拟黑多刺蚁肌细胞增强因子2与肌钙蛋白Ⅰ亚基基因的克隆及其在发育中的表达研究
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摘要
拟黑多刺蚁(Polyrhachis vicina Roger)是一种重要的经济昆虫,在分类学上隶属于节肢动物门、昆虫纲、膜翅目、胡蜂总科、蚁科、多刺蚁属。其个体发育过程中经历胚胎期、幼虫期、蛹期、成虫期等各个阶段,且是一种社会性昆虫,群体内存在严格的品级分化。拟黑多刺蚁资源较易获得,实验室养殖较为方便,因此是一种独特的进行个体发育及分化研究的实验材料。
本研究以拟黑多刺蚁为实验材料,通过RT-PCR、分子克隆、生物信息学分析、荧光实时定量RT-PCR等技术首次对与其肌肉发育及肌肉生理机能相关的肌细胞增强因子2(Mef2)基因和肌钙蛋白Ⅰ亚基(TnⅠ)基因分别进行了相关研究。主要获得了以下研究结果：
1通过实验获得了拟黑多刺蚁Mef2基因两种剪接体cDNA序列,其长度分别为2074bp和1793bp,主要在5'末端存在一定差异。通过ORF搜索发现,其编码区长度为1419bp,编码472氨基酸残基蛋白,且两种剪接体具有相同的ORF。将两种剪接体cDNA序列上传至美国国立生物技术信息中心(NCBI)GenBank数据库,获得登陆号分别为GQ386849和GQ386850。
2对预测的PvMEF2蛋白进行同源性和系统进化分析,发现其N末端具高度保守的MADS和MEF2结构域,保守区域与其它昆虫相关序列一致性可达100%。构建的系统进化树中,膜翅目昆虫拟黑多刺蚁与意大利蜜蜂(Apis mellifera)和金小蜂(Nasonia vitripennis)聚为一支,进一步反映了各类群之间的进化关系。
3 PvMEF2蛋白的理论等电点和分子量分别是6.83和50191.5Da,其分子式为C2168H3392N6528O714S16，原子总数为6918。对其翻译后修饰分析发现,共有40个可能O型糖基化修饰位点和2个可能N型糖基化修饰位点；磷酸化修饰位点分别包括丝氨酸的磷酸化位点30个,苏氨酸6个,酪氨酸6个；目前为止还未发现其信号肽序列；该蛋白无跨膜区,为非跨膜蛋白。
4蛋白结构预测结果表明,PvMEF2 N末端存在螺旋、折叠和无规则卷曲等多种形式,这可能与MEF2通过N末端的结构形式与其它蛋白结合而发挥一定生理功能有关。
5相对实时定量分析结果表明,在不同的发育阶段虫体及成虫不同品级中,PvMef2基因均有表达,说明其在拟黑多刺蚁发育过程中持续具有调控作用。但其表达量有一定差异。在幼虫期,一龄幼虫中表达量最高,接下来的儿期幼虫呈下降趋势,而至蛹期时,其表达量又略有上升。成虫中Mef2在雄蚁中的表达量要高于其它两个品级的。不同的表达量可能影响到对不同肌肉发生相关基因的调控,从而影响肌肉发生。两种剪接体均有表达,但其表达量有一定的差别,不同剪接体在肌肉发生中的具体调控作用有待于进一步研究。
6获得两条长度分别为1224bp和799bp的拟黑多刺蚁TnⅠ基因不同剪接体cDNA序列。两条序列在3'末端存在一定差异,其开放阅读框分别为660bp和615bp,分别编码219氨基酸和204氨基酸蛋白。将序列分别上传至美国国立生物技术信息中心(NCBI) GenBank数据库,获得登陆号分别为HM484304和HQ230044。
7获得的TNI蛋白序列均与意大利蜜蜂(Apis mellifera)等昆虫同源序列具有较高的一致性。系统进化树中膜翅目昆虫拟黑多刺蚁与意大利蜜蜂聚在一起,再与其它昆虫聚为一类,各分支反映了不同类群之间的亲缘关系与进化关系,但不能明确说明各蛋白亚型之间的关系。
8 TNI两种蛋白亚型理化性质接近,并与意大利蜜蜂的相似。其翻译后修饰可能位点较少,仅分别具有1个O型糖基化修饰及11个磷酸化位点。分析发现该蛋白无跨膜区,为非跨膜蛋白。
9 TNI两种蛋白亚型二级结构主要包括了螺旋和无规则卷曲等区域,而没有明显的折叠区。其三级结构具明显的3个螺旋区。
10实时定量分析表明,TnⅠ 基因在拟黑多刺蚁各个发育阶段均有表达,但不同阶段的表达量有一定差别,可能与各个时期的肌肉结构及生理机能特点有关。不同亚型TNI在各个阶段的存在情况及功能仍需进一步的研究。
相关基因研究表明,在拟黑多刺蚁不同发育阶段及不同品级中,其肌肉分化与肌肉功能的调控与Mef2基因及TnⅠ基因存在一定关系,为研究昆虫肌肉发生及肌肉机能提供了理论基础。但其具体调控机制及基因的确切功能有待于进一步研究。
The ant Polyrhachis vicina Roger is one of the important economic insects in China, which belongs to the genus of Polyrhachis (Hymenoptera, Formicidae). There are four main stages in the ontogenesis, described as embryo, larva, pupa and adult. Also because of the characteristic of caste differentiation, the ant is gradually becoming a special experimental material for the research of development and differentiation.
