携带苏云金芽胞杆菌cry1Ac10基因重组杆状病毒的构建及其杀虫活性

用Bac-to-Bac系统,构建了包含极晚期基因ph启动子驱动的带有全长苏云金芽胞杆菌cryIAc10基因和完整多角体基因的重组质粒pFCP, 用该重组质粒感染昆虫Sf9细胞,得到了带有多角体和能够表达cry1Ac10基因的重组杆状病毒vFcph,并在昆虫细胞中表达了Cry1Ac10蛋白.同时构建了含cry1Ac10的穿梭载体pHTC,并分别转化大肠杆菌、枯草杆菌和苏云金杆菌晶体缺陷型菌株,结果表明此三种工程菌均表达了分子量为133.3kDa的原毒素蛋白,其中在苏云金芽胞杆菌中的表达量最高.生物测定表明重组杆状病毒vFcph的表达产物具有杀虫活性,能增加杆状病毒力,加快杆状病毒杀虫速度,说明利用杆状病毒极晚期基因启动子驱动苏云金芽胞杆菌杀虫晶体蛋白表达,从而改善杆状病毒的杀虫特性是可行的.     

gfp基因标记的重组杆状病毒对棉铃虫幼虫的侵染历程

 用携带杆状病毒极晚期多角体蛋白基因启动子驱动gfp表达的重组病毒rHa FGP感染棉铃虫三龄幼虫 .在感染后不同时间取样 ,分离不同组织 ,制片 ,置于倒置荧光显微镜下观察基因的表达 .结果发现 ,随着感染时间的推移 ,荧光产生的部位出现更替 ,随后荧光强度也发生相应的变化 .从荧光出现的先后初步推断出杆状病毒对昆虫幼虫的侵染路线 :中肠上皮→血淋巴→气管系统→脂肪体 真皮 .在幼虫感染后 12h荧光即出现于中肠细胞中 ,表明此时已有极晚期蛋白表达 .说明利用杆状病毒极晚期基因启动子驱动苏云金芽孢杆菌杀虫晶体蛋白基因 (cry)表达 ,从而提高杆状病毒的杀虫毒力是可行的     

大肠杆菌中利用苏云金芽胞杆菌强启动子p1Ac指导Cry1Ac蛋白表达的特性分析

对利用苏云金芽胞杆菌(Bacillus thuringiensis,Bt)强启动子——cry1Ac基因启动子p1Ac指导cry基因在大肠杆菌中的表达进行了研究。结果显示,大肠杆菌中由启动子p1Ac指导表达的Cry1Ac蛋白与苏云金芽胞杆菌来源的Cry1Ac蛋白在碱溶性、胰蛋白酶活化、杀虫活性等方面有较好的一致性,从而解决了目前商业化载体大肠杆菌表达cry基因时形成不易溶解的包涵体问题。同时,还对p1Ac指导的cry1Ac基因在大肠杆菌中表达的发酵条件进行了初步探索。     

芽胞外壁基质组成蛋白的编码基因启动子PexsY指导的cry1Ac基因表达

【目的】利用非cry基因启动子PexsY(芽胞外壁基质组成蛋白编码基因启动子)表达Cry1Ac晶体蛋白,发现可用于cry基因表达的新元件,为高效工程菌的构建奠定基础。【方法】采用启动子融合lacZ技术,通过β-半乳糖苷酶活性分析了PexsY启动子和截短的PexsY启动子的转录活性;利用该启动子在苏云金芽胞杆菌(Bacillus thuringiensis,Bt)HD73菌株中表达了cry1Ac基因,通过透射电子显微镜观察晶体形态;蛋白定量、SDS-PAGE比较蛋白产量;生物活性测定进行功能验证。【结果】PexsY启动子在芽胞晚期转录活性很高,透射电镜观察到利用该启动子表达的cry1Ac基因形成了菱形晶体,SDS-PAGE分析可以检测到133kDa的Cry1Ac蛋白,且与cry3A启动子指导表达的蛋白产量相近,少于cry8E启动子指导表达的蛋白产量;生物活性测定表明PexsY指导表达Cry1Ac蛋白对玉米螟(Ostrinia furnacalis)具有杀虫活性。【结论】在Bt无晶体突变体中,非cry基因启动子PexsY可以正常表达133kDa的Cry1Ac蛋白,并形成晶体,具有在芽胞形成晚期表达cry基因的能力,该类启动子将在Bt工程菌构建中发挥重要作用。     

