小菜蛾鈣黏蛋白片段對(duì)Cry1Ac蛋白的增效作用

龔莉君,　彭　鵬,　袁哲明,2,　柳　傲,　楊中俠,2*

(1. 植物病蟲(chóng)害生物學(xué)與防控湖南省重點(diǎn)實(shí)驗(yàn)室, 湖南農(nóng)業(yè)大學(xué)植物保護(hù)學(xué)院, 長(zhǎng)沙　410128; 2. 湖南省生物農(nóng)藥與制劑加工工程技術(shù)研究中心, 長(zhǎng)沙　410128)

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小菜蛾鈣黏蛋白片段對(duì)Cry1Ac蛋白的增效作用

龔莉君1,彭鵬1,袁哲明1,2,柳傲1,楊中俠1,2*

(1. 植物病蟲(chóng)害生物學(xué)與防控湖南省重點(diǎn)實(shí)驗(yàn)室, 湖南農(nóng)業(yè)大學(xué)植物保護(hù)學(xué)院, 長(zhǎng)沙410128; 2. 湖南省生物農(nóng)藥與制劑加工工程技術(shù)研究中心, 長(zhǎng)沙410128)

根據(jù)已報(bào)道的昆蟲(chóng)毒素結(jié)合區(qū)鈣黏蛋白片段對(duì)Cry1Ac蛋白有增效作用,本文以3齡小菜蛾幼蟲(chóng)為研究對(duì)象,選取其鈣黏蛋白相同功能區(qū)的兩個(gè)片段,將兩個(gè)片段進(jìn)行克隆。通過(guò)pGEX-6p-1載體,在大腸桿菌中表達(dá)了功能區(qū)片段PxCAD1及PxCAD2。使用致死中濃度劑量的CrylAc蛋白(1 μg/mL)及較高濃度的PxCAD1(556 μg/mL)與PxCAD2(551.25 μg/mL)對(duì)小菜蛾幼蟲(chóng)進(jìn)行體外復(fù)配生測(cè),結(jié)果表明,PxCAD1可使小菜蛾幼蟲(chóng)的致死率上升為85.56%,PxCAD2則不能增強(qiáng)CrylAc蛋白的殺蟲(chóng)活性;而PxCAD1與PxCAD2本身對(duì)于小菜蛾幼蟲(chóng)并無(wú)毒性。研究結(jié)果為篩選有效的協(xié)同片段提供了理論依據(jù),對(duì)于揭示Bt殺蟲(chóng)蛋白的毒理機(jī)制和害蟲(chóng)對(duì)Bt殺蟲(chóng)蛋白的抗性機(jī)制具有重要意義。

小菜蛾;鈣黏蛋白片段;Cry1Ac;增效作用;表達(dá)

蘇云金芽胞桿菌(Bacillusthuringiensis,Bt)是目前世界上產(chǎn)量最大、應(yīng)用最廣泛、殺蟲(chóng)范圍最廣的微生物殺蟲(chóng)劑[12]。Bt晶體毒素被敏感昆蟲(chóng)取食后,其在幼蟲(chóng)腸道堿性環(huán)境和蛋白酶的作用下被激活,釋放出活性毒素蛋白?；钚远舅氐鞍纂S后與幼蟲(chóng)中腸上皮細(xì)胞的特異性受體結(jié)合形成毒素寡聚體,誘導(dǎo)毒素分子的空間構(gòu)象發(fā)生變化。多個(gè)毒性肽分子在細(xì)胞膜中聚集圍成外表面疏水、內(nèi)表面親水的膜離子通道,進(jìn)而破壞細(xì)胞滲透平衡、細(xì)胞吸脹破裂,導(dǎo)致腸壁破裂昆蟲(chóng)死亡[3]。害蟲(chóng)抗藥性的增強(qiáng)成為Bt毒素長(zhǎng)期有效使用的最大威脅,理解Bt的作用模型及害蟲(chóng)的抗性機(jī)制對(duì)于尋找策略提高Bt的功效及克服害蟲(chóng)抗性具有重要意義。

