CN105754882A - 用内源安全标记表达胃毒杀虫蛋白的转基因生防工程真菌及其构建方法 - Google Patents
用内源安全标记表达胃毒杀虫蛋白的转基因生防工程真菌及其构建方法 Download PDFInfo
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Abstract
本发明公开了用内源安全标记表达胃毒杀虫蛋白的转基因生防工程真菌及其构建方法。本发明是将杀虫蛋白Vip3Aa1的编码基因置于内源强启动子之下,构建带有该杀虫蛋白基因的表达载体。利用球孢白僵菌内源<i>ura3</i>营养缺陷型作为安全标记,将其携带上述表达载体的感受态芽生孢子转入球孢白僵菌野生菌株中,获得高表达杀虫蛋白Vip3Aa1的重组工程菌株。本发明公布的无外源抗性标记表达胃毒杀虫蛋白Vip3Aa1的转基因生防工程真菌为BbHUV5,具有球孢白僵菌天生缺乏的胃毒杀虫活性,整体杀虫效果大幅提高,而且不携带任何外源除草剂抗性基因标记的环境风险隐患。本发明采用的工程菌株构建思路及其工程菌株为开发增效且环境相容度高的转基因真菌杀虫剂开辟了新的技术途径。
Description
技术领域
本发明属于分子生物技术领域,涉及生物工程技术领域的DNA重组技术和应用,具体涉及一种用内源安全标记表达胃毒杀虫蛋白的转基因生防工程真菌及其构建方法。
背景技术
转基因工程技术已广泛应用于生防真菌的遗传改良中,是提高真菌生物防治潜能的杀手锏。一是通过对抗逆相关基因功能的研究分析,有目的地高表达内源抗逆基因或导入外源抗逆基因,以提高菌株的抗逆力,如抗氧化、耐高渗、耐高温、耐紫外辐射、抗杀菌剂、抗除草剂的能力。二是基于生防真菌一般经昆虫体壁侵染寄主的方式,将内源或外源杀虫蛋白基因导入生防真菌基因组中高表达,以拓宽菌株的杀虫谱或提高菌株对靶标害虫的毒力,或使真菌获得天生不具有的胃毒杀虫活性。然而,目前生防真菌基因工程中所用的筛选标记,仅限于除草剂草丁膦的抗性基因bar或氯嘧磺隆抗性基因sur,没有更多的选择。这些抗性标记虽然在真菌转化中是有效的,但所构建的工程菌除了表达靶标基因之外,也同时表达外源抗性标记基因,因而成为环境安全隐患的来源,由此增加了用于田间害虫防治的工程菌剂通过环境安全性评估的难度。
构建环境安全低风险的生防真菌工程菌,不仅要求被导入的靶标基因是环境安全的,而且要求用于转化子筛选的标记基因也是环境安全的。在生防真菌中一些引起特定营养缺陷型的基因,具有作为内源性安全筛选标记的潜力,但鲜见用于研究实践,以致于目前用于转化标记的基因都是外源的除草剂抗性基因。其它真菌转化中常用的潮霉素等抗生素标记,无一可用于生防真菌的转化。在球孢白僵菌功能基因的鉴定中,我们发现乳清酸核苷5-磷酸脱羧酶基因ura3的缺失能导致脲嘧啶营养缺陷型,而添加外源脲嘧啶可使ura3缺失株恢复正常生长,因而可用于白僵菌转化的新型安全筛选标记。这是因为ura3可调控细胞内尿嘧啶合成,其功能缺失使尿嘧啶合成受阻,因而无法正常生长。在以改良生防性状为目标的被整合靶标蛋白中,Vip3A是苏云金芽孢杆菌在营养生长期分泌的一类昆虫中肠特异性胃毒杀虫蛋白,其家族成员对鳞翅目害虫具有较广谱的杀虫活性,且对人畜安全无害。表达Vip3A蛋白抗棉铃虫的转基因棉花已在美国获准田间释放。本实验室先前使用bar标记基因成功地将Vip3Aa1(Vip3A家族的代表之一)基因转入白僵菌和绿僵菌中表达,使工程菌获得了野生株不具有的特异性胃毒杀虫活性,高表达Vip3Aa1的工程菌剂对田间甘蓝上多种害虫的防效可匹敌化学杀虫剂。
