of OcCHS1were determined by RT-PCR and qPCR. RNAi experiment was performed to explore the biological function. OcCHSl was expressed at all the developmental stages. Tissue-specific analysis showed that OcCHSl was highly expressed in the integument, followed in the trachea, and lowest in other tissues. RNAi-based gene silencing experiment showed that the fourth day of the4th instar nymphs injected by dsOcCHSJ could repress the transcript levels of OcCHSl by70.8%compared to those in the control nymphs. Moreover, dsRNA injections significantly increased abnormalities, such as stunted development, uncompleted molting, crimpy abdomen and eventually died. Consequentially,85.2%of mortalities were observed, while the mortality in the control nymphs was only7.4%. OcCHSl is essential for development and mainly responsible for chitin synthesis in cuticle and trachea of O. chinensis. Moreover, silencing of chitin synthase1gene by RNAi is lethal to O. chinensis.
引文
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