據(jù)報(bào)道,除殺蟲(chóng)晶體蛋白和外毒素具有殺蟲(chóng)作用外,還有許多與殺蟲(chóng)相關(guān)的增效物質(zhì)可增強(qiáng)Bt的殺蟲(chóng)功效。鞘翅目、鱗翅目及雙翅目昆蟲(chóng)中的幾種絲氨酸酶抑制劑(serine protease inhibitors)可增強(qiáng)Cry 毒素的毒性[45];增加昆蟲(chóng)中腸內(nèi)切幾丁質(zhì)酶(endochitinases)的水平或于Cry毒素制劑中增加外源幾丁質(zhì)酶(chitinase)的水平可將Cry毒素的效果增加10倍[67];Cyt蛋白(Cyt proteins)可與Cry4A、Cry4B 及 Cry11Aa等蛋白協(xié)同作用,克服致倦庫(kù)蚊(Culexpipiensquinquefasciatus)種群對(duì)Cry毒素的抗性[8];且研究表明Cyt1Aa通過(guò)作為膜結(jié)合受體,可增強(qiáng)Cry11Aa對(duì)致倦庫(kù)蚊的毒性等[9]。另有研究表明,煙草天蛾(Manducasexta)的鈣黏蛋白作為協(xié)同增效劑能顯著增強(qiáng)Bt Cry1A對(duì)鱗翅目幼蟲(chóng)小地老虎(Agrotisipsilon)、甜菜夜蛾(Spodopteraexigua)和玉米夜蛾(Helicoverpazea)的毒性[10]。Hua等[11]的研究表明鈣黏蛋白AdCad1是鞘翅目黑粉蟲(chóng)(Alphitobiusdiaperinus)幼蟲(chóng)中Bt毒素Cry3Bb的一個(gè)受體,且沉默AdCad1基因后可顯著降低黑粉蟲(chóng)幼蟲(chóng)對(duì)Cry3Bb的敏感性。

本研究依據(jù)Peng等[12]報(bào)道的棉鈴蟲(chóng)氨基酸毒素結(jié)合區(qū)HaCad1(GenBank登錄號(hào):AF519180)第1 217～1 461位氨基酸能增強(qiáng)Cry毒素活性,及Chen等[6]報(bào)道的對(duì)Bt毒素具有增效作用的煙草天蛾CR12MPED片段(GenBank登錄號(hào):AF319973)第1 362～1 567位序列,克隆并表達(dá)了2個(gè)小菜蛾鈣黏蛋白編碼基因相應(yīng)片段,并進(jìn)一步測(cè)定了原核表達(dá)并純化后的相應(yīng)蛋白PxCAD1及PxCAD2對(duì)小菜蛾的毒力,明確了PxCAD1對(duì)Cry1Ac殺蟲(chóng)蛋白的增效特性。

1　材料與方法

1.1供試?yán)ハx(chóng)、試劑、質(zhì)粒及菌株

小菜蛾(Plutellaxylostella)美國(guó)敏感品系(Cry1Ac-S),由中國(guó)農(nóng)業(yè)科學(xué)院蔬菜花卉研究所昆蟲(chóng)組惠贈(zèng),于室內(nèi)飼養(yǎng)多年,期間未施任何殺蟲(chóng)劑。

RNA抽提所用TRIzol、cDNA合成試劑盒、T4DNA連接酶購(gòu)自TaKaRa公司;限制性內(nèi)切酶EcoRI、XholI及SalI,購(gòu)自NEB及Thermo公司;TaqDNA聚合酶、瓊脂糖凝膠回收試劑盒購(gòu)自Tiangen公司;抗生素、溶菌酶及其他主要生化試劑購(gòu)自長(zhǎng)沙布蘭哲生物科技、上海生工生物工程有限公司和北京全式金生物技術(shù)有限公司;細(xì)菌蛋白抽提試劑、普通DNA產(chǎn)物純化試劑盒、SDS-PAGE凝膠制備試劑盒、Bradford蛋白測(cè)定試劑盒購(gòu)于康為世紀(jì)公司;pEASY-T1 Cloning Kit、表達(dá)載體pGEX-6p-1購(gòu)自全式金生物技術(shù)有限公司;SanPrep柱式質(zhì)粒DNA小量抽提試劑盒購(gòu)自上海生工生物工程有限公司;GST標(biāo)簽蛋白純化試劑盒購(gòu)自Merck Millipore公司。

1.2總RNA的抽提及檢測(cè)

按照TRIzoL抽提RNA法的說(shuō)明書(shū)提取小菜蛾幼蟲(chóng)中腸總RNA,抽提的總RNA純度符合試驗(yàn)要求后,存于-80℃?zhèn)溆谩?/p>

