象耳豆根結(jié)線蟲(chóng)纖維素結(jié)合蛋白Me-cbp-1基因的克隆與功能分析
發(fā)布時(shí)間：2018-08-30 20:11
【摘要】：纖維素結(jié)合蛋白(cellulose binding protein,CBP)是植物寄生線蟲(chóng)在侵染過(guò)程中分泌的重要效應(yīng)蛋白。為了獲得象耳豆根結(jié)線蟲(chóng)(Meloidogyne enterolobii)纖維素結(jié)合蛋白序列信息、并分析其在線蟲(chóng)寄生致病過(guò)程中的功能地位,本研究利用已構(gòu)建的象耳豆根結(jié)線蟲(chóng)二齡幼蟲(chóng)轉(zhuǎn)錄組數(shù)據(jù)庫(kù),結(jié)合cDNA末端快速擴(kuò)增(rapid-amplification of cDNA ends,RACE)技術(shù)克隆了象耳豆根結(jié)線蟲(chóng)纖維素結(jié)合蛋白基因Me-cbp-1(Gen Bank登錄號(hào):KU350655)cDNA全長(zhǎng)序列。生物信息學(xué)分析表明,Me-cbp-1 cDNA全長(zhǎng)809bp,其5'末端和3'末端的非編碼區(qū)長(zhǎng)度分別為43和139 bp,以及含有1個(gè)長(zhǎng)度為627 bp的完整開(kāi)放閱讀框(ORF),推導(dǎo)編碼208個(gè)氨基酸。預(yù)測(cè)蛋白Me-cbp-1含有起分泌功能的信號(hào)肽結(jié)構(gòu)和纖維素結(jié)合結(jié)構(gòu)域。同源性搜索比對(duì)分析表明,象耳豆根結(jié)線蟲(chóng)Me-cbp-1與南方根結(jié)線蟲(chóng)(M.incognita)、爪哇根結(jié)線蟲(chóng)(M.javanica)及花生根結(jié)線蟲(chóng)(M.arenaria)纖維素結(jié)合蛋白具有88%~91%的相似性。原位雜交顯示,Mecbp-1在象耳豆根結(jié)線蟲(chóng)二齡幼蟲(chóng)的亞腹食道腺細(xì)胞特異表達(dá)。利用RNAi技術(shù)對(duì)象耳豆根結(jié)線蟲(chóng)二齡幼蟲(chóng)的Me-cbp-1基因進(jìn)行沉默后,導(dǎo)致二齡幼蟲(chóng)的侵染率顯著降低。本實(shí)驗(yàn)結(jié)果初步證明了纖維素結(jié)合蛋白在象耳豆根結(jié)線蟲(chóng)二齡幼蟲(chóng)侵染寄主的過(guò)程中具有重要地位,推測(cè)其通過(guò)與細(xì)胞壁主要成分纖維素結(jié)合,促進(jìn)植物細(xì)胞壁的軟化和降解,從而有利于提高線蟲(chóng)的侵染和寄生能力。本研究為揭示象耳豆根結(jié)線蟲(chóng)分子寄生致病機(jī)制以及進(jìn)一步研究防治根結(jié)線蟲(chóng)病害新策略提供了理論依據(jù)。
[Abstract]:Cellulose binding protein (cellulose binding protein,CBP) is an important effector protein secreted by plant parasitic nematode. In order to obtain the sequence information of (Meloidogyne enterolobii) cellulose-binding protein of root-knot nematodes and to analyze its functional position in the parasitic process of nematode, the transcriptome database of the second instar larvae of root-knot nematode nematodes was constructed in this study. CDNA terminal rapid amplification (rapid-amplification of cDNA ends,RACE) technique was used to clone the full-length sequence of cellulose binding protein gene Me-cbp-1 (Gen Bank accession number: KU350655 of root-knot nematode. Bioinformatics analysis showed that the full length of Me-cbp-1 cDNA was 809bp.The non-coding region length of 5'terminal and 3'terminal was 43 and 139 bp, respectively, and a complete open reading frame (ORF), containing 627 bp in length was deduced to encode 208 amino acids. Predictive protein Me-cbp-1 contains signal peptide structures and cellulose-binding domains that play a secretory role. Homology analysis showed that the cellulose-binding proteins of Me-cbp-1 were similar to those of M.incognita, M.javanica and M.arenaria. The results showed that the cellulose-binding proteins of RKN were 88% and 91% similar to those of RKN (M.incognita), M.javanica (RKN) and M.arenaria (Peanut Root-knot nematode). In situ hybridization showed that Mecbp-1 was specifically expressed in the subventral esophageal gland of the second instar larvae of root-knot nematodes. The RNAi technique was used to silence the Me-cbp-1 gene of the second instar larvae of root-knot nematode, and the infection rate of the second instar larvae was significantly decreased. The results showed that cellulose binding proteins play an important role in the infection of the second instar larvae of root-knot nematode nematodes, and the cellulosic binding proteins can promote the softening and degradation of plant cell walls by binding with cellulose, the main component of cell wall. Thus, the ability of infection and parasitism of nematodes was improved. This study provides a theoretical basis for revealing the mechanism of molecular parasitism of root-knot nematodes and for further study on new strategies for the control of root-knot nematode diseases.
