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凡纳滨对虾mTOR信号通路中重要基因的克隆和功能的初步研究
辛芳
Subtype硕士
Thesis Advisor王雷
2016-05-13
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline生物工程
Keyword凡纳滨对虾 Mtor 雷帕霉素 氨基酸
Other Abstract       本研究通过对凡纳滨对虾(Litopenaeus vannamei)进行转录组测序,并结合模式生物中mTOR信号通路的已有研究结果,分析获得了33个可能参与凡纳滨对虾mTOR信号通路调控作用的基因片段。在此基础上,从凡纳滨对虾肌肉组织中克隆得到raptoreif4e2eif4e1a三个基因的cDNA序列。raptor基因cDNA序列全长5020 bp,开放阅读框3804 bp,编码1267个氨基酸残基;eif4e2基因cDNA序列全长1069 bp,开放阅读框699 bp,编码232个氨基酸残基;eif4e1a基因cDNA序列全长3579 bp,开放阅读框627 bp,编码208个氨基酸残基。氨基酸推断序列比对和进化分析均证实,凡纳滨对虾Raptor蛋白与其他已知Raptor蛋白为同源蛋白,eIF4E2和eIF4E1A为目前已知的eIF4E家族蛋白的同源蛋白。
       利用Realtime-PCR实时荧光定量的方法研究了torraptoreif4ebpeif4e2eif4e1a五个基因在凡纳滨对虾不同组织中的表达差异,结果表明:(1)凡纳滨对虾torraptoreif4ebpeif4e2eif4e1a五个基因在眼柄、肝胰脏、肠道、胃、鳃丝和肌肉等组织中均有表达。(2)tor基因在肝胰脏、肌肉和眼柄中的表达量显著高于其在肠道、胃和鳃丝中的表达量(p<0.05);在肝胰脏中表达量显著高于眼柄中的表达量,肝胰脏和肌肉中表达量之间没有显著差异,眼柄和肌肉中的表达量之间也没有显著差异;在肠道、胃和鳃丝中,tor基因表达量之间没有显著差异。(3)raptor基因在肝胰脏中表达量显著高于其他组织(p<0.05),肠道、胃和肌肉中的表达量之间没有显著差异,但肠道中表达量显著高于眼柄和鳃丝(p<0.05),同时眼柄、胃、鳃丝和肌肉中的表达量之间没有显著差异。(4)eif4ebp基因在肌肉中的表达量极显著高于其他组织(p<0.01),鳃丝中表达量次之,在眼柄、肝胰脏、肠道和胃中的表达量之间没有显著差异且表达量都相对较少。(5)eif4e2基因在肌肉中的表达量显著高于其他组织,eif4e1a基因在肠道和肌肉中都有较高表达量,且在肠道中的表达量显著高于其在肌肉中的表达量(p<0.05)。
利用Realtime-PCR实时荧光定量的方法研究了注射雷帕霉素或氨基酸后肌肉中torraptoreif4ebpeif4e2eif4e1a五个基因表达量的变化情况,探讨了torraptoreif4ebpeif4e2eif4e1a五个基因在mTOR信号通路中的信号传递调控机制及其在细胞生长中的调控作用。研究结果表明:(1)注射雷帕霉素后2个小时内肌肉中torraptoreif4e2eif4e1a基因表达量都出现显著下降,eif4ebp基因表达量显著上升(p<0.05),说明凡纳滨对虾mTOR信号通路对雷帕霉素敏感。(2)肌肉中torraptor基因的表达量在单独注射亮氨酸4个小时内均未出现变化;在注射精氨酸或者同时注射亮氨酸和精氨酸后,肌肉中torraptor基因的表达量均在半小时出现显著上升(p<0.05)。(3)肌肉中eif4e1a基因的表达量在单独注射亮氨酸或精氨酸4个小时内均未出现变化,但在同时注射亮氨酸和精氨酸后表达量均显著增加(p<0.05);肌肉中eif4e2基因在注射亮氨酸或精氨酸,以及同时注射亮氨酸和精氨酸后表达量都明显提高(p<0.05),且同时注射亮氨酸和精氨酸后,基因表达变量明显比单独注射亮氨酸或精氨酸后变化量大。以上研究结果表明,氨基酸能够激活凡纳滨对虾mTOR信号通路中基因的转录表达,且与小鼠、斑马鱼等模式生物相似,凡纳滨对虾eif4e2eif4e1a基因能够通过mTOR信号通路接收不同氨基酸信号来调控细胞生长。
     综上所述,凡纳滨对虾mTOR信号通路对雷帕霉素敏感,且可以接收氨基酸信号调控细胞生长。本研究首次克隆了凡纳滨对虾中参与mTOR信号通路的几个关键基因的全长cDNA序列,并对其功能进行了初步研究。不仅为mTOR信号通路的系统发生研究提供了参考,而且从分子水平了解对虾的生长和代谢调控机制,对于对虾饲料配方的优化也具有重要的意义。
; On the basis of transcriptome sequencing of Litopenaeus vannamei and the existing research results of mTOR signaling in model organisms, 33 gene fragments which posablelywere involved in mTOR signalingand had regulation effect in L. vannameiwere obtained.The full length cDNA sequencesof gene raptoreif4e2 and eif4e1afrom the muscle of L. vannamei were cloned. The cDNA sequence of raptor was 5020 bp, containing a 3804 bp open reading frame, which encoded 1267 amino acid residues.The cDNA of eif4e2 was 1069 bp, containing a 699 bp open reading frame, which encoded 232 amino acid residues. The cDNA of eif4e1a was 3579 bp, containing a 627 bp open reading frame, which encoded 208 amino acid residues. The deduced Raptor, eIF4E2 and eIF4E1A amino acids sequences of L. vannamei were separately compared with other known sequences. The results confirmed their homology.