1.3RT-PCR法克隆基因片段PxCAD1及PxCAD2

按照說(shuō)明書(shū)合成cDNA第一鏈。根據(jù)Peng等[12]報(bào)道的棉鈴蟲(chóng)氨基酸毒素結(jié)合區(qū)HaCad1第1 217～1 461位氨基酸能增強(qiáng)Cry毒素活性。在NCBI上下載其棉鈴蟲(chóng)(AF519180)及小菜蛾(EF541176)的氨基酸序列,通過(guò)DNAMAN軟件比對(duì),找出對(duì)應(yīng)的核苷酸序列,并以此為依據(jù)設(shè)計(jì)引物Cad-1f:ACGATCAGGGCCACCGACG;Cad-1r:GTACACCTTCACCTCCGCAC對(duì)PxCAD-1 (735 bp)片段進(jìn)行PCR擴(kuò)增。以合成的cDNA第一鏈為模板,進(jìn)行第2步PCR反應(yīng),反應(yīng)參數(shù)和程序?yàn)?94℃ 5 min;94℃ 1 min;62.2℃ 1 min;72℃ 1 min,30個(gè)循環(huán);72℃ 10 min。PCR產(chǎn)物經(jīng)電泳檢測(cè)后,回收產(chǎn)物,與pEASY-T1載體連接,轉(zhuǎn)化大腸桿菌,挑選陽(yáng)性克隆進(jìn)行酶切與PCR鑒定,后由上海生工生物工程公司進(jìn)行測(cè)序。

根據(jù)已發(fā)表的對(duì)Bt毒素具有增效作用的煙草天蛾鈣黏蛋白基因序列信息CR12MPED片段(序列登錄號(hào)AF319973),通過(guò)DNAMAN軟件比對(duì),找出小菜蛾(序列登錄號(hào)EF541176)相對(duì)應(yīng)的鈣黏蛋白核苷酸序列,并設(shè)計(jì)其引物Cad-2f:ATCAACAGGGAACTATTTACGG;Cad-2r:GCCCAACAGGTAGATGATGACG用于PxCAD2的PCR擴(kuò)增,產(chǎn)物大小約627 bp。以合成的cDNA第一鏈為模板,進(jìn)行第2步PCR反應(yīng),反應(yīng)參數(shù)和程序?yàn)?94℃ 5 min;94℃ 1 min;61.7℃ 1 min;72℃ 1 min,30個(gè)循環(huán);72℃ 10 min。PCR產(chǎn)物經(jīng)電泳檢測(cè)后,回收產(chǎn)物,與pEASY-T1載體連接,轉(zhuǎn)化大腸桿菌,挑選陽(yáng)性克隆進(jìn)行酶切與PCR鑒定,后由上海生工生物工程公司進(jìn)行測(cè)序。

1.4重組蛋白的表達(dá)及純化

目的片段先與pEASY-T1載體連接轉(zhuǎn)入大腸桿菌DH5α感受態(tài)細(xì)胞,挑選陽(yáng)性克隆提取質(zhì)粒后用雙酶切,回收目的片段,再與表達(dá)載體pGEX-6p-1連接,轉(zhuǎn)入大腸桿菌感受態(tài)BL21(DE3)中,挑取單菌落陽(yáng)性鑒定,將陽(yáng)性鑒定子加入含Chl及Amp抗生素的LB液體培養(yǎng)基中37℃過(guò)夜培養(yǎng)。經(jīng)最適溫度加入IPTG誘導(dǎo)表達(dá),檢測(cè)是否成功獲得與預(yù)測(cè)大小一致的總蛋白,后裂解檢測(cè)總蛋白為包涵體還是可溶性蛋白,確定可溶性蛋白后,通過(guò)改變誘導(dǎo)溫度、誘導(dǎo)IPTG濃度和誘導(dǎo)時(shí)間來(lái)篩選最優(yōu)的表達(dá)條件,盡可能多地獲得可溶性蛋白。最后用GST蛋白純化試劑盒純化篩選出單一的目的蛋白,操作方法按照試劑盒內(nèi)的說(shuō)明書(shū)進(jìn)行,先加入樹(shù)脂和洗滌緩沖液洗滌,再加入樣品懸浮樹(shù)脂進(jìn)行孵育結(jié)合后漂洗,最后加入洗脫液收集純化后的蛋白溶液。通過(guò)SDS-PAGE檢測(cè)后,用Brandford標(biāo)準(zhǔn)曲線法對(duì)蛋白進(jìn)行定量。

