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目的筛选并鉴定埃及伊蚊(Aedes aegypti)全基因组中的yellow基因,分析其基因结构、基因进化以及在不同发育时期和不同组织的表达谱。方法运用模式昆虫黑腹果蝇(Drosophila melanogaster)Dmyellow基因(Gen Bank登录号为AAF45497)王浆主蛋白(MRJP)结构域氨基酸序列,在埃及伊蚊全基因组数据库中筛选并鉴定埃及伊蚊yellow基因家族,采用Ex PASy在线相关工具包分析其理化性质和结构域,运用Signal P4.1预测信号肽,结合使用DNAstar、MAGA6.0和Gene Doc软件包进行同源性比对、保守性分析和系统进化树构建。提取埃及伊蚊总RNA,合成c DNA,利用实时荧光定量PCR(q RT-PCR)法对埃及伊蚊不同发育时期(卵、Ⅰ~Ⅳ龄幼虫、蛹以及未吸血雌蚊和雄蚊)和未吸血雌蚊不同组织(唾液腺、中肠、脂肪体和卵巢)的yellow基因表达谱进行相对定量分析。结果从埃及伊蚊全基因组中筛选出12条yellow基因(Aa-yellow、Aa-yellow-b、Aayellow-c、Aa-yellow-d、Aa-yellow-e、Aa-yellow-f2、Aa-yellow-fb、Aa-yellow-fc、Aa-yellow-g、Aa-yellow-g2、Aa-yellow-h和Aa-yellow-x)。生物信息学分析结果显示,12条yellow基因均具有MRJP结构域和信号肽序列。同源性比对结果显示,埃及伊蚊yellow基因家族成员间同源性较低,为15%~49%;但其保守域间同源性较高,可达60%。系统进化树分析结果显示,12个YELLOW蛋白分为5个亚家族,其中11个在黑腹果蝇中均有直系同源蛋白存在。q RT-PCR结果显示,Aa-yellow-d和Aa-yellow-x在雄蚊中高表达(P<0.01或P<0.05),相对表达量分别为0.018 9和0.023 5;Aa-yellow-fc在雌蚊和唾液腺中特异高表达(P<0.01),相对表达量分别为0.024 8和0.034 9;Aa-yellow-f2在Ⅱ龄幼虫中特异高表达(P<0.01),相对表达量为0.093 4;Aa-yellow、Aa-yellow-e和Aa-yellow-fb在Ⅲ龄幼虫中特异高表达(P<0.01),相对表达量分别为0.562 1、0.004 4和0.008 4;Aa-yellow、Aa-yellow-e、Aa-yellow-f2、Aayellow-fb、Aa-yellow-h和Aa-yellow-x等6条基因在卵巢中高表达(P<0.01或P<0.05),相对表达量分别为0.569 4、0.027 0、0.006 5、0.001 0、0.084 8和0.015 1;除Aa-yellow-c(0.004 0)和Aa-yellow-x(0.007 4)外,其余基因几乎不在中肠表达。结论在埃及伊蚊基因组中获得的12条yellow基因同源性较低,且在不同发育时期和不同组织中表达有差异。
Objective To screen and identify the yellow gene in the genome of Aedes aegypti and to analyze the gene structure, gene evolution and the expression profile in different developmental stages and different tissues. Methods The amino acid sequence of Drosophila melanogaster Dmyellow gene (GenBank accession number AAF45497) was used to screen and identify the Aedes aegypti yellow gene family in the Aedes aegypti genome-wide database , Analyzed its physico-chemical properties and domains using the Ex PASy online-related kit, predicted signal peptides using Signal P4.1, used homology alignments, conserved analyzes and phylogenetic analyzes using DNAstar, MAGA6.0 and Gene Doc software packages Tree construction. The total RNA of Aedes aegypti was extracted to synthesize c DNA. The expression of c DNA in Aedes albopictus was detected by real-time quantitative PCR (q RT-PCR) The yellow gene expression profile of different tissues (salivary gland, midgut, fat body and ovary) of the sucking female macaques was comparatively quantified. Results A total of 12 yellow genes (Aa-yellow, Aa-yellow-b, Aayellow-c, Aa-yellow-d, Aa-yellow-e, Aa-yellow-f2, Aa-yellow -fb, Aa-yellow-fc, Aa-yellow-g, Aa-yellow-g2, Aa-yellow-h and Aa-yellow-x). Bioinformatics analysis showed that all 12 yellow genes had MRJP domain and signal peptide sequence. Homology comparison showed that the homology of members of the Aedes aegypti yellow gene family was low from 15% to 49%, but their conserved homology was as high as 60%. Phylogenetic tree analysis showed that 12 YELLOW proteins were divided into 5 subfamilies, of which 11 had orthologous proteins in Drosophila melanogaster. q RT-PCR results showed that Aa-yellow-d and Aa-yellow-x were highly expressed in male mosquitoes (P <0.01 or P <0.05) with relative expression levels of 0.018 9 and 0.023 5, respectively. The expression of Aa-yellow-f2 was highly expressed in the second instar larvae (P <0.01), and the relative expression level was 0.093 4 ; Aa-yellow, Aa-yellow-e and Aa-yellow-fb were highly expressed in the third instar larvae (P <0.01), and the relative expression levels were 0.562, 0.004 4 and 0.008 4, Six genes of yellow-e, Aa-yellow-f2, Aayellow-fb, Aa-yellow-h and Aa-yellow-x were highly expressed in ovary (P <0.01 or P <0.05) , 0.027 0,0.006 5,0.001 0,0.084 8 and 0.015 1, except for Aa-yellow-c (0.004 0) and Aa-yellow-x (0.007 4). Conclusion The 12 yellow genes obtained from the Aedes aegypti genome have lower homology and have different expression in different developmental stages and different tissues.