苏云金芽胞杆菌Rpp39杀虫晶体蛋白基因的鉴定及cry2Aa12基因的克隆表达

[目的]本研究的目的是分析从四川生态条件下分离的苏云金芽胞杆菌Rpp39菌株的特性,从分子水平上揭示该菌株对鳞翅目高毒力的原因;进一步从中分离克隆cry2Aa基因,并对其进行初步的表达研究.[方法]本研究主要采用扫描电镜观察、PCR-RFLP鉴定法和SDS-PAGE分析法研究菌株的特性;采用PCR直接克隆法克隆cry2Aa全长基因,并亚克隆到原核表达载体pET-30a中,构建重组表达质粒pET-2Aa,再转入受体菌E.coli.BL21(DE3)中进行诱导表达;采用室内生物测定法测定表达产物对小菜蛾和水稻二化螟的毒力.[结果]经扫描电镜观察菌株Rpp39主要产生菱形、方形和圆形3种伴胞晶体;SDS-PAGE分析表明主要产生130 kDa和60 kDa左右2种蛋白;经PCR-RFLP鉴定,该菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Ia和cry2Aa五类杀虫晶体蛋白基因;1种cry2Aa类杀虫晶体蛋白全长基因被克隆,序列分析显示该基因的开放阅读框(ORF)为1902 bps,编码由634个氨基酸组成的蛋白质,氨基酸序列与Cry2Aa1蛋白同源性为99.7%,被国际Bt杀虫晶体蛋白基因命名委员会命名为cry2Aa12.重组表达质粒pET-2Aa在E.coli BL21(DE3)中,经IPTG诱导能正常表达,SDS-PAGE电泳验证含有65 kDa表达蛋白.生物活性测定表明表达的包涵体蛋白对小菜蛾和二化螟具有杀虫活性,LC50分别为5.4 μg/mL和22.3μg/mL.[结论]菌株Rpp39及从中分离克隆的cry2Aa12基因来自四川生态条件,丰富了菌株及基因的资源,在资源积累方面具有重要意义.     

苏云金芽胞杆菌sigK 基因插入失活突变体的特点及其对cry3A基因启动子活性的影响

摘要: 【目的】构建苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt) sigK 基因插入失活突变体,分析突变体特点并明确其对cry3A 基因启动子的影响。【方法】采用同源重组技术在苏云金芽胞杆菌HD-73 菌株sigK 基因中插入卡那霉素抗性基因,构建了sigK 基因插入失活突变体。通过生长曲线测定、扫描电子显微镜观察晶体、芽胞形成情况和芽胞计数及SDS-PAGE 等方法分析了突变体的特点; 构建了遗传恢复菌株对上述性状进行了功能验证; 利用启动子融合lacZ 技术检测了cry3A 基因启动子的转录活性。【结果】获得了苏云金芽胞杆菌HD-73 菌株sigK 基因突变体,生长曲线测定表明,突变体较出发菌株在稳定期后期生长较慢; 扫描电子显微镜观察和芽胞计数分析显示,突变体丧失了形成芽胞和晶体的能力; SDS-PAGE 分析表明突变体中伴胞晶体蛋白的表达量明显低于出发菌株和恢复菌株。利用载体pHT315 携带sigK 基因及其启动子在突变株中表达,所获得的遗传恢复菌株恢复了突变株产生芽胞和晶体的能力; sigK 基因的突变可以提高cry3A 基因启动子在产胞后期的转录活性,对cry3A 启动子指导的Cry 蛋白表达量没有显著影响。【结论】本研究证明sigK 基因为苏云金芽胞杆菌芽胞形成所必需,并影响伴胞晶体蛋白的产量; sigK 基因功能的丧失有利于cry3A 基因启动子在产胞后期的转录。     

苏云金芽胞杆菌cry2Ad基因的克隆及其表达产物的活性分析

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)SBT2是我国新分离出的一株野生菌株.扫描电镜显示该菌株产生双锥体形晶体.琼脂糖凝胶电泳发现其质粒图谱含有5个条带.聚丙烯酰胺凝胶电泳显示此菌株产生130 kD晶体蛋白.利用PCR-RFLP法进行杀虫基因类型鉴定,发现其含有cry1Aa、cry1Da、cry1Hb、cry1Jb、cry1Ka 、cry1Ib、基因.Cry2Ad蛋白的活性至今未见研究报道,本研究克隆和测序了该基因.并对其进行了表达.生物活性测定结果表明其表达产物对舞毒蛾(Lymantria dispar)、棉铃虫(Helicoverpa armigera)、亚洲玉米螟(Ostrinia furnacalis)、小菜蛾(Plutella xylostella)有低活性;对大猿叶甲(Colaphellus bowringi)无活性.     