Real-time PCR was used to detect the tissue-specific expression of tor, raptor, eif4ebp, eif4e2 and eif4e1a in L. vannamei. The results showed that: (1) tor, raptor, eif4ebp,eif4e2 and eif4e1a genes were expressed in all detected tissues including eyestalk, hepatopancreas, stomach, intestine, gill and muscle, which indicated that these genes were wildly expressed. (2) The relative expression level of torin hepatopancreas, muscle and eyestalk were significantly higher than that in stomach, intestine and gill; and there was no significantly difference in the relative expression level of torin stomach, intestine and gill. (3) The relative expression level of raptorin hepatopancreas was higherthan that in other tissues (p<0.05).(4) The relative expression level of eif4ebpin muscle was significantly higher than that in other tissues (p<0.01), and the relative expression level in gill was second (p<0.05).There was no significantly difference in the relative expression level of eif4ebpin eyestalk, hepatopancreas, stomach, intestine(p<0.05).(5) The relative expression level of eif4e2in muscle was significantly higher than that in other tissues (p<0.05). The relative expression level of eif4e1a in intestine and muscle were both higher than other tissues, and that in intestine was higher than in muscle (p<0.05).
Real-time PCR was used to detect the responses ofgene tor, raptor, eif4ebp,eif4e2 and eif4e1a in the muscle of L. vannamei to injection of rapamcyin and amino acids(leucine or arginine).We discussed these five genes involved in mTOR signaling and genes eif4ebp,eif4e2 and eif4e1a involved in the regulation of cell growth. The results showed that: (1) The relative expression levels of tor, raptor, eif4e2 and eif4e1a in muscle were reduced after injection of rapamcyin, however the relative expression levels of eif4ebp was incresed (p<0.05).It is indicated that the mTOR signaling in L. vannamei was sensitive to rapamycin. (2) The relative expression levels of tor and raptor unchanged during 4 hours after injection of unitary leucine,while which increased significantly after simultaneous injection of leucine and arginine or unitary injection of arginine during half an hour (p<0.05). (3) The relative expression level of eif4e1a unchanged during 4 hours after injection of unitary leucine or arginine, while which increased significantly after simultaneous injection of leucine and arginine (p<0.05). The relative expression level of eif4e2 increased significantly after injection of unitary leucine, unitary arginine or both leucine and arginine (p<0.05), and the relative expression level of eif4e2 after simultaneous injection of leucine and arginine was significantly higher than that after injection of unitary leucine or arginine. It is indicated that eif4e2 and eif4e1a can accept the signal of amino acid and regulate the growth of cell in muscle of L. vannamei, which is similar with that in model organisms, such as mice and zebrafish.
In conclusion, the mTOR signaling in L. vannamei was sensitive to rapamycin and could receive the signals from amino acids to regulate the growth of cell in animals. It was the first time to clone the full-length cDNA sequences of several key genes in the mTOR signaling of L. vannamei. These key genes were preliminaryly studied in this study. That provided a reference to the mTOR signaling system research. It is also very meaningful to understand the shrimp growth and metabolic regulation mechanism at the molecular level, which can help to optimize the feed formula of shrimp.
Subject Area生物学
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/112509
Collection实验海洋生物学重点实验室
Affiliation中国科学院海洋研究所
Recommended Citation
GB/T 7714
辛芳. 凡纳滨对虾mTOR信号通路中重要基因的克隆和功能的初步研究[D]. 北京. 中国科学院大学,2016.
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