苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26Aa和cry28Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26Aa和cry28Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26Aa在仅消除cry26Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26Aa所在质粒有关。     

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。     

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。     

苏云金芽胞杆菌G03 spoIVF操纵子敲除对芽胞和晶体形成的影响

spoIVF是一个普遍存在于芽胞杆菌中的操纵子。在枯草芽胞杆菌中,它编码的两个蛋白是芽胞形成所必需的。采用基因重组技术敲除了苏云金芽胞杆菌G03菌株中的spoIVF操纵子,构建了spoIVF缺失株G03(spoIVF-)。研究表明:该突变株丧失了形成芽胞和晶体的能力。lacZ基因与cry1Aa基因的启动子融合表达分析发现:突变株中的cry1Aa基因的活性严重降低。利用载体pSTK携带spoIVF操纵子在突变株中的表达,使突变株部分恢复了产胞和形成杀虫晶体蛋白的能力。这说明spoIVF操纵子是所必需的,同时该操纵子还影响σ^E因子控制的cry1Aa基因表达。     

转化crylC基因对苏云金芽胞杆菌杀虫活性的影响

将含基因crylC的质粒 ,通过电脉冲法转入含基因CrylC的质粒 ,通过电脉冲法转入含基因CrylAb、CrylAc和cry2的对小菜蛾具有高毒力的苏云金芽胞杆菌野生菌株YBT-803-1中 ,得到转化子BMBY-003。PCR和SDS PAGE分析显示 ,CrylC可在其中正常复制、表达 ,但使受体菌部分内源质粒发生丢失。生物测定结果表明 ,转化子BMBY-003既对甜菜夜蛾有毒力 ,LC₅₀ 值     

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-cyl-homoserine lactones,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pHT304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cryⅡ基因的克隆和表达

分离的苏云金芽胞杆菌(Bacillus-thuringiensis)YBT-791对鳞翅目小菜蛾(Plutellaxylostella)有毒力,将其质粒DNA提纯,经HindⅢ酶切后与杀虫晶体蛋白cryⅡ基因探针杂交,显示出分子量分别为5kb和9．4kb两条DNA阳性片段。把5kb的DNA阳性片段克隆到pUCl8的HindⅢ位点上并转化大肠杆菌TGl,经酶切和杂交检测,证明斑点杂交阳性克隆子中含有5kb的cryI片段。把这个含cryⅡ基因的5kbHindⅢ片段进行亚克隆,将4kb的BamHI－Pstl酶切片段插入穿梭载体pXl61中,并用电脉冲法克隆于苏云金杆菌不产伴胞晶体的突变株中,得到产生单一cry Ⅱ基因编码的杀虫晶体蛋白的克隆菌株M一5。经电镜观察,该克隆菌株能形成出发菌YBT－791多种形态伴胞晶体中的一种方形伴胞晶体;经免疫双扩试验,它只能与Cry Ⅱ晶体蛋白抗血清形成沉淀线;经SDS—PAGE电泳,克隆菌的伴胞晶体只含有一种65kDa的晶体蛋白。生物测定结果表明它既对鳞翅目小菜蛾(Piutella xylostella)有毒性,又对双翅目致倦库蚊(Culex quinquefasciatus)有毒性。     

苏云金芽胞杆菌的鉴定及对椰子织蛾的致死作用

【背景】利用采集到的样品分离出苏云金芽胞杆菌菌株,针对椰子织蛾幼虫进行室内生物活性测定,以期获得对椰子织蛾幼虫具有高毒力菌株,并为椰子织蛾的生物防控提供理论和技术依据,同时为转Bt基因作物提供新的基因资源。【方法】在海南岛椰子织蛾潜在分布区采集样品,利用温度法筛选苏云金芽胞杆菌并进行形态学和分子生物学鉴定,采用浸叶法进行Bt菌株对椰子织蛾幼虫的生物活性测定。【结果】海南省保亭县土壤样品中筛选出8株菌株,电子显微镜下观察到该批菌株含有菱形、球形、方形晶体;通过分子生物学鉴定明确了6株菌株含有cry基因,2株没有鉴定出cry基因;利用相同浓度菌悬液对椰子织蛾幼虫进行了生物活性测定,结果表明,50μg·m L-1的BAT10菌株杀虫晶体蛋白对椰子织蛾幼虫的致死率达100%,同样浓度的29-15-4与BAT20菌株杀虫晶体蛋白对椰子织蛾幼虫的致死率超过80%,其余5株Bt的杀虫晶体蛋白致死率较低,致死率小于50%。【结论与意义】本试验筛选出1株高毒力菌株和2株效果较好的Bt菌株,可应用于椰子织蛾的生物防控。     