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。     

苏云金芽胞杆菌cry2Ab、cry9Ea基因的表达及活性分析

旨在为农业害虫防治提供更多的苏云金芽胞杆菌基因资源,从中国吉林市龙潭山土壤样品中分离得到野生菌株命名为Bt LTS-7,扫描电镜显示该菌株产生晶体形状为双锥体和正方体,聚丙烯酰胺凝胶电泳显示该菌株产生130 kD和71 kD的晶体蛋白,通过PCR鉴定出该菌株中含有cry2Ab和cry9Ea基因,并成功克隆到了这两个新基因,并被Bt国际命名委员会正式命名为cry2Ab28和cry9Ea9,将两个基因分别在大肠杆菌Rosetta( DE3)中表达,并进一步对其表达蛋白进行杀虫活性测定.结果显示,Cry2Ab28蛋白对棉铃虫(Helicoverpa armigera)初孵幼虫具有杀虫活性,LC50为32.45 μg/mL.Cry9Ea9蛋白对小菜蛾初孵幼虫(Plutella xylostella)具有较高杀虫活性,LC50为0.77μg/mL.     

苏云金芽胞杆菌V4菌株cry杀虫基因的鉴定、表达及杀虫活性分析

旨在给生物防治提供更多的基因资源,以从本实验室分离的300株苏云金芽胞杆菌基因组为模板,利用cry1类全长通用引物进行PCR cry1类基因鉴定。经过PCR-RFLP鉴定出菌株V4含有cry1Ea基因,进一步研究发现该菌株还含有cry2Aa基因,并成功克隆到了这两个新基因,被Bt国际命名委员会正式命名为cry1Ea12和cry2Aa16。将两种基因在大肠杆菌Rosetta(DE3)中表达,表达蛋白进行杀虫活性测定,结果表明两种蛋白杀虫活性不理想,但对试虫存在明显的体重抑制,而V4菌株蛋白具有较高的杀虫活性。     

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     

苏云金芽胞杆菌杀线虫伴胞晶体蛋白基因cry6Aa的克隆与表达

通过对晶体蛋白N-末端氨基酸测序,设计简并探针,从对根结线虫高毒力苏云金芽胞杆菌YBT-1518菌株中克隆到1个含有杀线虫晶体蛋白基因的片段。序列测定表明该序列含有两个ORF(orf1和orf2),其中orf1与基因cry6Aa1同源性为98%,已在GenBank上登录(Acc.NO.AF499736),并被命名为cry6Aa2。将克隆的该片段克隆到穿梭载体pHT304上,并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株可形成米粒状伴胞晶体。生物测定表明,表达的毒素蛋白对北方根结线虫的LC50为9.47μg/mL,毒力与出发菌株(10.74μg/mL)相当。     

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.     

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cry1Ac10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。     

Two novel delta-endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp. finitimus.

Genes cry26Aal and cry28Aal were cloned from Bacillus thuringiensis ssp. finitimus strain B-1166 VKPM. This strain forms insecticidal crystal bodies either outside or inside the exosporium. The deduced amino acid sequence of the cry26Aal gene product included seven residues determined to be an N-terminal part of a chymotrypsin-treated delta-endotoxin isolated from the same strain. Earlier this protein was detected in both free and spore-associated types of crystals [Revina et al., Biokhimia (1999) in press]. Neither BtI nor BtII promoter sequences were found upstream of the open reading frames in both genes. Southern hybridization has shown that the surroundings of both genes at least 3 kb upstream and downstream of the open reading frames are unique. We suggest that the protein Cry26Aal in both types of crystal bodies is synthesized under the control of one and the same genomic locus.     

Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis delta-endotoxin nomenclature committee.     