转基因工程菌剂的注册必须通过严苛的环境安全评估。为了降低其安全风险,发明人尝试利用内源的脲嘧啶营养缺陷型基因ura3作为安全筛选标记,将Vip3Aa1导入球孢白僵菌中表达,并与先前的工程菌株比较目标基因和蛋白的表达水平,以构建更为安全的转基因工程菌技术平台。由于ura3源于白僵菌本身,这一技术实为无外源标记的基因操作技术。
发明内容
本发明要解决的技术问题是找到并提供一种内源安全标记,能表达昆虫胃毒杀虫蛋白,赋予生防真菌不具有的胃毒侵染杀虫机制和杀虫活性显著增强的杀虫蛋白超高表达的转基因工程菌株构建方法,并获得相应的工程菌株。
为了解决上述技术问题,本发明提供一种用内源ura3基因作为安全标记构建高表达胃毒杀虫蛋白的转基因生防工程真菌BbHUV5,该工程菌株的保藏名称为:球孢白僵菌Beauveriabassiana;保藏单位:中国微生物菌种保藏管理委员会普通微生物中心;保藏地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所;保藏日期:2015年12月02日;保藏编号:CGMCCNo.11802。
所述的用内源安全标记表达胃毒杀虫蛋白的转基因生防工程真菌的构建方法,其特征是包括以下步骤:
(1)载体构建
根据目标基因vip3Aa1的ORF及其载体的序列确定合适的酶切体系,分别提取pAN52-Phydl-tl-Vip3A和p0380-ura3质粒,经XbaI和HindIII双酶切,回收正确酶切片段并通过T4连接酶连接而成新质粒,经转化、PCR鉴定、酶切鉴定,获得阳性大肠杆菌转化菌株。然后扩增ura3的ORF并测序确认,选取正确转化子即为新载体pAN52-Phydl-tl-vip3Aa1-ura3;
(2)ura3缺失株感受态芽孢子转化与筛选
将所构建的双元质粒经HindIII酶切线性化,再用PEG和LiAc介导的ura3缺失株芽生孢子转化体系将其转化到其基因组中,筛选出阳性克隆,进行转化子中vip3Aa1基因的转录水平分析;
(3)获得内源安全标记表达胃毒杀虫蛋白的转基因生防工程菌株
筛选转录水平最高的转化子,以BbV28作为对照,进行酶联免疫吸附测定即ELISA实验,以检测Vip3Aa1蛋白在转化子中的表达水平,获得内源安全标记表达胃毒杀虫蛋白的转基因生防工程菌株BbHUV5,保藏编号为CGMCCNo.11802。
所述的用内源安全标记表达胃毒杀虫蛋白的转基因生防工程真菌的构建方法,其特征是:所述步骤(3)中的生防真菌为球孢白僵菌。
所述的用内源安全标记表达昆虫胃毒蛋白的转基因生防工程真菌的用途,其特征是:用于胃毒杀虫,不需要外源标记,避免了原先用除草剂抗性基因所标记的工程菌株的环境风险隐患。
上述一种用内源安全标记表达胃毒杀虫蛋白的转基因生防工程真菌,将杀虫蛋白Vip3Aa1的编码基因置于内源强启动子之下,构建出带有该杀虫蛋白的超高表达的载体。将此载体转入球孢白僵菌内源ura3营养缺陷型的感受态芽生孢子中,获得超高表达杀虫蛋白的重组工程菌株,其整体杀虫效力大幅提高,不携带任何外源除草剂抗性基因作为标记的环境风险。本发明公布的用内源ura3安全标记的高表达胃毒杀虫蛋白Vip3Aa1的转基因生防工程真菌BbHUV5,因具有天然白僵菌不具有的胃毒杀虫活性而使其杀虫活性大幅增强和杀虫谱显著拓宽。本发明采用的工程菌株构建思路及其工程菌株为开发杀虫增效且环境风险降低的真菌杀虫剂开辟了新的技术途径。
