松墨天牛磷酸丙糖异构酶基因的克隆及表达分析
发布时间：2018-07-17 22:29
【摘要】：松墨天牛,Monochamus alternatus Hope(Coleoptera:Cerambycidae),是我国的重要林业害虫,主要危害马尾松(Pinus massoniana)等松属植物,也是传播毁灭性病害松材线虫病(Bursaphelench xylophilus)的媒介昆虫。磷酸丙糖异构酶(Triosephosphate isomerase,TPI)是糖酵解酶的一种,广泛存在于自然界生物体中。磷酸丙糖异构酶参与机体细胞质内的葡萄糖分解代谢,催化二羟丙酮磷酸与3-磷酸甘油醛之间的可逆反应,并且在糖酵解、糖原异生、脂肪酸合成和戊糖磷酸等包含三糖磷酸脂代谢的途径中发挥着重要作用。本研究从松墨天牛cDNA文库中筛选出含有5′端的TPI基因片段,通过cDNA末端快速扩增技术(rapid amplification of c DNA ends,RACE)扩增该基因的3′端,通过软件进行拼接得到磷酸丙糖异构酶基因全长序列。本研究获得的松墨天牛磷酸丙糖异构酶基因全长为1450 bp,生物信息学软件分析表明其开放阅读框(ORF)为744 bp,编码247个氨基酸,命名为MaTPI(GenBank登录号:KX024698)。推测MaTPI分子量为26.71 kDa,等电点为5.07。MaTPI分子式为C1192H1892N320O363S6,属于亲水蛋白,不含有跨膜区域和信号肽,含有7个丝氨酸磷酸化位点和4个苏氨酸磷酸化位点。对MaTPI蛋白的二级结构及三级结构预测,发现该蛋白以α螺旋结构和β折叠结构居多。对该蛋白的结构域进行预测,发现MaTPI蛋白含有完整的磷酸丙糖异构酶结构域,这表明MaTPI编码蛋白属于TPI家族。同源序列比对显示MaTPI与赤拟谷盗(Tribolium castaneum)、大黄粉虫(Tenebrio molitor)有80%的最高同源性,与棉铃虫(Helicoverpa armigera)等12种昆虫的同源性在70-73%之间,并含有TPI活性功能位点序列——AYEPVWAIGTG。系统发育树显示松墨天牛与赤拟谷盗和大黄粉虫在同一分枝上,而与小火蚁(Wasmannia auropunctata)等昆虫的遗传距离较远。荧光定量PCR分析显示:MaTPI在成虫的表达量高于蛹和幼虫,三者表达量具有显著性差异(P0.05);MaTPI在成虫腹部的相对表达量显著高于头部、胸部、足、触角和翅(P0.05);MaTPI在幼虫各组织中的表达量是:脂肪体体壁血淋巴中肠马氏管(P0.05)。用11种农药处理松墨天牛幼虫后,发现有3种农药(白僵菌、啶虫脒、马拉硫磷)处理后的松墨天牛MaTPI表达量出现了上调表达,有8种农药(Bt、绿僵菌、印楝素、噻虫啉、灭多威、毒死蜱、杀虫双、敌杀死)处理的表达量出现下调表达。
[Abstract]:Monochamus alternatus Hope (Coleoptera: Cerambycidae) is an important forest pest in China. It mainly endangers Pinus massoniana and other pine plants, and is also a vector of Bursaphelench xylophilus. Triosephosphate isomerase (TPI) is a kind of glycolytic enzyme, which widely exists in natural organisms. Propranose phosphate isomerase participates in the glucose catabolism in the cytoplasm of the body, catalyzes the reversible reaction between dihydroxyacetone phosphoric acid and glyceraldehyde 3-phosphate, and in glycolysis, glycogen isomerization, Fatty acid synthesis and pentose phosphoric acid play an important role in the metabolism of triose phosphate. In this study, the TPI gene fragment containing 5 'terminal was screened from the cDNA library of Pinus mongolicus, and the 3'end of the gene was amplified by rapid amplification technique of (rapid amplification of c DNA endsrace. The full-length gene sequence of propanose phosphate isomerase gene was obtained by software splicing. In this study, the total length of the gene was 1450 BP, and the open reading frame (ORF) was 744 BP, encoding 247 amino acids, named MaTPI (GenBank accession number: KX024698). The molecular weight of MaTPI is 26.71 kDa. the isoelectric point of MaTPI is 5.07.The molecular formula of MaTPI is C1192H1892N320O363S6, which belongs to hydrophilic protein, does not contain transmembrane region and signal peptide, and contains 7 serine phosphorylation sites and 4 threonine phosphorylation sites. The secondary and tertiary structures of MaTPI protein were predicted. It was found that the 伪 helix structure and 尾 -fold structure were the majority of MaTPI protein. It was found that MaTPI protein contained a complete domain of propanose phosphate isomerase, which indicated that MaTPI coding protein belonged to TPI family. The homologous sequence alignment showed that MaTPI had the highest homology with Tribolium castaneum), molitor (Tenebrio molitor) and had 70-73% homology with Helicoverpa armigera, and had TPI active function locus AYEPVWAIGTG. the results showed that MaTPI had the highest homology with Tribolium castaneum), (Tenebrio molitor), and had the highest homology with Helicoverpa armigera. Phylogenetic tree showed that Pinochamnus japonicus was on the same branch as A. albophora and T. chinensis, but had a long genetic distance with Wasmannia auropunctata and other insects. Fluorescence quantitative PCR analysis showed that the expression of MaTPI in adults was higher than that in pupae and larva (P0.05) the relative expression of MaTPI in the abdomen of adult was significantly higher than that in head, chest and foot. The expression of MaTPI of antennae and wings (P0.05) in larval tissues was as follows: adipose body wall hemolymph mesenteric Markov tube (P0.05). After treated with 11 pesticides, it was found that three pesticides (Beauveria bassiana, acetamiprid, malathion) had up-regulated MaTPI expression, and 8 pesticides (BT, Metarhizium anisopliae, azadirachtin, thiazoline) were found. The expression of methomyl, chlorpyrifos, insecticide and trichlorfon) was down-regulated.