Methods of RT-PCR, molecular cloning, bioinformatics and real-time quantitative PCR are first used to study the Mef2 and TnI of Polyrhachis vicina to understand the development or the physiological function of muscles. The main results are as follows:
1) Two isoforms (isoform A and B) of PvMef2 cDNA sequence were obtained which are 2074bp and 1793bp respectively. The two sequences which are only different in 5'-UTR possess a same ORF of 1419bp encoding a 472-amino acid protein. All of the two sequences were submitted to the GenBank and assigned the accession numbers:GQ386849 and GQ386850.
2) The deduced protein of PvMef2 holds highly conserved MADS and MEF2 domains which share about 100% identity with the corresponding regions of some other insects. Results of phylogenetic analysis show that PvMEF2 is most closely related to MEF2 of Apis mellifera and Nasonia vitripennis and the tree illustrates the evolutionary relationship of different selected animals.
3) The theoretical isoelectric point (PI) and molecular weight of MEF2protein were 6.83 and 50191.5 Da respectively. Its chemical formula is C2168H3392N628O714S16, comprising 6918 atoms. Analysis of post-translational modification shows that there are 40 possible O-glycosylation sites,2 possible N-glycosylation sites and 42 phosphorylation sites including 30 Serine sites,6 threonine sites and 6 Tyrosine sites, and no signal peptide and transmembrane domain in the sequence.
4) Prediction of protein structure shows that N-terminal of PvMEF2 contains helix, sheet and coil,which is related to the physiological function of PvMEF2 by mean of combining other factors.
5) Results of real-time quantitative RT-PCR show expression levels of different stages change constantly with distinctly high levels in first instars relative to the following stages, while in the adult ants the PvMef2 gene was highly expressed in males compared with workers and females. Different expression levels will affect the regulation to different Mef2-regulated genes.The difference in expression levels of two PvMef2 mRNA isoforms can also be detected according to the results. Those results suggest that PvMef2 is developmentally and caste-specifically regulated at the level of transcription.
6) Two isoforms (isoform A and B) of PvTnl cDNA sequence were obtained which are different in 3'-UTR. The two sequences of 2074bp and 1793bp in length contain 660bp and 615bp ORFs respectively, encoding 219 and 204 amino acid protein. All the sequences were submitted to the GenBank and assigned the accession numbers:HM484304 and HQ230044.
7) The obtained Tnl protein sequences share high identity with some insects such as Apis mellifera. Phylogenetic tree shows that Polyrhachis vicina is grouped with Apis mellifera, and then they were grouped with other insects, showing the relationship among different groups. But the relationship among the protein subtypes of different species cannot be learned from the tree.
8) There is closed physicochemical property between different TNI subtypes which is also similar to that of Apis mellifera. Few posttranslational modification sites could be found besides 1 O-glycosylation site and 11 phosphorylation sites, and no transmembrane domain was found.
9) The secondary structure of the two TNI subtypes contains helix and coil, but no distinct sheet. And 3 helix regions can be found respectively in their tertiary structures.