【作者單位】： 海南省農(nóng)業(yè)科學(xué)院植物保護(hù)研究所/海南省植物病蟲(chóng)害防控重點(diǎn)實(shí)驗(yàn)室;中國(guó)熱帶農(nóng)業(yè)科學(xué)院環(huán)境與植物保護(hù)研究所;
【基金】：國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(973)項(xiàng)目前期基礎(chǔ)研究專項(xiàng)(No.2014CB160307) 國(guó)家自然科學(xué)基金(No.31401717和No.31360432)
【分類號(hào)】：S432.45
本文編號(hào)：2214159
[Abstract]:Cellulose binding protein (cellulose binding protein,CBP) is an important effector protein secreted by plant parasitic nematode. In order to obtain the sequence information of (Meloidogyne enterolobii) cellulose-binding protein of root-knot nematodes and to analyze its functional position in the parasitic process of nematode, the transcriptome database of the second instar larvae of root-knot nematode nematodes was constructed in this study. CDNA terminal rapid amplification (rapid-amplification of cDNA ends,RACE) technique was used to clone the full-length sequence of cellulose binding protein gene Me-cbp-1 (Gen Bank accession number: KU350655 of root-knot nematode. Bioinformatics analysis showed that the full length of Me-cbp-1 cDNA was 809bp.The non-coding region length of 5'terminal and 3'terminal was 43 and 139 bp, respectively, and a complete open reading frame (ORF), containing 627 bp in length was deduced to encode 208 amino acids. Predictive protein Me-cbp-1 contains signal peptide structures and cellulose-binding domains that play a secretory role. Homology analysis showed that the cellulose-binding proteins of Me-cbp-1 were similar to those of M.incognita, M.javanica and M.arenaria. The results showed that the cellulose-binding proteins of RKN were 88% and 91% similar to those of RKN (M.incognita), M.javanica (RKN) and M.arenaria (Peanut Root-knot nematode). In situ hybridization showed that Mecbp-1 was specifically expressed in the subventral esophageal gland of the second instar larvae of root-knot nematodes. The RNAi technique was used to silence the Me-cbp-1 gene of the second instar larvae of root-knot nematode, and the infection rate of the second instar larvae was significantly decreased. The results showed that cellulose binding proteins play an important role in the infection of the second instar larvae of root-knot nematode nematodes, and the cellulosic binding proteins can promote the softening and degradation of plant cell walls by binding with cellulose, the main component of cell wall. Thus, the ability of infection and parasitism of nematodes was improved. This study provides a theoretical basis for revealing the mechanism of molecular parasitism of root-knot nematodes and for further study on new strategies for the control of root-knot nematode diseases.
【作者單位】： 海南省農(nóng)業(yè)科學(xué)院植物保護(hù)研究所/海南省植物病蟲(chóng)害防控重點(diǎn)實(shí)驗(yàn)室;中國(guó)熱帶農(nóng)業(yè)科學(xué)院環(huán)境與植物保護(hù)研究所;
【基金】：國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(973)項(xiàng)目前期基礎(chǔ)研究專項(xiàng)(No.2014CB160307) 國(guó)家自然科學(xué)基金(No.31401717和No.31360432)
【分類號(hào)】：S432.45
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