带cry3Aa启动子的aiiA基因在苏云金芽胞杆菌中的表达

N 乙酰高丝氨酸内酯 (N acyl homoserinelactones,AHLs) ,是一类数量感知 (Quorum sensing)系统中的信号分子 ,它参与诱导调控许多植物病原菌致病基因的表达。苏云金芽胞杆菌的AiiA蛋白能降解这类AHLs分子 ,进而可减弱病原菌致病基因表达产生的病害。苏云金芽胞杆菌杀虫晶体蛋白基因cry3Aa的启动子是一种不依赖芽胞形成的启动子 ,它相对于其它cry类基因的启动子有启动基因转录时间早 ,转录时间长的优点。通过重叠延伸PCR ,用杀虫晶体蛋白基因cry3Aa启动子替换编码AiiA蛋白的基因aiiA自身的启动子 ,构建了融合基因pro3A aiiA。将融合基因装入穿梭载体pHT3 0 4的BamHI SphI位点 ,得到重组质粒pBMB686并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株BMB686的AiiA蛋白表达量在各个生长时期均高于对照菌株 ,对AHLs分子的降解活性和对胡萝卜软腐欧文氏菌感染马铃薯产生病害的抑制能力也明显优于对照菌株     

马尾松毛虫质型多角体病毒多角体蛋白基因的表达与定位

为了表达马尾松毛虫质型多角体病毒(DpCPV)多角体蛋白基因(S10片段)并探讨多角体蛋白在真核细胞中的定位,从DpCPV中分离出S10,与pET-28a载体连接成重组表达质粒pET28-S10;将S10克隆到杆状病毒转座载体pFASTBAC HTb中,依次筛选出重组转座质粒pFASTBAC S10,重组穿梭质粒Bacmid S10,重组杆状病毒Ac S10.多角体蛋白基因表达后,用SDS-PAGE、Western-blot和免疫荧光技术对表达产物进行了检测.结果表明S10原核表达质粒、重组杆状病毒成功获得;在昆虫细胞中表达的质型多角体蛋白主要定位于细胞质,同时有少量产物定位于细胞核.     

苏云金芽胞杆菌cry1Ac与烟草几丁质酶tchiB双价基因克隆表达及其杀虫增效作用研究

将苏云金芽胞杆菌(Bacillus thuringiensis,Bt)4.0718菌株质粒上的cry1Ac基因和烟草几丁质酶tchiB基因(去掉信号肽或去信号肽再加肠激酶位点)构建了重组基因。经过双酶切和亚克隆,将带有cry1Ac基因上游启动序列和下游终止序列的重组基因片段克隆至穿梭载体pHT315,分别构建重组质粒pHUAccB6、pHUAccB7,在大肠杆菌中扩增后,将两个重组质粒分别电转化苏云金芽胞杆菌无晶体突变株XBU001中,获得重组菌株HAccB6和HAccB7。经液体双相胞晶分离提取离心后,将晶体和上清液分别进行SDS-PAGE分析,双价基因重组与cry1Ac基因在无晶体突变株中表达量相比较,几丁质酶活性提高5.2倍,双价重组蛋白表达量显著提高,主要产生130kDa蛋白条带。经定量分析:双价重组目的晶体蛋白占总蛋白量的61.38%;Cry1Ac蛋白占总蛋白量的42%。发酵上清液经60%硫酸铵沉淀,显示出一条分子量为18kDa新蛋白条带。经原子力显微镜和电子显微镜观察,表达后的重组蛋白呈菱形或椭原形晶体,其规格约为1.5×3.0μm;经生测分析,重组菌株HAccB6和HAccB7毒力相近,与HAc菌株比较毒力提高4.5倍,对棉铃虫(Helicourpa armigora)具有高效杀虫活性,其3dLC50值分别为9.1μg/mL和11.34μg/mL。研究结果表明,烟草几丁质酶与cry1Ac双价基因重组表达产物具有杀虫增效作用。     

