【摘要】：松墨天牛,Monochamus alternatus Hope(Coleoptera:Cerambycidae),是我國的重要林業(yè)害蟲,主要危害馬尾松(Pinus massoniana)等松屬植物,也是傳播毀滅性病害松材線蟲病(Bursaphelench xylophilus)的媒介昆蟲。磷酸丙糖異構(gòu)酶(Triosephosphate isomerase,TPI)是糖酵解酶的一種,廣泛存在于自然界生物體中。磷酸丙糖異構(gòu)酶參與機體細胞質(zhì)內(nèi)的葡萄糖分解代謝,催化二羥丙酮磷酸與3-磷酸甘油醛之間的可逆反應(yīng),并且在糖酵解、糖原異生、脂肪酸合成和戊糖磷酸等包含三糖磷酸脂代謝的途徑中發(fā)揮著重要作用。本研究從松墨天牛cDNA文庫中篩選出含有5′端的TPI基因片段,通過cDNA末端快速擴增技術(shù)(rapid amplification of c DNA ends,RACE)擴增該基因的3′端,通過軟件進行拼接得到磷酸丙糖異構(gòu)酶基因全長序列。本研究獲得的松墨天牛磷酸丙糖異構(gòu)酶基因全長為1450 bp,生物信息學(xué)軟件分析表明其開放閱讀框(ORF)為744 bp,編碼247個氨基酸,命名為MaTPI(GenBank登錄號:KX024698)。推測MaTPI分子量為26.71 kDa,等電點為5.07。MaTPI分子式為C1192H1892N320O363S6,屬于親水蛋白,不含有跨膜區(qū)域和信號肽,含有7個絲氨酸磷酸化位點和4個蘇氨酸磷酸化位點。對MaTPI蛋白的二級結(jié)構(gòu)及三級結(jié)構(gòu)預(yù)測,發(fā)現(xiàn)該蛋白以α螺旋結(jié)構(gòu)和β折疊結(jié)構(gòu)居多。對該蛋白的結(jié)構(gòu)域進行預(yù)測,發(fā)現(xiàn)MaTPI蛋白含有完整的磷酸丙糖異構(gòu)酶結(jié)構(gòu)域,這表明MaTPI編碼蛋白屬于TPI家族。同源序列比對顯示MaTPI與赤擬谷盜(Tribolium castaneum)、大黃粉蟲(Tenebrio molitor)有80%的最高同源性,與棉鈴蟲(Helicoverpa armigera)等12種昆蟲的同源性在70-73%之間,并含有TPI活性功能位點序列——AYEPVWAIGTG。系統(tǒng)發(fā)育樹顯示松墨天牛與赤擬谷盜和大黃粉蟲在同一分枝上,而與小火蟻(Wasmannia auropunctata)等昆蟲的遺傳距離較遠。熒光定量PCR分析顯示:MaTPI在成蟲的表達量高于蛹和幼蟲,三者表達量具有顯著性差異(P0.05);MaTPI在成蟲腹部的相對表達量顯著高于頭部、胸部、足、觸角和翅(P0.05);MaTPI在幼蟲各組織中的表達量是:脂肪體體壁血淋巴中腸馬氏管(P0.05)。用11種農(nóng)藥處理松墨天牛幼蟲后,發(fā)現(xiàn)有3種農(nóng)藥(白僵菌、啶蟲脒、馬拉硫磷)處理后的松墨天牛MaTPI表達量出現(xiàn)了上調(diào)表達,有8種農(nóng)藥(Bt、綠僵菌、印楝素、噻蟲啉、滅多威、毒死蜱、殺蟲雙、敵殺死)處理的表達量出現(xiàn)下調(diào)表達。
[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 偽 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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S763.38

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