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S763.38
本文编号：2131128
[Abstract]:Monochamus alternatus Hope (Coleoptera: Cerambycidae) is an important forest pest in China. It mainly endangers Pinus massoniana and other pine plants, and is also a vector of Bursaphelench xylophilus. Triosephosphate isomerase (TPI) is a kind of glycolytic enzyme, which widely exists in natural organisms. Propranose phosphate isomerase participates in the glucose catabolism in the cytoplasm of the body, catalyzes the reversible reaction between dihydroxyacetone phosphoric acid and glyceraldehyde 3-phosphate, and in glycolysis, glycogen isomerization, Fatty acid synthesis and pentose phosphoric acid play an important role in the metabolism of triose phosphate. In this study, the TPI gene fragment containing 5 'terminal was screened from the cDNA library of Pinus mongolicus, and the 3'end of the gene was amplified by rapid amplification technique of (rapid amplification of c DNA endsrace. The full-length gene sequence of propanose phosphate isomerase gene was obtained by software splicing. In this study, the total length of the gene was 1450 BP, and the open reading frame (ORF) was 744 BP, encoding 247 amino acids, named MaTPI (GenBank accession number: KX024698). The molecular weight of MaTPI is 26.71 kDa. the isoelectric point of MaTPI is 5.07.The molecular formula of MaTPI is C1192H1892N320O363S6, which belongs to hydrophilic protein, does not contain transmembrane region and signal peptide, and contains 7 serine phosphorylation sites and 4 threonine phosphorylation sites. The secondary and tertiary structures of MaTPI protein were predicted. It was found that the 伪 helix structure and 尾 -fold structure were the majority of MaTPI protein. It was found that MaTPI protein contained a complete domain of propanose phosphate isomerase, which indicated that MaTPI coding protein belonged to TPI family. The homologous sequence alignment showed that MaTPI had the highest homology with Tribolium castaneum), molitor (Tenebrio molitor) and had 70-73% homology with Helicoverpa armigera, and had TPI active function locus AYEPVWAIGTG. the results showed that MaTPI had the highest homology with Tribolium castaneum), (Tenebrio molitor), and had the highest homology with Helicoverpa armigera. Phylogenetic tree showed that Pinochamnus japonicus was on the same branch as A. albophora and T. chinensis, but had a long genetic distance with Wasmannia auropunctata and other insects. Fluorescence quantitative PCR analysis showed that the expression of MaTPI in adults was higher than that in pupae and larva (P0.05) the relative expression of MaTPI in the abdomen of adult was significantly higher than that in head, chest and foot. The expression of MaTPI of antennae and wings (P0.05) in larval tissues was as follows: adipose body wall hemolymph mesenteric Markov tube (P0.05). After treated with 11 pesticides, it was found that three pesticides (Beauveria bassiana, acetamiprid, malathion) had up-regulated MaTPI expression, and 8 pesticides (BT, Metarhizium anisopliae, azadirachtin, thiazoline) were found. The expression of methomyl, chlorpyrifos, insecticide and trichlorfon) was down-regulated.
【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S763.38
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