CN116515649A - 一种提高球孢白僵菌抗热胁迫的转基因方法 - Google Patents
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Abstract
本发明公开了一种提高球孢白僵菌抗热胁迫的转基因方法,属于生物技术领域。本发明在球孢白僵菌中超量表达草菇Vvmapk基因,获得超表达Vvmapk的球孢白僵菌菌株。该超表达Vvmapk的球孢白僵菌菌株在草菇3‑磷酸甘油醛脱氢酶基因启动子驱动下实现超量表达草菇Vvmapk基因,通过试验验证发现:该菌株的分子孢子具有良好的耐热性能,分生孢子悬液在45℃环境中热处理60min仍然具有76.3%左右的萌发率,相较野生型32.9%左右的萌发率具有较大的优势。这为延长球孢白僵菌分生孢子粉剂的货架期和提高实际应用价值具有重要意义。
Description
技术领域
本发明涉及生物技术领域,特别是涉及一种提高球孢白僵菌抗热胁迫的转基因方法。
背景技术
球孢白僵菌是广泛应用于农林害虫防治的病原真菌,主要以分生孢子进行无性繁殖,分生孢子萌发长出菌丝,菌丝在光、空气等外界环境刺激下分化为分生孢子梗,进一步分化为分生孢子,分生孢子随风和动物移动进行扩散。当分生孢子附着在昆虫体壁上时,通过萌发形成的附着胞和侵染钉,并分泌水解酶穿透昆虫体壁,在宿主体内形成虫菌体来逃避宿主的免疫反应。当宿主体内营养物质被耗尽时,球孢白僵菌以菌丝体的形式穿出体壁,继续分化为分生孢子。因此,分生孢子是发育的起点,对宿主毒力的强弱至关重要。但球孢白僵菌分生孢子在扩散和实际应用过程中会遇到温度、湿度、紫外线等外界环境,限制其应用范围,分生孢子的抗逆性成为其杀虫毒力的重要制约因素。
MAPK途径参与真菌二态的转变、厚垣孢子的形成、白色素和细胞壁的合成以及毒力的改变。虫生真菌MAPK级联途径接受外界信号,通过Mapk1、Slt2和Hog1等三条途径分别调控菌体的生长分化、细胞壁完整性和高渗胁迫反应。球孢白僵菌Hog1途径调控多种胁迫反应。绿僵菌Fus3-MAPK、Hog1-MAPK和Slt2-MAPK级联途径都可以调控分生孢子发育,级联途径中相应成分被删去后,菌株的分生孢子梗受损或发育推迟,导致分生孢子的产量减少,而且Hog1-MAPK磷酸化水平受到Slt2-MAPK的调控,当hog1被敲除后,分生孢子发育中心途径因子Mr-AbaA、Mr-WetA、以及色素合成相关基因的转录水平均显著降低。蛋白磷酸化也可能参与酿酒酵母对冷胁迫的细胞信号传导,涉及到基因转录、蛋白质折叠和降解、细胞周期调节和形态发生等过程。在烟曲霉(Aspergillus fumigatus)中也发现此途径参与冷和热等多种胁迫反应。但关于草菇磷酸化途径是否参与冷热胁迫反应及发生机理未见报道。
发明内容
本发明的目的是提供一种提高球孢白僵菌抗热胁迫的转基因方法,以解决上述现有技术存在的问题,通过在草菇3-磷酸甘油醛脱氢酶基因启动子驱动下在球孢白僵菌种超量表达草菇Vvmapk基因,构建的转基因菌株相比野生球孢白僵菌显著提高抗热胁迫能力。
为实现上述目的,本发明提供了如下方案:
本发明提供一种提高球孢白僵菌抗热胁迫的转基因方法,包括:在球孢白僵菌中超量表达草菇基因Vvmapk,获取超表达Vvmapk的球孢白僵菌菌株的步骤。
优选的是,所述转基因方法具体包括以下步骤:
(1)分别克隆草菇基因Vvmapk以及驱动该基因表达的草菇3-磷酸甘油醛脱氢酶基因启动子后,进行基因融合;
(2)步骤(1)得到的融合片段导入表达载体,构建重组载体;
(3)将所述重组载体转入农杆菌进行遗传转化,筛选出具有抗性的农杆菌,之后再将具有抗性的农杆菌和球孢白僵菌等体积混合,经培养、筛选,获取超表达Vvmapk的球孢白僵菌菌株。
优选的是,步骤(1)中,克隆草菇基因Vvmapk所用引物如SEQ ID NO:1-2所示,克隆草菇3-磷酸甘油醛脱氢酶基因启动子所用引物如SEQ ID NO:3-4所示。
优选的是,步骤(2)中,所述表达载体包括pk2(bar)。
优选的是,步骤(3)中,通过电击方法将所述重组载体转入农杆菌,所述电击的条件为:2200V,4~5ms。
