本文選題：禾谷孢囊線蟲　切入點(diǎn)：果膠酸裂解酶　出處：《中國農(nóng)業(yè)科學(xué)》2017年19期

【摘要】：【目的】禾谷孢囊線蟲(Heterodera avenae)是一種嚴(yán)重危害麥類作物的重要植物病原線蟲,對農(nóng)業(yè)生產(chǎn)造成巨大的經(jīng)濟(jì)損失,然而其致病機(jī)制和有效防控方法還有待進(jìn)一步研究。通過克隆禾谷孢囊線蟲的果膠酸裂解酶新基因Ha-pel-1,并對其表達(dá)特性進(jìn)行分析,為后續(xù)探究Ha-pel-1的基因功能及其與寄主的互作提供理論依據(jù),并為探討禾谷孢囊線蟲防控途徑提供新思路�！痉椒ā坎捎猛纯寺〗Y(jié)合RACE技術(shù)從禾谷孢囊線蟲中克隆出一個(gè)新的果膠酸裂解酶基因;采用DNAMAN、Clustal、Signal P 4.0 Server和GSDS等相關(guān)生物信息學(xué)軟件和在線工具分析該基因的核苷酸和氨基酸序列,并使用MEGA 5.0構(gòu)建系統(tǒng)進(jìn)化樹;采用原位雜交和半定量PCR方法確定該基因的在禾谷孢囊線蟲中的表達(dá)部位及其在線蟲不同齡期中的表達(dá)情況�！窘Y(jié)果】從禾谷孢囊線蟲中成功克隆出一個(gè)果膠酸裂解酶基因Ha-pel-1(Gen Bank登錄號GQ998895),該基因c DNA全長1 717 bp,包含一個(gè)長度為1 563 bp的開放閱讀框,編碼一個(gè)長度為521個(gè)氨基酸殘基的蛋白,其理論分子量為57.5 k D,理論等電點(diǎn)為8.52。從線蟲基因組DNA中擴(kuò)增獲得長度為7 199 bp的Ha-pel-1基因組全長,基因結(jié)構(gòu)顯示分析發(fā)現(xiàn),Ha-pel-1基因組包含14個(gè)外顯子和13個(gè)內(nèi)含子,除第3個(gè)內(nèi)含子剪接位點(diǎn)是GC-AG外,其余12個(gè)內(nèi)含子都符合真核生物基因剪接位點(diǎn)GT-AG規(guī)則。同源比對結(jié)果表明,預(yù)測蛋白Ha-PEL-1的C端與大豆孢囊線蟲果膠酸裂解酶HG-PEL-1、甜菜孢囊線蟲果膠酸裂解酶HS-PEL-1均有67%的一致性和83%的相似性;此外,其N端信號肽后比報(bào)道的其他植物寄生線蟲果膠酸裂解酶多出一段長度為254個(gè)氨基酸殘基的序列,這段序列中,靠近N端的184個(gè)氨基酸殘基與數(shù)據(jù)庫中的蛋白均無相似性,而靠近C端有70個(gè)氨基酸殘基(Lys205—Glu274)與韋塞爾斯布朗病毒NS5蛋白(注冊號3ELD)的甲基轉(zhuǎn)移酶區(qū)域有32%的一致性和47%的相似性。氨基酸序列分析發(fā)現(xiàn),預(yù)測蛋白Ha-PEL-1包含一個(gè)長度為20個(gè)氨基酸殘基的信號肽和4個(gè)果膠酸裂解酶第3家族(PL3)的高度保守區(qū)域以及多個(gè)保守的半胱氨酸殘基;系統(tǒng)進(jìn)化分析發(fā)現(xiàn),Ha-pel-1及其他已報(bào)道的線蟲果膠酸裂解酶基因與細(xì)菌和真菌來源的PEL聚在一個(gè)大的分支中;原位雜交結(jié)果顯示Ha-pel-1主要在禾谷孢囊線蟲亞腹食道腺中表達(dá);半定量RT-PCR確定Ha-pel-1在寄生前和寄生后的2齡幼蟲中大量表達(dá)�！窘Y(jié)論】通過對禾谷孢囊線蟲中一個(gè)新果膠酸裂解酶基因Ha-pel-1的克隆和表達(dá)特征分析,揭示該基因與禾谷孢囊線蟲的侵染和寄生過程密切相關(guān)。
[Abstract]:[objective] Heterodera avenaeae is an important plant nematode that seriously endangers wheat crops, which causes huge economic losses to agricultural production. However, its pathogenic mechanism and effective control methods need to be further studied.A new Pectinate lyase gene Ha-pel-1 was cloned and its expression characteristics were analyzed in order to provide theoretical basis for further study on gene function of Ha-pel-1 and its interaction with host.Methods A new gene of Pectinic acid lyase was cloned from C. graminearum by homologous cloning combined with RACE technique.The nucleotide and amino acid sequences of the gene were analyzed by using the bioinformatics software such as DNAMAN Clustalal signal P 4.0 Server and GSDS, and the phylogenetic tree was constructed by MEGA 5.0.In situ hybridization and semi-quantitative PCR were used to determine the expression site of the gene in cereal cyst nematode and its expression in different ages of nematode. [results] A pectinic acid was cloned successfully from cereal cyst nematode.Ha-pel-1(Gen Bank accession number GQ998895GQ998895A, c DNA is 1 717 BP long and contains an open reading frame of 1 563 BP.A protein encoding 521 amino acid residues with a theoretical molecular weight of 57.5 KD and a theoretical isoelectric point of 8.52.The length of Ha-pel-1 genome was 7 199 BP, which was amplified from the genomic DNA of nematode. The gene structure analysis showed that the Ha-pel-1 genome contained 14 exons and 13 introns, with the exception of the third intron splicing site (GC-AG).The other 12 introns all accord with the GT-AG rule of splicing site of eukaryotic gene.The results of homology comparison showed that the C terminal of the predicted protein Ha-PEL-1 was 67% consistent with that of soybean cyst nematode Pectinic acid lyase HG-PEL-1 and beet cyst nematode Pectinic acid lyase HS-PEL-1, and the similarity was 83% between the predicted protein and soybean cyst nematode Pectinic acid lyase HG-PEL-1.The N-terminal signal peptide produced a length of 254 amino acid residues from the Pectinic lyase of other plant parasitic nematodes. In this sequence, the 184 amino acid residues near the N-terminal were not similar to the proteins in the database.There was 32% agreement and 47% similarity between the 70 amino acid residues Lys 205-Glu274 and the methyltransferase region of Wessels Brown virus NS5 protein (registration number 3ELDD).Amino acid sequence analysis showed that the predicted protein Ha-PEL-1 contained a signal peptide with a length of 20 amino acid residues and a highly conserved region of the third family of Pectinic acid lyase, as well as a number of conserved cysteine residues.Phylogenetic analysis showed that Ha-pel-1 and other reported PEL genes of nematode Pectinase were clustered in a large branch of PEL from bacteria and fungi, and in situ hybridization showed that Ha-pel-1 was mainly expressed in the subventral esophagus of C. graminearum.Semi-quantitative RT-PCR confirmed the high expression of Ha-pel-1 in pre-parasitic and post-parasitic second instar larvae. [conclusion] the cloning and expression characteristics of a new Pectinic acid lyase gene Ha-pel-1 in cereal cyst nematode were analyzed.It was revealed that the gene was closely related to the infection and parasitism of cereal cyst nematode.
【作者單位】： 中國農(nóng)業(yè)科學(xué)院植物保護(hù)研究所植物病蟲害生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室;中國熱帶農(nóng)業(yè)科學(xué)院橡膠研究所;中國熱帶農(nóng)業(yè)科學(xué)院環(huán)境與植物保護(hù)研究所農(nóng)業(yè)部熱帶作物有害生物綜合治理重點(diǎn)實(shí)驗(yàn)室;
【基金】：國家“973”計(jì)劃(2013CB127502) 國家公益性行業(yè)(農(nóng)業(yè))科研專項(xiàng)(201503114)
【分類號】：S432.45

