【作者】 张政；

【导师】 张建珍； 刘晓健；

【作者基本信息】 山西大学 ， 细胞生物学， 2019， 硕士

【摘要】 昆虫的蜕皮由蜕皮激素(20-hydroxyecdysone,20E)和保幼激素(Juvenile hormone,JH)协同调控。蜕皮激素通过与蜕皮激素受体EcR(Ecdysteroid receptor)和超气门蛋白USP(完全变态昆虫中称为Ultraspiracle protein,渐变态昆虫中称为RXR,Retinoid X receptor,维甲类X受体)的复合物结合,进而调控一系列下游转录因子的表达,从而调控蜕皮和变态等生理过程。蜕皮激素受体EcR的亚型EcR-B1入核需要GTP酶Ran和核转运因子2(Nuclear Transport Factor 2,NTF2)的参与。而蜕皮过程中伴随几丁质的合成和降解,磷酸乙酰氨基葡萄糖变位酶(Phosphoacetylglucosamine mutase,PAGM)是几丁质合成的关键酶之一。本文以飞蝗(Locusta migratoria)为研究对象,对上述蜕皮发育过程中4个基因(LmRXR、LmRan、LmNTF2和LmPAGM)的分子特性及其生物学功能进行研究,主要研究结果如下:1)克隆获得LmRan和LmNTF2基因cDNA开放阅读框全长序列,分别编码215个氨基酸和130个氨基酸。通过RT-qPCR方法分析LmRan和LmNTF2在五龄飞蝗不同组织部位及不同发育龄期的mRNA表达,结果表明,LmRan在前肠、翅芽、精巢和卵巢中的表达量较高,且在每个龄期均表达。LmNTF2在五龄飞蝗第1天表达最高,且在胃盲囊、中肠及后肠高表达。运用RNAi技术研究这两个基因的功能,结果表明沉默LmRan致使飞蝗100%蜕皮前死亡,且在飞蝗若虫-若虫蜕皮和若虫-成虫变态过程中均具有重要作用。但是沉默LmNTF2并未观察到可见表型。注射dsLmRan后飞蝗体壁无皮层溶离,无新表皮的形成;体重较对照组显著下降,且胃盲囊严重萎缩。进一步利用RT-qPCR方法分析发现沉默LmRan 48 h后,参与20E合成的CYP家族基因LmCYP302a1、LmCYP315a1和LmCYP314a1及几丁质代谢关键基因LmGfat和LmCHT10的表达显著下调。2)根据NCBI已公开的LmRXR-L和LmRXR-S cDNA序列(GenBank登录号:AAQ55293.1和AAF00981.1),采用RT-qPCR方法分别对LmRXR-L和LmRXR-S的mRNA表达特性进行分析,结果表明,两者均在精巢和卵巢高表达,且在五龄第2-3天高表达。RNAi实验表明LmRXR在飞蝗蜕皮和变态过程中均起到重要作用。注射dsLmRXR飞蝗体壁仍经历皮层溶离,旧表皮降解新表皮形成的过程;且新表皮的几丁质层状结构与对照组相比无显著变化;几丁质含量亦无明显差异。进一步利用RT-qPCR方法分析发现沉默LmRXR 48 h后,除LmCYP302a1下调和LmFTZ-F1上调外,其他基因的表达均未受到影响。3)克隆获得LmPAGM基因的cDNA开放阅读框全长序列,编码497个氨基酸。mRNA表达分析表明LmPAGM在五龄飞蝗不同组织均衡表达且在第8天显著高表达。RNAi实验表明注射dsLmPAGM组中LmPAGM的表达较对照组显著下调,并且约30%的飞蝗无法成功羽化为成虫,表明LmPAGM对飞蝗蜕皮起重要作用。本文的研究结果为深入解析飞蝗蜕皮发育的分子调控机制奠定了基础数据,同时为飞蝗防治提供了新的分子靶标。更多还原