1.5表達(dá)蛋白的生物活性測(cè)定

小菜蛾的生物測(cè)定方法同Yang等[13]。將定量后的PxCAD1(556 μg/mL)蛋白與Cry1Ac混合液、PxCAD2(551.25 μg/mL)蛋白與Cry1Ac混合液、Cry1Ac(1 μg/mL)溶液、PxCAD1(556 μg/mL)蛋白溶液及PxCAD2(551.25 μg/mL)蛋白溶液,分別采用葉片浸漬法對(duì)小菜蛾幼蟲(chóng)進(jìn)行毒力測(cè)定,以表達(dá)載體pGEX-6p-1與Cry1Ac毒素的混合液為對(duì)照。藥后72 h觀察并記錄死亡蟲(chóng)數(shù),每個(gè)蛋白的生物測(cè)定重復(fù)3次,數(shù)據(jù)統(tǒng)一采用SPSS 11.5統(tǒng)計(jì)軟件進(jìn)行分析。

2　結(jié)果與分析

2.1PxCAD1及PxCAD2基因片段的獲得

PCR擴(kuò)增得到了大小為735 bp與627 bp的PxCAD1及PxCAD2特異性DNA片段(圖1),前者編碼245個(gè)氨基酸,后者編碼209個(gè)氨基酸序列。為明確棉鈴蟲(chóng)及煙草天蛾源增效位置片段與得到的小菜蛾相應(yīng)位置片段PxCAD1與PxCAD2的異同,故將得到的小菜蛾P(guān)xCAD1與棉鈴蟲(chóng)毒素結(jié)合區(qū)相對(duì)應(yīng)的序列(序列登錄號(hào)AF519180)比對(duì),結(jié)果發(fā)現(xiàn)具有50.83%的相似性(圖2);PxCAD2片段為根據(jù)已報(bào)道的煙草天蛾毒素結(jié)合區(qū)相對(duì)應(yīng)的序列進(jìn)行克隆,故將得到的小菜蛾P(guān)xCAD2與煙草天蛾毒素結(jié)合區(qū)相對(duì)應(yīng)的序列(序列登錄號(hào)AF319973)比對(duì),結(jié)果發(fā)現(xiàn)具有55.56%的相似性(圖3)。

圖2　小菜蛾P(guān)xCAD1片段與棉鈴蟲(chóng)鈣黏蛋白片段序列比對(duì)Fig.2　Sequence alignment between PxCAD1 fragment and cadherin fragment of Helicoverpa armigera

圖3　小菜蛾P(guān)xCAD2片段與煙草天蛾鈣黏蛋白片段序列比對(duì)Fig.3　Sequence alignment between PxCAD2 fragment and cadherin fragment of Manduca sexta

2.2小菜蛾鈣黏蛋白毒素結(jié)合區(qū)PxCAD1及PxCAD2的表達(dá)與純化

2.2.1目的片段與載體pEASY-T1重組質(zhì)粒的雙酶切

根據(jù)Primer 5.0軟件找到合適的酶切位點(diǎn),PxCAD1片段用EcoRI和SalI進(jìn)行雙酶切,結(jié)果其重組質(zhì)粒幾乎被酶完全切開(kāi),獲得了含有黏性末端的735 bp的PxCAD1目的片段和剩下切開(kāi)的線性質(zhì)粒片斷,PxCAD2用EcoRI和XhoI進(jìn)行雙酶切,結(jié)果其重組質(zhì)粒大部分被酶切成功,少量酶切失敗,獲得了大部分含黏性末端的627 bp的PxCAD2目的片段、剩下已切開(kāi)的大部分的線性質(zhì)粒片段及少量的未切開(kāi)的環(huán)狀重組質(zhì)粒(圖4)。

圖4　PxCAD1(a)和PxCAD2(b)重組質(zhì)粒的雙酶切Fig.4　Double enzyme digestion of the recombinant plasmid with PxCAD1(a) and PxCAD2(b) fragment