利用苏云金芽胞杆菌转座子Tn4430构建含cry1Ac10基因的解离载体

将cry1Ac10基因和苏云金芽胞杆菌的复制起始区连 接在一起,并在其两侧按相同方向各连接一个来自苏云金芽胞杆菌转座子Tn4430的解离位点,构成转移单位。再将革兰氏阳性细菌的抗性标记基因和大肠杆菌克隆载体pUC19与之连接,获得含cry1Ac10基因的解离载体pBMB801。将其转化苏云金芽胞杆菌无晶体突变体,再导入含解离酶基因的辅助质粒pBMB1200。在解离酶作用下,两个解离位点间发生重组,消除基在操作中的抗性标记基因等非必需基因片段,获得仅保留有完整cry1Ac10基因和来 自苏云金芽胞杆菌质粒复制起始区,在无抗生素选择压力下能稳定遗传的重组质粒pBMB801B 。     

苏云金芽胞杆菌标记重组菌株的构建与杀虫基因水平转移

利用SOE法将构建的绿色荧光蛋白基因gfp和苏云金芽胞杆菌的杀虫晶体蛋白基因cry1Ac10的嵌合基因克隆到穿梭载体pAD4412上获得重组质粒pBMBZGC10,再通过电转化法导入苏云金芽胞杆菌无质粒突变株CryB中获得重组菌株CryB(pBMBZGC10).将重组菌株CryB(pBMBZGC10)的发酵液按30、60和90 ml共3个浓度梯度,菌数约为10.7～10.8·ml^-1,分次喷洒供试植株小白菜、蕹菜和番茄,结果表明,cry1Ac10基因没有向供试土壤细菌、真菌和放线菌转移,也未在供试植物根、茎和叶中检测到该基因.     

苏云金芽胞杆菌中荧光分析融合杀虫晶体蛋白的形成

为研究苏云金芽胞杆菌杀虫晶体蛋白在细胞中的定位及在细胞中的形成, 构建了Cry1Ac-GFP融合蛋白, 大小约为160 kD. 将携带cry1Ac启动子的cry1Ac-gfp融合基因片段克隆到pHT304载体上, 获得融合表达载体pHTcry1Ac-gfp. pHTcry1Ac-gfp转化到无晶体突变株HD-73 cry-中, 获得融合表达菌株HD-73-(pHTcry1Ac-gfp). gfp基因通过同源重组插入到HD-73内源大质粒pHT73上cry1Ac基因的3′端, 获得原位融合表达菌株HD-73Φ(cry1Ac-gfp)3534. 激光共聚焦显微镜和Western杂交分析表明, 不对称隔膜形成时, HD-73-(pHTcry1Ac-gfp)和HD-73Φ(cry1Ac-gfp)3534细胞中检测到Cry1Ac-GFP融合蛋白的表达. 融合蛋白颗粒在细胞中的聚集存在一定的极性, 分布于母细胞不对称隔膜附近. Cry1Ac-GFP和Cry1Ac蛋白对小菜蛾的杀虫活性在95%置信区间内没有明显差异.     

苏云金芽胞杆菌cry1Ac与烟草几丁质酶tchiB双价基因克隆表达及其杀虫增效作用研究

将苏云金芽胞杆菌(Bacillus thuringiensis,Bt)4.0718菌株质粒上的cry1Ac基因和烟草几丁质酶tchiB基因 (去掉信号肽或去信号肽再加肠激酶位点)构建了重组基因。经过双酶切和亚克隆,将带有cry1Ac基因上游启动序列和下游终止序列的重组基因片段克隆至穿梭载体pHT315,分别构建重组质粒pHUAccB6、pHUAccB7,在大肠杆菌中扩增后,将两个重组质粒分别电转化苏云金芽胞杆菌无晶体突变株XBU001中,获得重组菌株HAccB6和HAccB7。经液体双相胞晶分离提取离心后,将晶体和上清液分别进行SDS-PAGE分析,双价基因重组与cry1Ac基因在无晶体突变株中表达量相比较,几丁质酶活性提高5.2倍,双价重组蛋白表达量显著提高,主要产生130kDa蛋白条带。经定量分析：双价重组目的晶体蛋白占总蛋白量的61.38%;Cry1Ac蛋白占总蛋白量的42%。发酵上清液经60％硫酸铵沉淀,显示出一条分子量为18kDa新蛋白条带。经原子力显微镜和电子显微镜观察,表达后的重组蛋白呈菱形或椭原形晶体,其规格约为1.5×3.0μm;经生测分析,重组菌株HAccB6和 HAccB7毒力相近,与HAc菌株比较毒力提高4.5倍,对棉铃虫（Helicourpa armigora）具有高效杀虫活性,其3d LC₅₀值分别为9.1μg/mL和11.34μg/mL。研究结果表明,烟草几丁质酶与cry1Ac双价基因重组表达产物具有杀虫增效作用。     