转cry1Ab/cry1Ac基因水稻对大螟幼虫的取食、生长及存活的影响

本文采用活体生测的方法测定了转cry1Ab/cry1Ac基因水稻对大螟Sesamia inferens(Walker)幼虫的取食、生长以及存活的影响。结果表明,以转cry1Ab/cry1Ac基因水稻为食的大螟3龄和5龄幼虫的累计取食量和相对摄食率与对照相比都显著的减少。取食转Bt水稻后,大螟3龄和5龄幼虫体重均出现负增长。随着取食转基因水稻时间的延长,大螟3龄幼虫的校正死亡率可以达到100%,5龄幼虫的校正死亡率达到85%。     

水稻中cry1Ah1基因密码子优化方案的比较

cry1Ah1基因是本实验室克隆的具有自主知识产权的模式基因,对鳞翅目害虫水稻二化螟等具有高毒力,具有较好的应用前景。为提高cry1Ah1基因在水稻中的表达量,探讨密码子使用频率对基因表达的影响,依据水稻密码子使用频率设计5种不同的优化方案,提高GC含量并去除剪切信号等不稳定因素后合成cry1Ah1基因杀虫活性区域。优化后的基因在大肠杆菌Rosetta(DE3)中正常表达了65 kDa蛋白,表达蛋白对2龄小菜蛾和水稻二化螟初孵幼虫都具有良好的杀虫活性。优化的基因转化水稻日本晴后,PCR阳性率达到87%以上,实时荧光定量RT-PCR和ELISA分析表明全部采用最高频率密码子的优化方案效果最好,Cry1Ah蛋白平均表达量占可溶性蛋白的0.104%。     

四种cry 基因启动子在spoIIID基因突变株中的活性比较

【目的】分析spoIIID基因突变对苏云金芽胞杆菌cry1、cry3、cry4和cry8基因启动子Pcry1Ac、Pcry3A、Pcry4A和Pcry8E的影响,比较以上启动子在无芽胞spoIIID基因突变体(HD-△SpoIIID)中的转录活性。【方法】分别构建了Pcry1Ac、Pcry3A、Pcry4A和Pcry8E与lacZ基因融合的转录分析载体,并导入HD-73野生型菌株和HD-△SpoIIID突变株中测定β-半乳糖苷酶活性;通过高温诱变方法在HD-△SpoIIID基础上筛选出缺失cry1Ac基因的HD--△SpoIIID突变体;构建了4种启动子与cry1Ac基因融合表达载体,分别将它们转入HD-ΔSpoIIID和HD--ΔSpoIIID中,分析Cry1Ac蛋白表达量及其生物活性。【结果】HD-73和HD-ΔSpoIIID菌株中四个启动子转录活性由高到低分别为:Pcry8E>Pcry1Ac>Pcry4A>Pcry3A;spoIIID基因的缺失未影响Pcry1Ac和Pcry8E转录活性,Pcry3A在HD-ΔSpoIIID菌株中转录活性略有升高,Pcry4A在HD-ΔSpoIIID菌株中转录活性在T5到T10略有降低。从翻译水平来看在HD-ΔSpoIIID中cry8E启动子略低于cry1Ac启动子,并高于Pcry4Aa,Pcry3A指导的Cry1Ac蛋白产量,生物活性测定结果与蛋白表达量相符。【结论】cry8E基因启动子Pcry8E在spoIIID突变体中在转录水平活性是最高的启动子,而cry1Ac启动子指导自身基因cry1Ac表达时,在翻译水平上略高于cry8E启动子指导的Cry1Ac产量。     

Cloning and nucleotide sequence of a novel cry gene from Bacillus thuringiensis

A cry1Ab-type gene was cloned from a new isolate of Bacillus thuringiensis by PCR. When restriction pattern was compared with that of known genes it was found to have additional restriction site for ClaI. Nucleotide sequencing and homology search revealed that the toxin shared 95% homology with the known Cry1Ab proteins as compared to more than 98% homology among the other reported Cry1Ab toxins. The gene encoded a sequence of 1,177 amino acids compared to 1,155 amino acids encoded by all the other 16 cry1Ab genes reported so far. An additional stretch of 22 amino acids after the amino acid G793 in the new toxin sequence showed 100% homology with several other cry genes within cry1 family. Homology search indicated that the new cry1Ab-type gene might have resulted by nucleotide rearrangement between cry1Ab and cry1Aa/cry1Ac genes.     

Cloning and analysis of the first cry gene from Bacillus popilliae.