2.2.2重組表達(dá)載體的構(gòu)建及其表達(dá)與純化

(1)PxCAD1可溶性蛋白誘導(dǎo)表達(dá)條件的優(yōu)化

首先選擇最適生長(zhǎng)溫度 37℃,轉(zhuǎn)速160 r/min,參考已發(fā)表文獻(xiàn)中所采用的IPTG終濃度1 mmol/L及誘導(dǎo)時(shí)間3 h進(jìn)行誘導(dǎo),SDS-PAGE結(jié)果如圖5,在3泳道55 kDa左右出現(xiàn)與預(yù)測(cè)GST-PxCAD1大小一致的目的條帶,可知重組蛋白PxCAD1成功表達(dá)。

圖5　37℃條件下誘導(dǎo)PxCAD1表達(dá)的總蛋白Fig.5　Expression of total PxCAD1 protein induced at 37℃

經(jīng)檢測(cè)后發(fā)現(xiàn)在上清中獲得的PxCAD1可溶性蛋白只有少量,為獲得更多的可溶性蛋白對(duì)誘導(dǎo)條件進(jìn)行了一系列的優(yōu)化。優(yōu)化條件為22℃、0.5 mmol/L IPTG、4 h時(shí),擴(kuò)大培養(yǎng),超聲波裂解菌體,純化篩選出單一目的蛋白,如圖6(a),用Brandford標(biāo)準(zhǔn)曲線法對(duì)蛋白進(jìn)行定量,得到PxCAD1的濃度為556 μg/mL。

(2)PxCAD2可溶性蛋白誘導(dǎo)表達(dá)條件的優(yōu)化

首先選擇最適生長(zhǎng)溫度 37℃下誘導(dǎo),參考已發(fā)表文獻(xiàn)中所采用的IPTG終濃度1 mmol/L,誘導(dǎo)時(shí)間6 h,轉(zhuǎn)速150 r/min進(jìn)行誘導(dǎo),SDS-PAGE結(jié)果如圖6(b),在2泳道51 kDa左右出現(xiàn)與預(yù)測(cè)GST-PxCAD2大小一致的目的條帶,可知重組蛋白PxCAD2成功得到表達(dá)。

圖6　PxCAD1(a)和PxCAD2(b)的SDS-PAGE檢測(cè)Fig.6　SDS-PAGE of PxCAD1(a)and PxCAD2(b)polypeptides

為獲得更多的可溶性蛋白,對(duì)誘導(dǎo)條件優(yōu)化后選擇乳糖自誘導(dǎo)法的表達(dá),并根據(jù)優(yōu)化條件擴(kuò)大培養(yǎng),超聲波裂解菌體,純化篩選出單一目的蛋白(圖7),用Brandford標(biāo)準(zhǔn)曲線法對(duì)蛋白進(jìn)行定量,得到PxCAD2的濃度為551.25 μg/mL。

圖7　PxCAD2的SDS-PAGE檢測(cè)Fig.7　SDS-PAGE of PxCAD2 peptide

2.3表達(dá)蛋白的生物測(cè)定

制備用作生物測(cè)定的經(jīng)過(guò)純化的Cry1Ac蛋白、PxCAD1及PxCAD2,并分別測(cè)定其濃度。以小菜蛾敏感品系3齡幼蟲(chóng)(LC50=1.873 μg/mL,見(jiàn)表1)作為生物測(cè)定對(duì)象,分別測(cè)定了在Cry1Ac致死中濃度(1 μg/mL),及較高濃度的PxCAD1(556 μg/mL)或PxCAD2(551.25 μg/mL)存在時(shí)的死亡率(圖8)。結(jié)果表明,1 μg/mL的Cry1Ac可引起小菜蛾幼蟲(chóng)46.7%的死亡率;當(dāng)加入濃度為556 μg/mL的PxCAD1時(shí),小菜蛾3齡幼蟲(chóng)的死亡率為85.6%,當(dāng)加入濃度為551.25 μg/mL的PxCAD2時(shí),死亡率為45%;而對(duì)照組在1 μg/mL的Cry1Ac中,加入濃度為300.27μg/mL的pGEX-6p-1載體時(shí),小菜蛾3齡幼蟲(chóng)的死亡率為45.6%,處理與對(duì)照之間差異顯著。濃度為556 μg/mL的GST-PxCAD1蛋白溶液與濃度為551.25 μg/mL的GST-PxCAD2蛋白溶液均不會(huì)顯著引起小菜蛾幼蟲(chóng)的死亡,表明表達(dá)后的PxCAD1與PxCAD2蛋白本身并無(wú)毒性,而PxCAD1蛋白可增強(qiáng)Cry1Ac蛋白的殺蟲(chóng)活性,而較高濃度的PxCAD2也無(wú)增效作用。

