【作者】 张迎春；

【导师】 王建军；

【作者基本信息】 扬州大学 ， 农药学， 2013， 硕士

【摘要】 电压门控钠离子通道在细胞兴奋性传导过程中具有重要作用,也是DDT、拟除虫菊酯和新型嗯二嗪类杀虫剂的主要作用靶标。目前,对昆虫钠离子通道与抗药性关系的研究主要集中于昆虫对拟除虫菊酯的击倒抗性。棉铃虫Helicoverpa armigera (Hubner)是一种重要的棉花害虫,许多国家的棉铃虫都对拟除虫菊酯类杀虫剂产生了严重的抗性。赤拟谷盗Tribolium castaneum是鞘翅目重要储粮害虫,也是除黑腹果蝇Drosophila melanogaster、冈比亚按蚊Anopheles gambiae、家蚕Bombyx mori外第4个被全基因组测序的昆虫。本文克隆了棉铃虫和赤拟谷盗钠离子通道基因,并分析了其在不同发育阶段的mRNA表达水平,比较了其基因组结构和选择性剪接位点,研究结果对于进一步明确昆虫钠离子通道的结构和功能关系以及新型高效杀虫剂的创制具有重要的理论意义和实践价值。通过RT-PCR技术克隆了棉铃虫和赤拟谷盗钠离子通道基因全长cDNA序列,HaNav和TcNav。HaNav开放阅读框长6168bp,编码2055个氨基酸残基；TcNav开放阅读框长6138bp,编码2045个氨基酸残基。HaNav和TcNav氨基酸序列的一致性为76%,HaNav、TcNav与黑腹果蝇钠离子通道基因para氨基酸序列的一致性都为75%。HaNav和TcNav与己知昆虫钠离子通道具有共同的结构特征,包括4个典型的同源结构域(Ⅰ-Ⅳ),每个结构域含6个跨膜片段(S1-S6)。决定离子选择性的重要基序DEKA(分别位于Ⅰ、Ⅱ、Ⅲ和Ⅳ同源结构域S5和S6连接环)在HaNav (D385, E997, K1517, A1810)和TcNav (D387, E988, K1492,A1785)中高度保守,而在钠离子通道快速失活中起着重要作用的位于同源结构域Ⅲ和Ⅳ之间的疏水性MFM基序也存在于HaNav(1584-1586)和TcNav (1559-1561)中。利用实时荧光定量PCR进一步对HaNav和TcNav的mRNA表达水平的分析发现,HaNav和TcNav在棉铃虫和赤拟谷盗不同发育阶段的表达水平存在一定差异。基因组结构分析发现,HaNav和鳞翅目昆虫烟芽夜蛾Heliothis virescens、小菜蛾Plutella xylostella及家蚕钠离子通道基因相似,含有33个外显子。与HaNav相比,TcNav含有26个外显子,对应HaNav外显子8-10,22-25,30-31,32-33分别合并为一个外显子。HaNav和TcNav的外显子序列一致性为43-87%。尽管HaNav和TcNav的15个外显子长度完全相同,但两者内含子长度差异较大,HaNav的内含子平均长度是TcNav的4.7倍。除了TcNav第12内含子5剪接位点为GC外,HaNav和TcNav内含子都具有保守的5’剪接位点GT和3’剪接位点AG。对cDNA克隆的多重序列比对发现,HaNav和TcNav存在多个选择性剪接位点。在HaNav中,对应着黑腹果蝇para选择性剪接位点j、i和f,外显子2,外显子11的3’端和外显子21的5’端分别发生了外显子跳跃、选择性3’剪接和选择性5’剪接,两对互斥外显子18a/b和26a/b分别与para的c/d和1/k相对应,对应para选择性剪接位点a和b的外显子12、16的5’端存在于所有克隆中,但e和h在HaNav中未发现。在TcNav中,对应着para选择性剪接位点j和i,外显子2,外显子9的3’端分别发生了外显子跳跃、选择性3’剪接,对应para选择性剪接位点a的外显子10的5’端存在于所有克隆中,但e和h在TcNav中未发现。与HaNav和para不同的是,只有d和1存在于TcNav的cDNA和基因组序列中。此外,内含子16、17、18和19发现于TcNav的多个cDNA克隆,表明发生内含子保留。其中,内含子16导致开放阅读框架(ORF)发生变化和终止密码子的出现,内含子17、18和19没有改变ORF,但内含子19导致终止密码子的出现。更多还原

