赤拟谷盗组蛋白乙酰转移酶Elp3基因的克隆及RNA干扰研究

发布时间：2018-04-09 16:47

本文选题：赤拟谷盗　切入点：组蛋白乙酰转移酶　出处：《扬州大学》2016年硕士论文

【摘要】：由组蛋白乙酰转移酶(histone acetyltransferase, HATs)和组蛋白去乙酰化酶(histone deacetylases, HDACs)共同调节的组蛋白乙酰化是表观遗传调控的一种重要机制。作为RNA聚合酶II(RNAP-II)延伸复合体的催化亚基,Elp3属于GNAT家族组蛋白乙酰转移酶,能够在体内和体外乙酰化组蛋白H3和H4。赤拟谷盗Tribolium castaneum(鞘翅目拟步甲科)是一种危害严重的世界性农业粮食害虫,也是所有昆虫中系统性RNA干扰最有效的昆虫。本文克隆了赤拟谷盗组蛋白乙酰转移酶Elp3基因,分析了Elp3基因在赤拟谷盗不同发育阶段的mRNA表达水平,在此基础上利用RNA干扰(RNA interference,RNAi)技术研究了其功能,研究结果对于进一步深入了解组蛋白乙酰化在昆虫生长发育中的功能和昆虫对逆境胁迫的适应机制具有重要的科学意义。通过反转录多聚酶链式反应技术(RT-PCR)克隆了赤拟谷盗Elp3基因全长cDNA序列,命名为cElp3。TcElp3开放阅读框长1776 bp,编码591个氨基酸残基,预测蛋白质分子质量为67.74kDa,等电点为8.66。高度保守的GNAT-type HAT结构域(Pfam accession no. PF00583)位于羧基端493-539位氨基酸；另一个保守的Radical SAM(S-adenosylmethionine)结构域(Pfam accession no.PF04055)位于氨基端98-346位氨基酸。多重序列比对表明,TcElp3与同为鞘翅目的中欧山松大小蠹Dendroctonus ponderosae同源基因的氨基酸序列一致性最高,为81.36%,与意大利蜜蜂Apis mellifera同源基因的氨基酸序列一致性为76.48%,与小菜蛾Plutella xylostella、黑腹果蝇Drosophila melanogaster、豌豆蚜虫Acyrthosiphon pisum和褐飞虱Nilaparvata lugens同源基因的氨基酸序列一致性分别为75.59%、75.30%、74.62%和74.54%。系统发生分析也表明,TcElp3基因与中欧山松大小蠹同源基因的亲缘关系最近。通过荧光定量PCR研究了TcElp3基因在赤拟谷盗1日龄和3日龄卵、1天、5天和20天幼虫、1天和5天蛹、1天和7天雌虫、1天和7天雄虫等不同发育阶段的mRNA表达动态。结果表明,TcElp3基因在各个发育阶段都能表达,其中3日龄卵的表达水平最低,在5天幼虫中的表达水平最高,蛹期和成虫期的表达水平无显著差异。RNA干扰实验发现,当TcElp3基因在晚期幼虫被抑制表达时,所有干扰组的赤拟谷盗幼虫都能成功进入化蛹阶段,但是大部分虫体在蛹期发育迟缓,并出现翅的畸形,98.33%的虫体在蛹期死亡。少数羽化的成虫出现后翅折叠的异常,并在次日全部死亡。RNAi后第6天,分别对干扰组、IB组和CK组幼虫进行过氧化氢处理,48小时后干扰组死亡率高达84.72%,而CK组和IB组死亡率分别为25%和26.39%。进一步研究发现,干扰组的两种过氧化氢酶基因(TcCatl和TcCat2)的表达水平相对于对照都有显著降低,其中TcCatl基因降低了49.65%,而TcCat2基因降低了85.17%。这些研究结果表明TcElp3基因在赤拟谷盗的生长发育过程中以及对氧化胁迫的适应性方面起到重要作用。
[Abstract]:The catalytic subunit Elp3, which acts as an extension complex of RNA polymerase II RNAP-II, belongs to the GNAT family of histone acetyltransferases, and can acetylated histone H3 and H4 in vivo and in vitro.Tribolium castaneum (Coleoptera Caraneum) is one of the most harmful agricultural food pests in the world. It is also the most effective insect with systematic RNA interference in all insects.In this paper, the Elp3 gene of histone acetyltransferase (Elp3) was cloned, and the expression level of Elp3 gene was analyzed at different developmental stages, and its function was studied by RNA interference RNA interference RNAi technique.The results have important scientific significance for further understanding the function of histone acetylation in insect growth and development and the adaptation mechanism of insect to stress.The full-length cDNA sequence of Elp3 gene was cloned by reverse transcriptase polymerase chain reaction (RT-PCR). It was named cElp3.TcElp3 open reading frame 1776 BP, encoding 591 amino acid residues. The predicted molecular weight was 67.74 kDa and the isoelectric point was 8.66.The highly conserved GNAT-type HAT domain Pfam accession no.PF00583) is located at the carboxyl terminal 493-539 amino acids, and another conserved Radical SAM S-adenosylmethionine domain Pfam accession no. PF04055) is located at the amino terminal 98-346 amino acids.Multiple sequence alignment showed that TcElp3 had the highest amino acid sequence identity with Dendroctonus ponderosae homologous gene of Coleoptera.The amino acid sequence of the homologous gene was 76.48, 75.59% and 74.54% with Plutella xylostella xylostella, Drosophila melanogaster, Acyrthosiphon pisum and Nilaparvata lugens, respectively.Phylogenetic analysis also showed that TcElp3 gene was most closely related to the homologous gene of Prunus sibiricus.The mRNA expression dynamics of TcElp3 gene were studied by fluorescence quantitative PCR at different developmental stages of 1 day and 3 day old eggs at 1 and 20 days of age, 1 day and 5 days, 1 day and 7 days of female larvae, respectively.The results showed that TcElp3 gene could be expressed in all stages of development, and the expression level of TcElp3 gene was the lowest in 3-day-old eggs and the highest in 5-day larva, but there was no significant difference between pupae stage and adult stage. RNA interference test showed that the expression level of TcElp3 gene was the lowest in 3-day-old eggs.When the expression of TcElp3 gene was inhibited in the late stage of larva, all the interfering larvae could successfully enter the stage of pupation, but most of them were stunted in pupal stage, and 98.33% of them died in pupal stage.A few adult Eclosion worms showed abnormal folding of their hind wings and died 6 days after all the death of RNAi the next day.After treated with hydrogen peroxide for 48 hours, the mortality rate of interference group was 84.72, while that of CK group and IB group was 25% and 26.39%, respectively.Further studies showed that the expression levels of TcCatl and TcCat2) of the two catalase genes in the interference group were significantly lower than those in the control group. The TcCatl gene decreased by 49.65 and the TcCat2 gene decreased by 85.17.These results suggest that the TcElp3 gene plays an important role in the growth and development process and the adaptability to oxidative stress.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S379.5

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