10) Expression of Tnl gene can be detected but different in developmental stages of Polyrhachis vicina by means of real-time quantitative RT-PCR. The expression level may be related to the muscular structure and their physiological function of different stages. But all need farther studies.
The regulation of muscle differentiation and function may be related to Mef2 and Tnl in different developmental stages and different castes. Some theoretical foundation may be learned from our reports, but more researches are needed to investigate their specific mechanism and function.
本研究以拟黑多刺蚁为实验材料,通过RT-PCR、分子克隆、生物信息学分析、荧光实时定量RT-PCR等技术首次对与其肌肉发育及肌肉生理机能相关的肌细胞增强因子2(Mef2)基因和肌钙蛋白Ⅰ亚基(TnⅠ)基因分别进行了相关研究。主要获得了以下研究结果：
1通过实验获得了拟黑多刺蚁Mef2基因两种剪接体cDNA序列,其长度分别为2074bp和1793bp,主要在5'末端存在一定差异。通过ORF搜索发现,其编码区长度为1419bp,编码472氨基酸残基蛋白,且两种剪接体具有相同的ORF。将两种剪接体cDNA序列上传至美国国立生物技术信息中心(NCBI)GenBank数据库,获得登陆号分别为GQ386849和GQ386850。
2对预测的PvMEF2蛋白进行同源性和系统进化分析,发现其N末端具高度保守的MADS和MEF2结构域,保守区域与其它昆虫相关序列一致性可达100%。构建的系统进化树中,膜翅目昆虫拟黑多刺蚁与意大利蜜蜂(Apis mellifera)和金小蜂(Nasonia vitripennis)聚为一支,进一步反映了各类群之间的进化关系。
3 PvMEF2蛋白的理论等电点和分子量分别是6.83和50191.5Da,其分子式为C2168H3392N6528O714S16，原子总数为6918。对其翻译后修饰分析发现,共有40个可能O型糖基化修饰位点和2个可能N型糖基化修饰位点；磷酸化修饰位点分别包括丝氨酸的磷酸化位点30个,苏氨酸6个,酪氨酸6个；目前为止还未发现其信号肽序列；该蛋白无跨膜区,为非跨膜蛋白。
4蛋白结构预测结果表明,PvMEF2 N末端存在螺旋、折叠和无规则卷曲等多种形式,这可能与MEF2通过N末端的结构形式与其它蛋白结合而发挥一定生理功能有关。
5相对实时定量分析结果表明,在不同的发育阶段虫体及成虫不同品级中,PvMef2基因均有表达,说明其在拟黑多刺蚁发育过程中持续具有调控作用。但其表达量有一定差异。在幼虫期,一龄幼虫中表达量最高,接下来的儿期幼虫呈下降趋势,而至蛹期时,其表达量又略有上升。成虫中Mef2在雄蚁中的表达量要高于其它两个品级的。不同的表达量可能影响到对不同肌肉发生相关基因的调控,从而影响肌肉发生。两种剪接体均有表达,但其表达量有一定的差别,不同剪接体在肌肉发生中的具体调控作用有待于进一步研究。
6获得两条长度分别为1224bp和799bp的拟黑多刺蚁TnⅠ基因不同剪接体cDNA序列。两条序列在3'末端存在一定差异,其开放阅读框分别为660bp和615bp,分别编码219氨基酸和204氨基酸蛋白。将序列分别上传至美国国立生物技术信息中心(NCBI) GenBank数据库,获得登陆号分别为HM484304和HQ230044。
7获得的TNI蛋白序列均与意大利蜜蜂(Apis mellifera)等昆虫同源序列具有较高的一致性。系统进化树中膜翅目昆虫拟黑多刺蚁与意大利蜜蜂聚在一起,再与其它昆虫聚为一类,各分支反映了不同类群之间的亲缘关系与进化关系,但不能明确说明各蛋白亚型之间的关系。
8 TNI两种蛋白亚型理化性质接近,并与意大利蜜蜂的相似。其翻译后修饰可能位点较少,仅分别具有1个O型糖基化修饰及11个磷酸化位点。分析发现该蛋白无跨膜区,为非跨膜蛋白。
9 TNI两种蛋白亚型二级结构主要包括了螺旋和无规则卷曲等区域,而没有明显的折叠区。其三级结构具明显的3个螺旋区。
10实时定量分析表明,TnⅠ 基因在拟黑多刺蚁各个发育阶段均有表达,但不同阶段的表达量有一定差别,可能与各个时期的肌肉结构及生理机能特点有关。不同亚型TNI在各个阶段的存在情况及功能仍需进一步的研究。
相关基因研究表明,在拟黑多刺蚁不同发育阶段及不同品级中,其肌肉分化与肌肉功能的调控与Mef2基因及TnⅠ基因存在一定关系,为研究昆虫肌肉发生及肌肉机能提供了理论基础。但其具体调控机制及基因的确切功能有待于进一步研究。
The ant Polyrhachis vicina Roger is one of the important economic insects in China, which belongs to the genus of Polyrhachis (Hymenoptera, Formicidae). There are four main stages in the ontogenesis, described as embryo, larva, pupa and adult. Also because of the characteristic of caste differentiation, the ant is gradually becoming a special experimental material for the research of development and differentiation.