附图说明
图1为质粒pAN52-Phyd1-tl-Vip3A-ura3的构建示意图。
图2为球孢白僵菌疑似转化子的PCR鉴定;上为表达的vip3Aa1基因,下为回补的ura基因.泳道1为相应的质粒阳性对照。
图3为无抗性标记的球孢白僵菌工程菌株(BbHUV)中胃毒杀虫基因vip3Aa1的转录水平图。
图4为无抗性标记的球孢白僵菌工程菌株(BbHUV)中胃毒杀虫基因vip3Aa1的蛋白表达水平图。
具体实施方式
下面结合实验对利用本发明方法得到的重组工程菌株进行详细说明。
球孢白僵菌的野生株Bb2860、ura3敲除株△ura3、表达Vip3Aa1的工程菌株BbV28和BbHV8均为本实验室保藏或构建的菌株。BbV28的构建使用了真菌通用启动子即构巢曲霉的PgpdA驱动vip3Aa1在Bb2860中表达,BbHV8的构建则使用了经优化的内源疏水蛋白基因(hyd1)启动子Phydl-tl驱动vip3Aa1在Bb2860中高表达,二者的筛选标记都是草丁膦抗性基因bar。质粒:pAN52-Phydl-tl-Vip3Aa1和p0380-ura3,均为本实验室前期研究中所构建。ELISA试剂TMBcomponent购自Amresco(Solon,OH,USA),Vip3Aa1蛋白的兔血清多克隆抗体由浙江大学农业与生物技术学院沈志成教授提供。
工程菌株的构建方法
步骤1,根据目标基因vip3Aa1的开放阅读框(OpenReadingFrame,ORF)及其载体的序列进行分析,确定合适的酶切体系。分别提取pAN52-Phydl-tl-Vip3A和p0380-ura3质粒,经XbaI和HindIII双酶切,回收正确酶切片段并通过T4连接酶连接而成新质粒(图1)。经转化、PCR鉴定、酶切鉴定,获得阳性大肠杆菌转化菌株。然后,扩增ura3的ORF并测序确认,选取正确转化子即为本发明所用的新载体pAN52-Phydl-tl-vip3Aa1-ura3。
步骤2,ura3缺失株感受态芽生孢子的制备。将球孢白僵菌△ura3敲除株接种于萨氏培养基(SDAY)平板上,在25℃下培养至产孢。然后刮取适量分生孢子粉到1.5mL含0.05%吐温80的无菌水中,涡旋震荡分散。将分生孢子液接种于20mL的萨氏培养液SDB中,25℃下150r/min震荡培养2天后,吸取5mL培养液转入50mL限氮GM培养液(4%葡萄糖、0.3%NH4NO3、0.3%KH2PO4及0.3%MgSO4)中震荡培养1天,即得芽生孢子。在超净条件下过滤培养液,所获芽生孢子重悬于25mL无菌水中,,在4℃下3000r/min离心5min,弃上清,再重悬于25mL双蒸水洗涤,再4℃离心5min,弃上清。洗涤后的芽生孢子转入1mL0.1MLiAc中并吹匀,冰浴30min以增加细胞壁通透性。将以上芽生孢子液转移到1.5mL的EP管中,于4℃下6000~8000r/min离心5s,弃上清。最后加入0.5mL0.1MLiAc和0.15mL60%甘油,并按60μL每管分装到-80℃预冷30min的EP管中,-80℃冰箱保存备用。