【Abstract】 The ecdysis and metamorphosis are jointly regulated by 20E(20-hydroxyecdysone)and JH(Juvenile hormone)in insects.20 E binds to ecdysteroid receptor and Ultraspiracle protein(USP in holometabolous insects),which is called RXR(Retinoid X receptor)in hemimetabolous insects.Furthermore,it regulates the expression of a series of downstream transcription factors,thus regulating physiological processes such as ecdysis and metamorphosis.Nucleation of Ec R-B1,a subtype of Ec R,requires the involvement of the GTPase Ran and Nuclear Transport Factor 2(NTF2).Phosphoacetylglucosamine mutase(PAGM)is one of the key enzymes in the synthesis of chitin.In this paper,the molecular characteristics and biological functions of four genes(Lm RXR,Lm Ran,Lm NTF2 and Lm PAGM)in ecdysis and metamorphosis were studied in Locusta migratoria.The research results are as follows:1)The full-length ORF c DNA sequences of Lm Ran and Lm NTF2 genes were obtained by searching the L.migratoria transcriptome database,which encode 215 and 130 amino acids,respectively.RT-q PCR was used to analyze the m RNA expression of these two genes in different tissues and different developmental stages.The results showed that Lm Ran highly expressed in foregut,wing,testisand ovary,and was expressed in different stages.Lm NTF2 was most highly expressed on the first day of the fifth nymph of L.migratoria,and was highly expressed in gastric caecum,midgut and hindgut.The functions of these two genes were studied with RNAi technology.The result showed that after ds Lm Ran injection at 4th and 5th stages,the expression of Lm Ran in ds Lm Ran-injected locusts was significantly decreased compared with that in the control group,and all died before molting,indicating that Lm Ran gene played an important role in the metamorphosis of L.migratoria nymph to nymph molting and nymph to adult molting.After the silencing of Lm NTF2,both the experimental group and the control group were able to develop into adults.Further section histological analysis of the integument was carried out.H&E staining results showed that there was no degradation of old cuticle or formation of new cuticle after silencing Lm Ran.In addition,after silencing Lm Ran,the weight of L.migratoria was significantly lower than that of the control group,and anatomy showed that the gastriccaecum of L.migratoria was severely atrophic in the ds Lm Ran group.RT-q PCR was used to analyze the expressions of CYP family genes,20 E signaling pathway genes and key genes involved in chitin metabolism,and it was found that after silencing Lm Ran for 48 hours,the expressions of Lm CYP302a1,Lm CYP315a1 and Lm CYP314a1 of CYP family genes involved in 20 E synthesis and Lm Gfat and Lm CHT10 of key genes involved in chitin metabolism were significantly down regulated.2)According to the Lm RXR-L and Lm RXR-S c DNA sequences published in NCBI(Gen Bank number: AAQ55293.1 and AAF00981.1),specific PCR amplification primers were designed for Lm RXR-L and Lm RXR-S.RT-q PCR was used to study m RNA expression characteristics,and the results show that the Lm RXR-L and Lm RXR-S are highly expressed in testis and ovary,followed by integument,foregut,midgut,gastric caecum,hidgut,fat body,malpighian tube and wing.Both Lm RXR-L and Lm RXR-S were highly expressed at the second and third day of the fifth stages.The function of Lm RXR gene was studied by RNAi technology,and the results showed that after ds Lm RXR injection at the fourth and fifth stages,the expression of Lm RXR in the experimental group was significantly decreased compared with that in the control group,and 90% of the locusts were finally unable to successfully molting and died,and the remaining 10% of the locusts died before molting without developmental delay.However,there is no remarkably difference in the histological structure of cuticle between ds Lm RXR injected nymphs and control.The results of transmission electron microscope and chitin contents showed that there was no significant change in the chitin lamellar structure and chitin contents of the old cuticle and the new cuticle of the experimental group compared with that of the control group.The weight of ds Lm RXR-injected group was lower than the control group,and the gastric caecum was severely atrophic.RT-q PCR was used to analyze the expression of CYP family genes,20 E signaling pathway genes and key genes of chitin metabolism.The results showed that after silencing Lm RXR for 48 hours,the expression of all genes was not affected except for Lm CYP302a1,which is down regulated and Lm FTZ-F1,which is up regulated.3)The full-length ORF c DNA sequence of Lm PAGM was obtained by analyzingthe transcriptome data of L.migratoria,which encode 497 amino acids.RT-q PCR was used to analyze the expression of Lm PAGM in different tissues and different days of the fifth stages.The results showed that Lm PAGM was expressed in integument,foregut,midgut,hindgut,gastric caecum,malpighian tube,fat body and wing,and the expression increased significantly on the eighth day of the fifth stage.The biological function of Lm PAGM was explored by RNAi.The expression of ds Lm PAGM-injected group was significantly down regulated compared with the control group,and about30% of locust could not successfully molt into adults.The results of this study lay a foundation for further understanding the molecular regulatory mechanism of migratory locust molt development,and provide new molecular targets for locust control.更多还原