Methods of RT-PCR, molecular cloning, bioinformatics and real-time quantitative PCR are first used to study the Mef2 and TnI of Polyrhachis vicina to understand the development or the physiological function of muscles. The main results are as follows:
1) Two isoforms (isoform A and B) of PvMef2 cDNA sequence were obtained which are 2074bp and 1793bp respectively. The two sequences which are only different in 5'-UTR possess a same ORF of 1419bp encoding a 472-amino acid protein. All of the two sequences were submitted to the GenBank and assigned the accession numbers:GQ386849 and GQ386850.
2) The deduced protein of PvMef2 holds highly conserved MADS and MEF2 domains which share about 100% identity with the corresponding regions of some other insects. Results of phylogenetic analysis show that PvMEF2 is most closely related to MEF2 of Apis mellifera and Nasonia vitripennis and the tree illustrates the evolutionary relationship of different selected animals.
3) The theoretical isoelectric point (PI) and molecular weight of MEF2protein were 6.83 and 50191.5 Da respectively. Its chemical formula is C2168H3392N628O714S16, comprising 6918 atoms. Analysis of post-translational modification shows that there are 40 possible O-glycosylation sites,2 possible N-glycosylation sites and 42 phosphorylation sites including 30 Serine sites,6 threonine sites and 6 Tyrosine sites, and no signal peptide and transmembrane domain in the sequence.
4) Prediction of protein structure shows that N-terminal of PvMEF2 contains helix, sheet and coil,which is related to the physiological function of PvMEF2 by mean of combining other factors.
5) Results of real-time quantitative RT-PCR show expression levels of different stages change constantly with distinctly high levels in first instars relative to the following stages, while in the adult ants the PvMef2 gene was highly expressed in males compared with workers and females. Different expression levels will affect the regulation to different Mef2-regulated genes.The difference in expression levels of two PvMef2 mRNA isoforms can also be detected according to the results. Those results suggest that PvMef2 is developmentally and caste-specifically regulated at the level of transcription.
6) Two isoforms (isoform A and B) of PvTnl cDNA sequence were obtained which are different in 3'-UTR. The two sequences of 2074bp and 1793bp in length contain 660bp and 615bp ORFs respectively, encoding 219 and 204 amino acid protein. All the sequences were submitted to the GenBank and assigned the accession numbers:HM484304 and HQ230044.
7) The obtained Tnl protein sequences share high identity with some insects such as Apis mellifera. Phylogenetic tree shows that Polyrhachis vicina is grouped with Apis mellifera, and then they were grouped with other insects, showing the relationship among different groups. But the relationship among the protein subtypes of different species cannot be learned from the tree.
8) There is closed physicochemical property between different TNI subtypes which is also similar to that of Apis mellifera. Few posttranslational modification sites could be found besides 1 O-glycosylation site and 11 phosphorylation sites, and no transmembrane domain was found.
9) The secondary structure of the two TNI subtypes contains helix and coil, but no distinct sheet. And 3 helix regions can be found respectively in their tertiary structures.
10) Expression of Tnl gene can be detected but different in developmental stages of Polyrhachis vicina by means of real-time quantitative RT-PCR. The expression level may be related to the muscular structure and their physiological function of different stages. But all need farther studies.
The regulation of muscle differentiation and function may be related to Mef2 and Tnl in different developmental stages and different castes. Some theoretical foundation may be learned from our reports, but more researches are needed to investigate their specific mechanism and function.
引文
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