步骤3,质粒的芽生孢子转化。将所构建的双元质粒经HindIII酶切线性化,再用PEG和LiAc介导的芽生孢子转化体系将其转化到野生株Bb2860的基因组中。转化过程如下:取1管制备好的感受态芽生孢子,冰浴融化后于4℃下8000r/min离心5s,弃上清。在芽生孢子沉淀中依次加入240μL50%聚乙二醇(PEG4000)、35μL1MLiAc、25μL4g/L热变性的蛙鱼精DNA(ssDNA)、10μL线形化的pAN52-Phydl-tl-vip3Aa1-ura3质粒以及35μL1M二硫苏糖(DTT)。将悬液剧烈震荡充分混匀后,冰浴30min后转到42℃热激20min,然后再冰浴片刻。随后,在6000~8000r/min离心15s,弃上清,并以500μL含0.5mg/mL尿嘧啶的M-100培养液重悬芽生孢子,吹匀后转入已灭菌的试管中。在20℃下150r/min预培养2~3h后,取孢子悬液重新离心15s,充分去上清,防止尿嘧啶残留。再以无菌水重悬,取100μL孢子液均匀涂布于无抗性标记的M-100培养基上,置于25℃下保湿培养6~7天。挑取正常生长的单孢菌落转移至SDAY培养基多孔培养板中进行复筛。
步骤4,转化子的鉴定及转录水平筛选。挑取多孔培养板上生长良好的转化子菌丝,进行KOD鉴定。再从菌丝中提取基因组DNA进行PCR鉴定。以各转化子的基因组DNA为模板,用vip3AaI基因的引物Vip3Aa1-F/R(表1)进行PCR鉴定。然后,再以vip3AaI基因阳性的转化子基因组为模板,以ura3基因的引物进行PCR扩增反应,进一步鉴定基因组中ura3基因的存在。上述PCR扩增反应程序为:94℃预变性5min后进入35个扩增循环,每循环依次94℃变性30s,60℃退火30s,72℃延伸1.5mins,循环结束后再72℃延伸7min。
步骤5,转化子中vip3Aa1基因的转录水平分析。提取上述PCR鉴定为双阳性转化子的RNA,反转录成cDNA,进行实时定量PCR(qRT-PCR)检测。以BbV28作为对照,以18SrRNA为内参,qRT-PCR定量分析vip3Aa1基因在各转化子中的转录水平,并以高表达vip3Aa1的BbHV8作为参照。
表1用于球孢白僵菌工程株构建及鉴定的成对引物.
步骤6,目标蛋白在转化子中表达水平的酶联免疫吸附测定。筛选目标基因转录水平最高的转化子,以BbV28作为对照,进行酶联免疫吸附测定即ELISA实验,以检测Vip3Aa1蛋白在转化子中的表达水平。具体步骤如下:将菌丝或分生孢子的蛋白抽提物用包被缓冲液(0.05M碳酸盐缓冲液、0.16%Na2CO3及0.29%NaHCO3;pH9.6)稀释至适当浓度,各取0.1mL加入酶标板反应孔中,4℃静置过夜。弃去孔内液体,用洗涤缓冲液(0.15MPBS,0.8%NaCI、0.02%KC1、0.29%Na2HPO4、0.02%KH2PO4及0.05%吐温20;pH7.4)洗板3次,每次3min。在各反应孔内加入0.1mL封闭液(包被液加1%牛血清白蛋白BSA),37℃孵育1h。然后,再用洗涤缓冲液洗板3次,每次3min。加入适当稀释的抗Vip3Aa1的兔血清抗体(一抗)0.1mL,37℃孵育1h后倾去孔内液体,再用洗涤缓冲液洗板3次,每次3min。随后,在各反应孔中加入适当稀释、与辣根氧化酶交联的羊抗兔Ig抗体(二抗),37℃孵育1h后洗板。最后于各反应孔中加入0.1mL的TMBcomponent,37℃下黑暗反应15min后立即加入0.1mL的2MH2SO4终止反应。最后将处理后的酶标板放入Model680酶标仪(Bio-Rad,Hercules,CA,USA)中测定各孔的OD450。反应以BbV28为阳性对照,Vip3Aa1蛋白的表达量用各待测转化子与阳性对照株中目标蛋白含量的比值来衡量,同样以高表达Vip3Aa1蛋白的BbHV8作为参照。