类似S-层蛋白的苏云金芽胞杆菌伴胞晶体蛋白基因的克隆

芽胞杆菌CTC菌株被鉴定为苏云金芽胞杆菌 ,鞭毛血清型H2 ,幕虫亚种 ;产生卵圆形伴胞晶体 ,伴胞晶体蛋白为 1 0 0kD ;测定了该蛋白的N 末端序列 ,该序列与炭疽芽胞杆菌的细胞表面S 层蛋白具 92 %～ 93%相似性 ;根据Southern杂交制作了该晶体蛋白基因ctc所在位置的限制性酶切图谱 ,分别克隆了该基因 5′和 3′端所在的 2 9kbXbaI片段和 3 1kbClaIDNA片段 ,彼此间具 0 6kb重叠 ,通过拼接获得含完整ctc基因的克隆。含该基因的大肠杆菌与表达S 层蛋白的大肠杆菌具相似生长特征。初步表明CTC菌株的伴胞晶体由细胞表面S 层蛋白组成。苏云金芽胞杆菌区别于蜡状芽胞杆菌和炭疽芽胞杆菌的唯一标准是能形成伴胞晶体 ,由于S 层是细胞表面的结构成分 ,本文对CTC菌株鉴定为苏云金芽胞杆菌以及伴胞晶体作为苏云金芽胞杆菌鉴别的唯一标准提出了质疑     

苏云金芽胞杆菌CTC菌株的S-层蛋白可以形成伴胞晶体

苏云金芽胞杆菌(Bacillus thuringiensis)CTC菌株产生卵圆形伴胞晶体,晶体蛋白分子量为100kD;透射电子显微镜观察结果表明该菌株有S层结构,而且在母细胞内可以形成伴胞晶体和S层的初体结构;其蛋白基因导入苏云金芽胞杆菌无晶体突变株BMB171后,扫描电子显微镜观察结果表明转化子能形成晶体,而其形状与CTC菌株的相同;转化子晶体蛋白的分子量大小也与CTC菌株的相同,为100kD。以上实验结果结合以前晶体蛋白N末端测序和基因核苷酸序列,表明苏云金芽胞杆菌CTC菌株的S层蛋白可以形成伴胞晶体。     

A novel negative regulatory factor for nematicidal Cry protein gene expression in Bacillus thuringiensis

A 3-kb HindIII fragment bearing the cry6Aa2 gene and the adjacent and intergenic regions was cloned from Bacillus thuringiensis strain YBT-1518. Two open reading frames (ORFs), namely, orf1 (termed cry6Aa2) and orf2 that were separated by an inverted-repeat sequence were identified. orf1 encoded a 54-kDa protein that exhibited high toxicity to the plant-parasitic nematode Meloidogyne hapla. The orf2 expression product was not detected by SDS-PAGE, but its mRNA was detected by RT-PCR. The orf2 coexpressed with orf1 at a high level in the absence of the inverted-repeat sequence, whereas, the expression level of orf1 was decreased. When orf2 was mutated, the level of orf1 expression was enhanced obviously. In conclusion, the inverted-repeat sequence disturbs orf2 expression, and the orf2 downregulates orf1 expression. This is an example of novel negative regulation in B. thuringiensis and a potential method for enhancing the expression level of cry genes.     