【Abstract】 Voltage-gated sodium channels play an important role in the generation and propagation of action potentials in excitable cells, and are primary targets of several insecticides including DDT, pyrethroids and oxadiazines. At present, rsearches on the relationship between insect sodium channels and insecticide resistance mainly focus on the knockdown resistance (kdr) of pyrethroid insecticides. The cotton bollworm, Helicoverpa armigera (Hubner) is an important pest in cotton, and has developed serious resistance to pyrethroids in many countries. The red flour bettele, Tribolium castaneum, is an important pest in stored grain and is the forth pest that is whole-genome sequenced. In this study, the sodium chanmel genes from H. armigera and T. castaneum were cloned, and the genomic structure and alternative splicing of these two genes were also studied. The results of the research might assist in understanding the structure-function relationships of insect sodium channels as well as the development of novel insecticides.The full-length cDNA sequences of voltage-gated sodium channel a-subunits homologous to the para gene from Drosophila melanogaste were cloned from Helicoverpa armigera (named as HaNav) and Tribolium castaneum (TcNav) using RT-PCR. The composite HaNav contains an ORF of6168bp encoding a protein of2055amino acid residues. The ORF of TcNav contains6152bp and encodes2045amino acid residues. An amino acid sequence alignment shows that HaNav and TcNav share76%identity with each other, and are75%identical with D. melanogaster para. Both HaNav and TcNav proteins share homologous overall organization with all known sodium channel subunits, including four large hydrophobic domains, each composed of six membrane-spanning segments. Specifically, the predominantly negatively charged DEKA motif (D, E, K, and A, respectively, in the putative pore positions in the short segments connecting S5and S6of domains I-IV), which forms the ion selectivity filter and is possessed by all functional sodium channel proteins, is completely conserved and positioned in each of the four domains in the HaNav (D385, E997, K1517, A1810) and TcNav (D387, E988, K1492, A1785). The conserved tripeptide MFM motif for fast inactivation in loop3(L3) in both insects and arachnids is also detected in HaNav at positions1584-1586and TcNav1559-1561. Further expression analysis using qRT-PCR showed that the mRNA expression levels of HaNav and TcNav varied among different developmental stages.Genomic structures of HaNav and TcNav were predicted by comparison of the composite cDNA sequences with the genomic sequences. The HaNav was split into33exons including four mutually exclusive exons, which shares nearly identical genomic organization with previously identified NaV from three Lepitopteran species, Heliothis virescens, Plutella xylostella, and Bombyx mori. In comparison to HaNav, the TcNav has26exons, and exons corresponding to8-10,22-25,30-31,32-33in HaNav are combined into single exons. Pairwise comparison showed that the identity between each pair of exons of the HaNav and TcNav genes was43-87%. The length of15exons is strongly conserved in both HaNav and TcNav, while the lengths of introns vary considerably, on average, HaNav introns are4.7times longer than TcNav ones. The5’donor and3’acceptor site sequences in both HaNav and TcNav are in agreement with the GT/AG consensus sequence, except5’donor sequence (GC) for intron12in HaNav.The alignment of multiple cDNA clone sequences verified several alternative splice sites in both HaNav and TcNav. Exon2,3’portion of exon11,5’portion of exon21were found to be optionally included in HaNav (optional exons), which correspond to optional exons j, i and f of para respectively. Two pairs of mutually exclusive exons were found in HaNav including18a/18b and26a/26b, which correspond to mutually exclusive exons c/d and k/1of para, respectively. While optional exons e and h of para were not identified in HaNav, sequences in5’ portion of exon12and16, correspondind to optional exons a and b of para, respectively, were found in all cDNA clones. In TcNav, Exon2,3’portion of exon9were found to be optionally included in transcripts, which correspond to optional exons j and i of para respectively, and sequence in5’portion of exon10, correspondind to optional exon a of para, was found in all cDNA clones. Similar to HaNav, optional exons e and h of para were not identified in TcNav. In contrast to HaNav and para, which have two pairs of mutually exclusive exons, only exons corrending to d and1of para were identified in both cDNA and genomic sequences of TcNav. While the number of alternative splicing sites in TcNav is much lower than that in HaNav, four short introns16,17,18and19were found in several cDNA clones (transcripts), indicating the occurrence of intron retention. Interesting, while the intron16caused the shift of the open reading frame and resulted in premature termination codons (PTC) in the sequence downstream of the alternative exon, the transcripts containing symmetrical introns17,18and19are translated in frame, but retention of intron19leads to in-frame PTC.更多还原