工程菌株胃毒杀虫活性的生物测定
将野生菌株Bb2860与工程菌株BbHU5在萨氏培养基平板上所产的分生孢子,以含0.02%吐温80的无菌水配制成4×106、2×107和1×108个孢子/mL悬液。斜纹夜蛾二、三龄幼虫每30~40头一组置于荷叶圆片(直径13cm)上并转至自动喷塔的样品台上,将各孢子悬液1mL喷到载虫的叶片上。然后,将载虫叶片转移到直径15cm的培养皿中,在25℃和光周期12:12h条件下饲养8天。期间,每隔1~2天更换事先喷雾相同菌液的新鲜叶片供试虫取食。沉降到叶片和虫体上的孢子密度,用一盖玻片置于叶片上收集自喷塔中沉降的孢子,在显微镜下将孢子密度确定为孢子数/cm2。饲养期间每天观察记录幼虫死亡数,虫尸移致饱和湿度条件下培养3~5天,死得较慢且体表长出特征供试菌的虫尸视为死于正常的体壁感染,死得很快且不能长出正常菌物的虫尸则视为死于摄入分生孢子所释放的胃毒杀虫蛋白Vip3Aa1的作用。
实验结果
转化子的PCR鉴定
以引物idUra3-F/R和idVip3A-F/R(表1)对球孢白僵菌的疑似转化子进行PCR鉴定。结果显示,大多数转化子的基因组DNA中都出现目标基因vip3Aa1的条带,仅有少数转化子中未见目标基因的条带,而所有转化子中都出现ura3的条带(图2)。结果表明,将ura3缺失株感受态芽生孢子导入野生株中,使球孢白僵菌在获得外源胃毒杀虫蛋白基因vip3Aa1的同时,原缺失的ura3基因被成功回补。
被导入目标基因及其编码蛋白的表达水平
将PCR鉴定呈阳性的转化子进一步进行qRT-PCR鉴定,检测各转化子中vip3Aa1基因相对于BbV28的转录水平。结果显示,随机抽取的10个转化子,其目标基因的转录水平较BbV28提高约3~25倍,但不及BbHV8(图3)。将qRT-PCR检测中目标基因转录水平最高的三个转化子进一步进行ELISA检测,其菌丝和分生孢子中胃毒杀虫蛋白Vip3Aa1的表达水平也都显著高于BbV28中的表达水平,但也不及BbHV8(图4)。
胃毒杀虫活性的生物测定结果
生物测定数据经时间-剂量-死亡率模拟分析,估计出死亡50%所需的孢子剂量LD50。野生菌株致死试虫缓慢,处理后第4、5天对二龄幼虫的LD50分别为1022和451个孢子/cm2,表现为经体壁侵染的正常毒力;对三龄幼虫的的LD50分别高达5494和2617个孢子/cm2,远超过最高喷雾浓度下的孢子沉降量(表2),说明野生株对较老的三龄幼虫的体壁侵染能力大幅减弱。与此形成鲜明对比的是,工程菌株BbHU5致死试虫很快,取食喷菌叶片后的前2天大量死亡,三齝幼虫的大量死亡有所延后,以致处理后第4、5天对二龄幼虫的LD50分别仅为47和26个孢子/cm2,对三龄幼虫的LD50分别仅为261和147个孢子/cm2。工程菌株如此低的LD50不可能是经体壁侵染所致,而是孢子随叶片摄入后释放到消化道的胃毒杀虫蛋白所致。野生菌株的分生孢子中无胃毒杀虫蛋白表达,只能通过体壁侵染对二龄幼虫产生缓慢的致死作用。
表2球孢白僵菌野生菌株与表达胃毒杀虫蛋白Vip3Aa1的工程菌株BbHU5对斜纹夜蛾幼虫口服毒力的比较