优选的是,步骤(3)中,依次用IM固体培养基以及添加头孢噻肟钠抗生素和除草剂PPT的CZA培养基进行培养筛选,其中,IM固体培养基包括以下组分:7.8g MES,5mL 1M葡萄糖,0.3g NaCl,0.3g K2HPO4,0.3g MgSO4·7H2O,5mL甘油,15g琼脂,100μL 200mM乙酰丁香酮,加ddH2O补充至1000mL。
本发明还提供草菇基因Vvmapk在构建提高球孢白僵菌抗热胁迫能力中的应用,通过在球孢白僵菌中超量表达草菇基因Vvmapk,提高球孢白僵菌抗热胁迫能力。
本发明还提供包含草菇基因Vvmapk的重组载体在提高球孢白僵菌抗热胁迫能力中的应用,通过将包含表达草菇基因Vvmapk的重组载体在球孢白僵菌中超量表达,提高球孢白僵菌抗热胁迫能力。
本发明还提供包含所述的重组载体的宿主菌在提高球孢白僵菌抗热胁迫能力中的应用。
本发明公开了以下技术效果:
本发明首次发现并通过试验验证草菇Vvmapk基因在球孢白僵菌中超量表达,能提高球孢白僵菌分生孢子的抗热处理能力,具体当构建超量表达Vvmapk基因的球孢白僵菌分生孢子悬浮液在45℃条件下被热处理60min时,分生孢子的萌发率呈现出下降的趋势,但和WT(野生型)相比,超表达菌株的分生孢子萌发率极显著高于WT(P<0.01)。说明通过草菇Vvmapk基因在球孢白僵菌中超量表达可以显著提高其抗热胁迫能力,而耐热性能是延长球孢白僵菌分生孢子粉剂的货架期和提高实际应用价值的重要因素,因此,本发明获取的超表达Vvmapk基因的球孢白僵菌具有实际应用价值。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为pK2(bar)-Vvgpd:mapk载体构建示意图;
图2为超表达载体的构建和转化株PCR验证;A:Vvgpd基因启动子的扩增;B:pK2(bar)-OEVvmapk载体用EcoRΙ酶切验证;C:重组载体转化农杆菌的PCR验证,以Vvgpd-F/PtrpC为引物;D:球孢白僵菌OEVvmapk转化子的PCR验证,以Vvmapk-F/PtrpC为引物;Marker代表标准DNA分子;1、2、3代表不同菌株;
图3为OEVvmapk菌株Vvmapk的mRNA水平;WT代表野生株系;#1、#2、#3、#4分别代表不同株系;
图4为OEVvmapk菌株分生孢子对热胁迫的反应;**:P<0.01。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1
1、在球孢白僵菌超量表达草菇Vvmapk
1.1草菇Vvmapk基因和Vvgpd启动子融合
(1)草菇DNA模板的制备
用灭菌牙签挑取在PDA培养基上生长的草菇菌丝,浸没于5μL 0.3mol/L的NaOH溶液中,在PCR仪中95℃加热3min进行菌丝裂解,然后加入106μL中和液混合均匀,混合液即为DNA粗提液。中和液配方:5mL 1M Tris-HCl(pH=8.0),20mL 0.3M HCl,用ddH2O定容至400mL。
(2)草菇Vvgpd基因启动子的PCR扩增
PCR反应体系为:10μL 2×KeyPo Master Mix(Dye Plus)反应混合液,各1μL Vvgpd-F/R引物(见表1),8μL DNA模板,总体积为20μL。
反应程序为:98℃/3min,98℃/10s,55℃/5s,72/5s,72/5min,其中第2~4步进行30个循环。
表1试验所用的引物
(3)草菇Vvmapk基因的PCR扩增
从草菇菌丝体中提取RNA,经过反转录获得cDNA,以cDNA为模板进行PCR扩增。PCR扩增体系为:10μL 2×KeyPo Master Mix(Dye Plus)反应混合液,各1μL Vvmapk-F/R引物(见表1),1μL cDNA模板,补足ddH2O使反应总体积为20μL。反应程序为:98℃/3min,98℃/10s,55℃/5s,72/10s,72/5min,其中第2~4步进行30个循环,获得草菇Vvmapk基因(GenBank:FJ906769.1)。