Bt4.0718 cry1Ac基因的克隆与表达分析

在基因库中比对14种cry1Ac基因序列,发现了同源性很高的上游启动子区域和下游终止子区域。根据这一同源序列设计引物,从B t4.0718中扩增出包含双启动子和终止子的4.2 kb片段,用PCR-RFLP检测确定其中含有cry1Ac基因。然后将此片段克隆到穿梭载体pHT304中,转化大肠杆菌DH5α和B t无晶体突变株XZM-101。同时,利用原子力显微镜观察发现重组菌株BXZM34能够产生菱形晶体。     

苏云金芽胞杆菌高效表达载体的构建

[目的]通过比较cry1A、cry3A、cry4A和cry8E四个基因的启动子转录活性,筛选出一个强启动子,利用强启动子构建一个苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt)高效表达载体.[方法]利用启动子融合lacZ技术检测了4种启动子的转录活性.通过扫描电子显微镜观察晶体、SDS-PAGE、蛋白定量和生物活性测定等方法对新建高效表达载体进行功能验证.[结果]构建了Pcry1A、Pcry3A、Pcry4A和Pcry8E4个启动子融合报告基因lacZ的表达载体,经β-半乳糖苷酶活性分析得知,启动子活性从高到低依次为Pcry8E＞Pcry1A＞Pcry4A＞Pcry3A.选取cry8E启动子,以pHT315作为基础载体构建苏云金芽胞杆菌高效表达载体pHT315-8E21b,将cry1Ac基因连接到pHT315-8E21b和广泛应用的cry3A启动子指导的pSXY-422b上,分别转入无晶体突变株HD-73-,获得菌株HD-8E1Ac和HD-422-1Ac.扫描电子显微镜观察显示,HD-8E1Ac菌株可以形成菱形晶体,说明正确表达了cry1Ac基因.SDS-PAGE分析结合蛋白定量实验表明pHT315-8E21b表达效率高于pSXY-422b.对小菜蛾(Plutella xylostella)的生物活性测定表明HD-8E1Ac菌株对小菜蛾有生物活性,且菌株活性高于HD-422-1Ac.[结论]利用强启动子Pcry8E构建了一个能在Bt中高效表达的穿梭载体pHT315-8E21b,该载体可正确表达cry1Ac基因,其表达效率高于被广泛应用的pSXY422b.     

Preliminary comparing the toxicities of the hybrid cry1Acs fused with different heterogenous genes provided guidance for the fusion expression of Cry proteins

In order to provide guidance for selecting suitable heterogenous gene that can efficiently enhance toxicity or broaden insecticidal spectrum of Cry1Ac through fusion expression, two hybrid cry1Acs fused with chitinase-encoding gene tchiB and neurotoxin gene hwtx-1 respectively were constructed and their toxicities were compared. A Bacillus thuringiensis strain harboring the cry1Ac gene in vector pHT315 was used as control. Bioassay revealed that LC₅₀ (after 72 h) of Cry1Ac protoxin was 41.01 μg mL⁻¹, while the hybrid cry1Acs fused with tchiB and hwtx-1 were 4.89 and 23.14 μg mL⁻¹, which were 8.23- and 1.77-fold higher than Cry1Ac protoxin in terms of relative toxicity respectively. Both fusion crystals had a higher toxicity than the original Cry1Ac protein and the toxicity of hybrid cry1Acs fused with hwtx-1 experienced a more significant increase than that fused with tchiB.     