以上结果表明,真菌转化通用启动子PgpdA虽能驱动vip3Aa1在白僵菌中表达,但表达水平远不如经优化的内源疏水蛋白基因启动子Phydl-tl的驱动效果,但表达vip3Aa1的工程菌BbV28对斜纹夜蛾的胃毒杀虫效果很显著,超高表达的工程菌BbHV8的胃毒杀虫效果进一步大幅提高,其孢子制剂的田间杀虫效果甚至可匹敌化学杀虫剂。本发明也使用了内源启动子Phydl-tl驱动同一杀虫基因在Bb2860野生株中表达,目标基因和蛋白的表达水平在选出的转化子中都大大高于BbV28,但与BbV28相比仍有差距,说明仍有进一步优化筛选的必要。然而,以ura3营养缺陷型取代草丁膦抗性基因bar作为转化子筛选标记的工程菌构建新技术平台已搭建成功,对降低转基因工程菌的环境安全隐患、助推工程菌剂通过环境评估具有重要意义。
最后,值得注意的是,以上列举的仅是本发明的具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (4)
1.用内源安全标记表达胃毒杀虫蛋白的转基因生防工程真菌,其特征是:菌株BbHUV5的保藏名称为:球孢白僵菌Beauveriabassiana;保藏单位:中国微生物菌种保藏管理委员会普通微生物中心;保藏地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所;保藏日期:2015年12月02日,保藏编号:CGMCCNo.11802。
2.根据权利要求1所述的用内源安全标记表达胃毒杀虫蛋白的转基因生防工程真菌的构建方法,其特征是包括以下步骤:
(1)载体构建
根据目标基因开放阅读框(ORF)及其载体的序列确定合适的酶切体系,分别提取pAN52-Phydl-tl-Vip3A和p0380-ura3质粒,经XbaI和HindIII双酶切,回收正确酶切片段并通过T4连接酶连接而成新质粒,经转化、PCR鉴定、酶切鉴定,获得阳性大肠杆菌转化菌株,然后,扩增ura3的ORF并测序确认,选取正确转化子即为新载体pAN52-Phydl-tl-vip3Aa1-ura3;
(2)ura3缺失株感受态芽孢子转化与筛选
将所构建的双元质粒经HindIII酶切线性化,再用PEG和LiAc介导的ura3缺失株经芽生孢子转化体系将其转化到其基因组中,筛选出阳性克隆,进行转化子中vip3Aa1基因的转录水平分析;
(3)获得内源安全标记表达昆虫胃毒蛋白的转基因生防工程菌株
筛选转录水平最高的转化子,以BbV28作为对照,进行酶联免疫吸附测定即ELISA实验,以检测Vip3Aa1蛋白在转化子中的表达水平,获得内源安全标记表达昆虫胃毒杀虫蛋白的转基因生防工程菌株BbHUV5,保藏编号为CGMCCNo.11802。
3.根据权利要求2所述的用内源安全标记表达昆虫胃毒杀虫蛋白的转基因生防工程真菌的构建方法,其特征是:所述步骤(3)中的生防真菌为球孢白僵菌。
4.根据权利要求1所述的用内源安全标记表达胃毒杀虫蛋白的转基因生防工程真菌的用途,其特征是:用于胃毒杀虫,不需要外源标记,避免了原先除草剂标记下工程菌株的环境风险。
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SHENG-HUA YING等: "Use of uridine auxotrophy (ura3) for markerless transformation of the mycoinsecticide Beauveria bassiana", 《APPL MICROBIOL BIOTECHNOL》 * |
贾盘兴等编著: "《微生物遗传学实验技术》", 30 September 1992, 科学出版社 * |
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