表1　小菜蛾對(duì)Cry1Ac毒素的敏感性測(cè)定

圖8　PxCAD1與PxCAD2對(duì)Cry1Ac殺蟲(chóng)活性的增效作用Fig.8 Enhancement of PxCAD1 and PxCAD2 of Plutella xylostella to Cry1Ac insecticidal activity

3　討論

鈣黏蛋白屬依賴于鈣離子的跨膜蛋白家族中的一類,在多細(xì)胞間起連接和保持細(xì)胞完整性的作用,還在細(xì)胞增殖、分化過(guò)程中起連接蛋白的作用,并能調(diào)節(jié)胞外結(jié)構(gòu)域與胞質(zhì)結(jié)構(gòu)域的信號(hào)轉(zhuǎn)導(dǎo)途徑[14]。其作為Bt Cry毒素的初級(jí)受體,介導(dǎo)Bt Cry毒素毒殺害蟲(chóng)的過(guò)程,在鱗翅目[12,1518,22]、鞘翅目[8, 23]及雙翅目[10]等多種昆蟲(chóng)中鈣黏蛋白的氨基酸序列均已確定。昆蟲(chóng)的鈣黏蛋白從N端至C端依次是信號(hào)肽、由9～12個(gè)重復(fù)子(cadherin repeats,CR)組成的重復(fù)區(qū)、近膜區(qū)(membrane proximal extracellular domain,MPED)、跨膜區(qū)(transmembrane domain)及胞質(zhì)區(qū)(intracellular domain)[11, 18]5個(gè)功能區(qū)域。鈣黏蛋白已被證實(shí)為鱗翅目害蟲(chóng)Cry1Ac毒素的首選靶標(biāo)[18]。

已有的報(bào)道證明,煙草天蛾、煙芽夜蛾及甜菜夜蛾體內(nèi)的鈣黏蛋白毒素結(jié)合片段,在Bt毒素反應(yīng)過(guò)程中起著協(xié)同作用,且在煙草天蛾中無(wú)論鈣黏蛋白片段大小,均能在不同水平上影響毒素毒性的增強(qiáng)[10]。Pacheco等[24]的分析表明,殺蟲(chóng)活性的增強(qiáng)是由于鈣黏蛋白片段CR12與Cry1A毒素結(jié)合促進(jìn)了低聚物的形成。除此之外,他還指出鈣黏蛋白片段CR7和CR11增強(qiáng)了Cry1Ac、Cry1Ab毒素對(duì)煙草天蛾幼蟲(chóng)的殺蟲(chóng)活性,但是效率不如CR12片段。

Peng等[19]的研究結(jié)果表明棉鈴蟲(chóng)鈣黏蛋白片段毒素結(jié)合區(qū)HaCad1顯著增強(qiáng)了Cry1A毒素的毒性。本研究選取了小菜蛾鈣黏蛋白的相同位置序列PxCAD1,測(cè)序后與棉鈴蟲(chóng)的該段序列氨基酸比對(duì)的相似性為50.83%,經(jīng)表達(dá)純化后,加入Cry1Ac毒素對(duì)小菜蛾進(jìn)行室內(nèi)毒力測(cè)定,致死率與對(duì)照有顯著差異,由此表明,該肽段對(duì)Cry1Ac毒素有顯著增效作用。

Chen等[1]的研究結(jié)果表明,煙草天蛾鈣黏蛋白片段毒素結(jié)合區(qū)CR12MPED顯著增強(qiáng)了Cry1A毒素的毒性,并發(fā)現(xiàn)CR12MPED 片段能夠高親和力地結(jié)合煙草天蛾中腸上皮細(xì)胞膜(Kd = 32 nM),并認(rèn)為增效作用的產(chǎn)生是由于CR12MPED 片段在昆蟲(chóng)中腸BBMV 上增加了毒素結(jié)合位點(diǎn),這些結(jié)合位點(diǎn)能夠吸引Cry1A毒素與中腸上皮細(xì)胞膜結(jié)合[1]。本研究選取了小菜蛾鈣黏蛋白的相同位置序列PxCAD2,其與煙草天蛾的該段序列氨基酸比對(duì)的相似性為55.56%,經(jīng)表達(dá)純化后,加入Cry1Ac毒素并對(duì)小菜蛾進(jìn)行室內(nèi)毒力測(cè)定,結(jié)果發(fā)現(xiàn)處理組與對(duì)照組對(duì)小菜蛾的致死率并無(wú)顯著差異,顯然該肽段對(duì)Cry1Ac毒素并無(wú)增效作用。

