本文選題：棉蚜 + 溶菌酶�。� 參考：《昆蟲學(xué)報》2017年02期

【摘要】：【目的】克隆棉蚜Aphis gossypii Glover溶菌酶基因并進行原核表達,分析其在不同日齡和不同寄主植物上棉蚜體內(nèi)的表達特征,為進一步研究溶菌酶的功能及其與棉蚜共生菌間的關(guān)系奠定基礎(chǔ)。【方法】以無翅棉蚜成蟲總RNA為模板,采用RT-PCR與RACE方法獲得棉蚜溶菌酶基因的cDNA全長序列;將信號肽序列剪切后的棉蚜溶菌酶基因片段連接到原核表達載體pET-30a,并轉(zhuǎn)化到大腸桿菌Eschericha coli中進行誘導(dǎo)表達;采用定量PCR方法測定該基因在不同日齡(1-19日齡)以及西葫蘆、黃瓜和豇豆等不同寄主植物上棉蚜體內(nèi)的相對表達量�！窘Y(jié)果】克隆獲得棉蚜溶菌酶基因,命名為AgLYS(Gen Bank登錄號:KY575341),cDNA全長為680 bp,開放閱讀框為516bp,編碼172個氨基酸殘基。AgLYS的N-末端含有長度為36個氨基酸殘基的信號肽序列。系統(tǒng)發(fā)育分析表明,棉蚜溶菌酶屬于i型溶菌酶,并且與豌豆蚜Acyrthosiphon pisum i型溶菌酶基因的氨基酸序列一致性達90%。AgLYS可在大腸桿菌中誘導(dǎo)表達,表達蛋白分子量為20 k D左右。AgLYS在1-19日齡的棉蚜體內(nèi)均有表達,但在1-9日齡棉蚜體內(nèi)表達量較低,11日齡后的棉蚜體內(nèi)表達量極顯著升高(P0.01)。同時,在西葫蘆上飼養(yǎng)的棉蚜體內(nèi)AgLYS的表達量極顯著高于在黃瓜和豇豆上飼養(yǎng)的棉蚜體內(nèi)的表達量(P0.01)。【結(jié)論】從棉蚜中克隆獲得了i型溶菌酶基因AgLYS,并在大腸桿菌中誘導(dǎo)表達。棉蚜AgLYS基因的表達量受蚜蟲日齡及所取食寄主種類的影響,推測棉蚜i型溶菌酶AgLYS可能與棉蚜體內(nèi)共生菌種群密度的調(diào)控相關(guān)。
[Abstract]:[objective] to clone and express the lysozyme gene of Aphis gossypii Glover in cotton aphid, and analyze its expression characteristics in different days and host plants. In order to further study the function of lysozyme and the relationship between lysozyme and symbiotic bacteria, the full-length cDNA sequence of lysozyme gene of cotton aphid was obtained by RT-PCR and race. The lysozyme gene fragment of APHIS gossypii was ligated into the prokaryotic expression vector pET-30a and transformed into Escherichia coli for induction expression. Relative expression of cotton aphid on cucumber and cowpea. [results] lysozyme gene of cotton aphid was cloned. The full length of the cDNA is 680bp.The open reading frame (ORAF) is 516bp.The N- terminal of AgLYYS encodes 172 amino acid residues. The N-terminal of AgLYS contains 36 amino acid residues. Phylogenetic analysis showed that the lysozyme of cotton aphid belongs to type I lysozyme, and the amino acid sequence of the lysozyme gene of aphid Acyrthosiphon pisum i is 90. AgLYS can be induced and expressed in Escherichia coli. The molecular weight of the expressed protein was about 20kD. AgLYS was expressed in cotton aphids of 1-19 days old, but the expression of Ag-LYS in cotton aphids at 1-9 days old was significantly higher than that in cotton aphids after 11 days of age. At the same time, the expression of AgLYS in cotton aphid raised on zucchini was significantly higher than that in cotton aphid raised on cucumber and cowpea. [conclusion] I type lysozyme gene AgLYS was cloned from cotton aphid and expressed in Escherichia coli. The expression of AgLYS gene in cotton aphids was affected by the aphids' age and host species. It was suggested that the type I lysozyme AgLYS might be related to the density of symbiotic species in cotton aphids.
【作者單位】： 南京農(nóng)業(yè)大學(xué)植物保護學(xué)院;
【基金】：國家自然科學(xué)基金項目(31672034) 江蘇省自然科學(xué)基金項目(BK20141368)
【分類號】：S433
，

本文編號：1993236