(4)将草菇Vvmapk基因和Vvgpd基因(GenBank:KF528323.1)启动子(起始密码子ATG前1200bp)进行PCR融合连接。以Vvgpd-F/Vvmapk-R为引物,各1μL步骤(2)和(3)回收的片段为模板进行PCR扩增。反应程序同步骤(2)。
1.2构建重组载体
具体方法为:将融合片段通过同源重组的方法连接在pk2(bar)载体上,反应体系按照ClonExpress MultiS One Step Cloning Kit(南京诺唯赞生物科技股份有限公司)说明书推荐的体系进行:2μL 5×反应缓冲液,1μL经过EcoRΙ酶切后回收的载体,3μL融合片段,1μL连接酶,补足ddH2O为10μL,在PCR仪中按照37℃/30min进行连接,转化大肠杆菌。经过提取质粒、酶切和测序确认重组载体pK2(bar)-OEVvmapk的正确性。
1.3农杆菌转化
通过电击转化的方法将重组载体pk2(bar)-OEVvmapk转入农杆菌AGL-1,电击条件为2200V,4~5ms,在添加卡那抗生素(工作浓度为50μg/mL)的YEB培养基上筛选。
1.4培养单菌落
把上述筛选出的农杆菌,在添加卡那抗生素(工作浓度为50μg/mL)的YEB液体培养基上培养至OD600=0.5,12000r/min离心1min收集沉淀,弃去上清,然后用IM液体培养基调整至浓度为OD600=0.15,在28℃、200r/min条件下继续培养6h。把培养好的农杆菌和球孢白僵菌分生孢子悬液(浓度为1×105个/mL)以等体积混合,涡旋振荡。按照100μL/平板的量把混合菌液涂布在提前铺有微孔滤膜的IM固体培养基上,26℃避光培养48h,然后把滤膜转移到加有头孢噻肟钠抗生素(工作浓度为500μg/mL)和除草剂PPT[18%(wt%)的草铵膦原溶液用无菌水稀释4倍,然后按照1:1000的比例添加]的CZA培养基上培养5~7d,直至有单菌落的出现。
其中,上述试验步骤中IM液体培养基配方为:7.8g MES,10mL 1M葡萄糖,0.3gNaCl,0.3g K2HPO4,0.3g MgSO4·7H2O,5mL甘油,100μL 200mM AS(乙酰丁香酮,Acetosyringone),加ddH2O定容为1000mL,调pH=5.3。注意葡萄糖以及AS需要过滤后,在培养基冷却到55℃后再加入。IM固体培养基配方:5mL 1M葡萄糖,15g琼脂粉,其他成分同液体培养基。
1.5筛选阳性转化子
用牙签挑取单菌落接种至具有同样抗性的48孔板上进行筛选。再次用无菌牙签挑取生长在48孔板中的菌落,以菌丝DNA为模板,进行PCR扩增验证潜在的阳性转化子。把阳性转化子进行转接和扩增,用做下步的试验材料。
1.6利用定量PCR方法筛选超量表达菌株OEVvmapk的表达情况
使用带有SYBR Green(Bio-Rad)的iCycler iQ多色实时PCR检测系统进行实时定量PCR。5μL反应混合液,0.5μL上下游引物(序列见表1中qVvmapk-F/R和qactin-F/R,浓度为10μmol/L),3μL稀释20倍的cDNA模板,1μL ddH2O,总体积为10μL,短暂离心混合均匀;95℃/5min进行预变性;然后按照95℃/15s;60℃/15s;72℃/30s的程序进行39个循环的扩增。基因的相对表达量以actin的表达量为参考进行确定。
1.7测定分生孢子萌发率
45℃/60min对超量表达菌株的分生孢子进行热击处理。将分生孢子悬液调整浓度为1×108个/mL,在金属浴中对孢悬液进行45℃热击处理60min,然后涂布在CZA培养基上,26℃条件下培养24-76h,每隔12h用锋利的刀片切取1cm×1cm大小的培养基块,用倒置显微镜进行观察拍照,计算分生孢子的萌发率。
2、结果与分析
(1)重组载体构建
重组载体构建示意图见图1,通过将草菇Vvmapk基因的ORF框和Vvgpd启动子进行融合,通过设计引物的方法在融合片段的两端加上EcoRΙ酶切位点,通过同源重组的方法和pK2(bar)载体进行连接,构建超量表达载体pK2(bar)-OEVvmapk。