【相似文獻(xiàn)】

相關(guān)期刊論文 前2條

1 王衍桐,彭德良,陳受宜;灰布支黑豆對大豆孢囊線蟲(Heterodera glycines)14號小種的抗性遺傳[J];遺傳學(xué)報(bào);2000年02期

2 張淑齡;孢囊線蟲的陰門錐制片技術(shù)[J];動物學(xué)雜志;1988年02期

相關(guān)碩士學(xué)位論文 前5條

1 吳獨(dú)清;禾谷孢囊線蟲新效應(yīng)因子Ha-G20E03基因克隆及用于研究其功能的新模式系統(tǒng)的探索[D];中國農(nóng)業(yè)科學(xué)院;2015年

2 胡小斌;禾谷孢囊線蟲的抗性品種鑒定、互作表型及綜合防治研究[D];南京農(nóng)業(yè)大學(xué);2014年

3 崔思佳;湖南省旱稻孢囊線蟲rDNA-ITS序列特征及基于ISSR的遺傳多樣性分析[D];湖南農(nóng)業(yè)大學(xué);2016年

4 宋曉磊;禾谷孢囊線蟲(Heterodera avenae Wollenweber)在山東省的分布調(diào)查、孵化及分子鑒定[D];山東農(nóng)業(yè)大學(xué);2011年

5 呂蓓;受大豆孢囊線蟲侵染誘導(dǎo)表達(dá)的cDNA的克隆[D];中國農(nóng)業(yè)科學(xué)院;2001年

，

本文編號：1720116