An 80-kDa parasporal crystal protein was detected in protein extracts of sporangia of Bacillus popilliae isolated from a diseased larva of the common cockchafer (Melolontha melolontha L.). Amino acid analysis of tryptic peptides revealed significant homology to the Cry2Aa endotoxins of Bacillus thuringiensis. The gene cryBP1 (cry18Aa1), which codes for the parasporal crystal protein, was found in a putative cry operon on the bacterial chromosome, which contains at least one further (smaller) open reading frame, orf1. The 706-amino-acid-long CryBP1 (Cry18Aa1) protein has a predicted molecular mass of 79 kDa and shows about 40% sequence identity to the Cry2 polypeptides of B. thuringiensis. In the light of published observations which suggest that the parasporal crystal proteins of B. popilliae are slightly toxic to their grub hosts, we propose the following survival strategy of B. popilliae. As an obligate pathogen of grubs, B. popilliae germinates in the gut of a grub and the parasporal crystal proteins are released and activated. The activated protein does not cause colloid osmotic lysis but instead damages the gut wall somehow to allow the vegetative cells to enter the hemolymph more easily. By becoming a parasite, B. popilliae can continue to proliferate efficiently while the living grub provides a food supply. This process is in contrast to that of B. thuringiensis, which rapidly kills the insect and is then limited to growth on the larval carcass.     

转cry1Ab/cry1Ac基因籼稻对稻田节肢动物群落影响

将稻田节肢动物群落按营养关系划分为5个功能团,即植食类、寄生类、捕食类、腐食类和其它类,从功能团优势度、功能团内科组成及其优势度、群落主要参数及群落相异性等方面,经两年四点的调查就2个转cry1Ab/cry1Ac基因籼稻（Bt水稻）品系TT9.3和TT9.4对稻田节肢动物群落的影响作了较系统评价。植食类、寄生类和腐食类功能团内某些优势科的优势度在Bt水稻田与对照（IR72）田之间有时呈显著或极显著差异,如Bt水稻田中茧蜂或姬蜂科的优势度有时明显低于对照。但是,在大多情况下Bt水稻田与对照田之间功能团优势度、功能团内科组成及其优势度、群落主要参数（物种丰富度、Shannon-Wiener多样性指数、均匀性指数、优势集中性指数）及其时间动态基本无明显差异;Bt水稻田与对照田间植食类、寄生类、捕食类亚群落和整个节肢动物群落的相异性大多较低。可见,Bt水稻对稻田节肢动物群落基本无明显的负面影响。     

Transcriptional silencing and developmental reactivation of cry1Ab gene in transgenic rice

One transgenic rice line lacking CrylAb expression product was screened in the progenies of Agrobacterium-transformed transgenic rice variety Zhong 8215 with a cry1Ab gene under field releasing conditions by using GUS histochemical assay and Western blot. Molecular hybridization results revealed that the crylAb gene was silenced in the transgenic rice variety Zhong 8215 and two copies of ubiquitin promoter were integrated into the rice genome. The silencing of crylAb gene in transgenic rice was found to be due to the methylation of the ubiquitin promoter as revealed by methylation analysis. Meanwhile, different concentrations of demethylation reagent 5-azacytidine combining with different treatment time were employed to treat the silenced transgenic rice seeds. The results indicated that 5-azacytidine could reactivate 8%-30% of the silenced transgenic rice plants and the expression level of the reactivated cry1Ab transgene could reach as high as 0.147% of the total soluble protein. Treatment with low con     

Characterization of cry1, cry2, and cry9 genes in Bacillus thuringiensis isolates from China

Bacillus thuringiensis isolates from different ecological regions and sources of China were analyzed to study the distribution and diversity of cry genes and to detect the presence of novel cry genes. Strains containing cry1-type genes were the most abundant and represent 237 of the 310 B. thuringiensis isolates (76.5%). About 70 and 15.5% of the isolates contained a cry2 gene or cry9 gene, respectively, while 10.0% of the strains did not contain a cry1, cry2, or cry9 gene. Among the cry1 containing isolates, cry1A (67.7%), cry1I (60.6%), cry1C (43.9%), and cry1D (39.4%) genes were the most abundant. Forty-three different cry1 gene profiles were detected in this collection. Several cry1 genes were associated at a high frequency, such as the cry1C-cry1D and cry1A-cry1I gene combination. The cry1A and cry2 amplicons were digested with selected restriction enzymes to examine sequence diversity. Based on this RFLP analysis, one novel cry1A-type gene was observed.