(2)超表达载体的构建和转化株PCR验证
以草菇DNA为模板,Vvgpd-F/R为引物扩增Vvgpd基因启动子,扩增片段大小为1200bp左右(图2中A)。
以草菇cDNA为模板,Vvmapk-F/R为引物扩增Vvmapk基因,把获得的Vvmapk片段(1150bp)和Vvgpd基因启动子进行PCR融合连接,然后克隆到pK2(bar)载体上,经过酶切,可以切下2350bp大小的片段(图2中B),与预期片段大小相符。
进一步测序确认克隆片段的正确性。把获得的pK2(bar)-OEVvmapk重组载体转入农杆菌中,以Vvgpd-F/PtrpC-R为引物可以扩增出2350bp左右大小片段(图2中C),说明重组载体转入农杆菌中。通过农杆菌介导的方法把pK2(bar)-OEVvmapk重组载体转入球孢白僵菌中,以Vvmapk-F/PtrpC-R(见表1)为引物可以扩增出1200bp左右大小的片段,说明重组载体已经转入球孢白僵菌中(图2中D)。
(3)超量表达Vvmapk的不同株系进行定量PCR验证
选取超量表达的4个不同株系进行定量PCR验证。和WT相比,4个株系中Vvmapk的表达量都得到不同程度的升高,其中株系2#、3#、4#株系的基因表达量是WT的2.2-2.7倍(图3),以这3个株系作为后续的试验材料。
(4)检测萌发率
对分生孢子悬液进行45℃热击处理60min,在培养24h时并没有观察到孢子萌发情况,继续培养至48h观察分生孢子的萌发情况,并统计萌发率,发现超量表达菌株的平均萌发率为76.3%,显著高于野生型分生孢子的平均萌发率32.9%(见图4)。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (9)
1.一种提高球孢白僵菌抗热胁迫的转基因方法,其特征在于,包括:在球孢白僵菌中超量表达草菇基因Vvmapk,获取超表达Vvmapk的球孢白僵菌菌株的步骤。
2.根据权利要求1所述的转基因方法,其特征在于,所述转基因方法具体包括以下步骤:
(1)分别克隆草菇基因Vvmapk以及驱动该基因表达的草菇3-磷酸甘油醛脱氢酶基因启动子后,进行基因融合;
(2)步骤(1)得到的融合片段导入表达载体,构建重组载体;
(3)将所述重组载体转入农杆菌进行遗传转化,筛选出具有抗性的农杆菌,之后再将具有抗性的农杆菌和球孢白僵菌等体积混合,经培养、筛选,获取超表达Vvmapk的球孢白僵菌菌株。
3.根据权利要求2所述的转基因方法,其特征在于,步骤(1)中,克隆草菇基因Vvmapk所用引物如SEQ ID NO:1-2所示,克隆草菇3-磷酸甘油醛脱氢酶基因启动子所用引物如SEQID NO:3-4所示。
4.根据权利要求2所述的转基因方法,其特征在于,步骤(2)中,所述表达载体包括pk2(bar)。
5.根据权利要求2所述的转基因方法,其特征在于,步骤(3)中,通过电击方法将所述重组载体转入农杆菌,所述电击的条件为:2200V,4~5ms。
6.根据权利要求2所述的转基因方法,其特征在于,步骤(3)中,依次用IM固体培养基以及添加头孢噻肟钠抗生素和除草剂PPT的CZA培养基进行培养筛选,其中,IM固体培养基包括以下组分:7.8g MES,5mL 1M葡萄糖,0.3g NaCl,0.3g K2HPO4,0.3g MgSO4·7H2O,5mL甘油,15g琼脂,100μL 200mM乙酰丁香酮,加ddH2O补充至1000mL。
7.草菇基因Vvmapk在构建提高球孢白僵菌抗热胁迫能力中的应用,其特征在于,通过在球孢白僵菌中超量表达草菇基因Vvmapk,提高球孢白僵菌抗热胁迫能力。
8.包含草菇基因Vvmapk的重组载体在提高球孢白僵菌抗热胁迫能力中的应用,其特征在于,通过将包含表达草菇基因Vvmapk的重组载体在球孢白僵菌中超量表达,提高球孢白僵菌抗热胁迫能力。
9.包含权利要求8中所述的重组载体的宿主菌在提高球孢白僵菌抗热胁迫能力中的应用。
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