鄭曉旭等[26]用大腸桿菌進(jìn)行表達(dá)的條件,構(gòu)建了小菜蛾鈣黏蛋白片段基因的重組載體,優(yōu)化了一系列條件,但發(fā)現(xiàn)溫度的改變對(duì)融合蛋白的可溶性幾乎沒(méi)有影響,其得到的蛋白均為包涵體蛋白。本研究選擇了與其不同位置的鈣黏蛋白片段,并選用了適合的原核表達(dá)載體pGEX-6p-l,經(jīng)一系列優(yōu)化條件后,得到了大量的可溶性蛋白,并將其純化后對(duì)小菜蛾幼蟲(chóng)進(jìn)行體外復(fù)配生測(cè),本文首次針對(duì)小菜蛾的CAD1及CAD2兩個(gè)片段研究了它們的增效作用。另有研究結(jié)果表明,多肽片段的空間結(jié)構(gòu)是影響Cry毒素協(xié)同增效的因素之一,鈣黏蛋白片段對(duì)Cry毒素起增效作用的多肽片段大都為包涵體,處于展開(kāi)狀態(tài)[6],也有報(bào)道可溶性鈣黏蛋白片段對(duì)毒素呈減效作用[27]。本研究中,表達(dá)的PxCAD1及PxCAD2蛋白均為可溶性蛋白片段,其中較高濃度的PxCAD1蛋白片段表現(xiàn)出了增效活性,但PxCAD2蛋白片段即使高濃度對(duì)Cry1Ac毒素也沒(méi)有表現(xiàn)出增效或減效作用,具體原因尚需進(jìn)一步研究。該結(jié)果將為篩選有效的小菜蛾鈣黏蛋白增效片段即尋找Cry1A毒素新的增效因子提供理論基礎(chǔ)。

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(責(zé)任編輯：田喆)

Synergistic effects of two cadherin gene fragments in functional region fromPlutellaxylostellato CrylAc protein toxicity

Gong Lijun1,Peng Peng1,Yuan Zheming1,2,Liu Ao1,Yang Zhongxia1,2

(1. Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and Insect Pests, College of Plant Protection, Hunan Agricultural University, Changsha410128, China; 2. Hunan Provincial Engineering & Technology Research Center for Biopesticide and Formulation Processing, Changsha410128, China)

It was reported that peptide from toxin-binding region of cadherin receptor in some insects had a synergistic effect on Cry1Ac toxin. Two cadherin gene fragments (PxCAD1 andPxCAD2) fromPlutellaxylostellawere cloned. The functional region fragmentsPxCAD1 andPxCAD2 were expressed inEscherichiacoliby using vector pGEX-6p-1. Bioassays with CrylAc (1 μg/mL) in the presence ofPxCAD1(556 μg/mL) polypeptide andPxCAD2(551.25 μg/mL)polypeptide were performed againstP.xylostellalarvae. The results showed thatPxCAD1 caused a mortality of 85.56% to the larvae, while there was no significant enhancement of CrylAc toxicity toP.xylostellalarvae withPxCAD2 peptide. Meanwhile,PxCAD1 andPxCAD2 alone were non-toxic toP.xylostellalarvae. Our results provided theoretical basis for screening effective synergistic fragments, and had great significance in revealing the mechanism of Bt insecticidal protein and insect resistance to Bt.

Plutellaxylostella;cadherin fragment;CrylAc;synergism;expression

20150407

20150420

國(guó)家自然科學(xué)基金(31171861)；湖南省高等學(xué)?？茖W(xué)研究青年項(xiàng)目(12B065)

E-mail:yzxmichelle@aliyun.com

Q 966

A

10.3969/j.issn.05291542.2016.03.007