Characterization of the cry1Ac17 Gene from an Indigenous Strain of Bacillus thuringiensis subsp. kenyae

An indigenously isolated strain of Bacillus thuringiensis subsp. kenyae exhibited toxicity against lepidopteran as well as dipteran insects. The lepidopteran active cry1Ac protoxin gene coding sequence of 3.5 kb from this strain was cloned into vector pET28a(+). However, it could not be expressed in commonly used Escherichia coli expression hosts, BL21(DE3) and BL21(DE3)pLysS. This gene is classified as cry1Ac17 in the B. thuringiensis toxic nomenclature database. The coding sequence of this gene revealed that it contains about 3% codons, which are not efficiently translated by these expression hosts. Hence, this gene was expressed in a modified expression host, Epicurian coli BL21-Codonplus (DE3)-RIL. The expression of gene yielded a 130-kDa Cry1Ac17 protein. The protein was purified and its toxicity was tested against economically important insect pests, viz., Helicoverpa armigera and Spodoptera litura. LC₅₀ values obtained against these insects were 0.1 ng/cm³ and 1231 ng/cm², respectively. The higher toxicity of Cry1Ac17 protein, compared to other Cry1Ac proteins, toward these pests demonstrates the potential of this isolate as an important candidate in the integrated resistance management program in India.     

昆虫杆状病毒几丁质酶及其应用研究进展

杆状病毒几丁质酶基因（chitinase,,ChiA）是晚期表达的非必需基因,高度保守。表达产物几丁质酶分为3个区：N-端信号肽区,中部酶活性区和C-末端酶内质网结合区。该酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的几丁质,促进虫体液化;作为组织蛋白酶原（pro-V-Cath）的分子伴侣,参与其加工和运输过程; 影响多角体的形成,并与细胞的裂解有关;还与病毒侵染机制相关联。杆状病毒ChiA与细菌ChiA源于共同的祖先,而昆虫ChiA则可能直接来自杆状病毒。在害虫生物防治中,杆状病毒ChiA可直接作为杀虫剂,或作为苏云金杆菌和杆状病毒等微生物杀虫剂的增效剂使用;杆状病毒ChiA可转入植物,获得具有持续杀虫及抗病活性的转基因植物;将杆状病毒ChiA的内质网定位序列删除、突变,或在病毒基因组中插入外源ChiA,重组病毒的杀虫活性增强。通过基因工程手段,删除病毒基因组ChiA或V-Cath,可改善杆状病毒表达系统对分泌蛋白和膜结合蛋白的表达。     

Identification of cry-Type Genes on 20-kb DNA Associated with Cry1 Crystal Proteins from Bacillus thuringiensis

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.     

Comparative study on effect of different promoters on expression of cry1Ac in Bacillus thuringiensis chromosome

AIMS: The aim of this work was to investigate the effect of cry3A promoter on the expression of cry1Ac in Bacillus thuringiensis chromosome and stably enhance the production of different cry genes under the control of cry3A promoter. METHODS AND RESULTS: The cry1Ac gene, which is specific to Lepidopteran larvae, was integrated into the chromosome of a B. thuringiensis plasmid-free and acrystalliferous strain BMB171, under the control of cry3A promoter and cry1Ac promoter, respectively. The expression of cry1Ac genes in the chromosome of host strain was investigated. The results from sodium dodecyl sulfate-polyacrymide gel electrophoresis, crystal observation and bioassay showed that either integrated with cry3A promoter (cry3Apro-cry1Ac) or with its native promoter (cry1Acpro-cry1Ac), cry1Ac gene could efficiently and stably express in the chromosome. The production of cry3Apro-cry1Ac gene was higher than that of cry1Acpro-cry1Ac gene. CONCLUSIONS: The cry3A promoter enhanced the expression of cry1Ac gene efficiently either on the chromosome or on the plasmid in B. thuringiensis strain. SIGNIFICANCE AND IMPACT OF THE STUDY: So far, the comparative studies on cry3A promoter and other cry promoters were carried on B. thuringiensis plasmids. This system offers an additional method for potentially improving the efficacy of B. thuringiensis insecticidal proteins efficiently, stably and safely.