New Strategy for Isolating Novel Nematicidal Crystal Protein Genes from Bacillus thuringiensis Strain YBT-1518

We have developed a strategy for isolating cry genes from Bacillus thuringiensis. The key steps are the construction of a DNA library in an acrystalliferous B. thuringiensis host strain and screening for the formation of crystal through optical microscopy observation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. By this method, three cry genes—cry55Aa1, cry6Aa2, and cry5Ba2—were cloned from rice-shaped crystals, producing B. thuringiensis YBT-1518, which consists of 54- and 45-kDa crystal proteins. cry55Aa1 encoded a 45-kDa protein, cry6Aa2 encoded a 54-kDa protein, and cry5Ba2 remained cryptic in strain YBT-1518, as shown by SDS-PAGE or microscopic observation. Proteins encoded by these three genes are all toxic to the root knot nematode Meloidogyne hapla. The two genes cry55Aa1 and cry6Aa2 were found to be located on a plasmid with a rather small size of 17.7 kb, designated pBMB0228.     

苏云金芽孢杆菌的cry2A芽孢期启动子和分子伴侣ORF1-ORF2对Cry11Aa蛋白表达的影响

[目的]分析苏云金芽孢杆菌的cry2A型芽孢期启动子对晶体蛋白Cry11Aa的协调作用和分子伴侣ORF1-ORF2对Cry11Aa表达的促进功能.[方法]3个包括cry11Aa编码区的重组质粒pHcy1、pHcy2和pHcy4被构建并电激转化到苏云金芽孢杆菌晶体缺陷株4Q7中,其中pHcy1质粒携带cry11Aa基因自身启动子和分子伴侣p19基因,pHcy2携带cry2A型芽孢期启动子和分子伴侣orf1-orf2基因,pHcy4质粒在pHcy1的上游插入了cry2A型芽孢期启动子和分子伴侣orf1-orf2基因.SDS-PAGE分析了Cry11Aa蛋白在各重组苏云金菌株中的表达情况,并通过生物测定确定了其对蚊虫的生物活性.[结果]SDS-PAGE结果表明,Cry11Aa蛋白在4Q7(pHcy1)和4QT(pHcy4)均获得了表达,在4Q7(pHcy2)中未检测到Cry11Aa蛋白,推测晶体蛋白Cry11A不能利用cry2A型启动子进行表达调控;Cry11Aa蛋白在等体积4Q7(pHcy4)培养液中的表达量是4Q7(pHcy1)菌株的1.25倍,暗示着分子伴侣ORF1-ORF2在某种程度上能提高Cry11Aa的蛋白表达量.4Q7(pHcy1)和4Q7(pHcy4)形成的Cry11Aa蛋白晶体的形状和大小相似,两者对致倦库蚊的生物活性没有明显差异,LC50s分别为59.33 ng/mL和66.21 ng/mL,.[结论]推测晶体蛋白Cry11A能否成功表达与其使用启动子的类型和两者的协调配合有关.分子伴侣ORF1-ORF2虽然在某种程度上能提高Cry11Aa的蛋白表达量,但对提高Cry11Aa蛋白的杀蚊毒力没有显著性帮助.     

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.     

苏云金芽孢杆菌cry2Aa操纵元蛋白对杀虫蛋白晶体形成作用的研究

利用重叠PCR的方法,通过两次PCR扩增,分别获得cry2A10操纵元的orf1、orf1 orf2与cry2Ab5基因的融合片段。融合片段经BamHⅠ和EcoRⅠ双酶切与pHT315连接,分别构建了基因融合片段的原核表达载体pFU(orf1 2Ab)和pFU(orf1 orf2 2Ab),电转化Bt无晶体突变株4Q7后,扫描电镜下可观察到典型的方形晶体,通过SDS-PAGE可检测到60kD大小的蛋白表达带。结果表明,cry2Ab5可在cry2a0的启动子帮助下有效转录和表达,并在orf2产物帮助下形成蛋白晶体。     

An engineered Bacillus thuringiensis strain with insecticidal activity against Scarabaeidae (Anomala corpulenta) and Chrysomelidae (Leptinotarsa decemlineata and Colaphellus bowringi)

A genetically-engineered Bacillus thuringiensis (Bt) strain, 3A-HBF, with a broad insecticidal spectrum was constructed by introducing the recombinant plasmid pSTK-3A containing cry3Aa7 into the wild-type Bt strain HBF-1 containing the cry8Ca2 gene. The Cry3Aa7 protein produced by strain 3A-HBF was verified by SDS-PAGE and Western blotting. Flat rectangular crystals of Cry3Aa7 protein were observed besides spherical crystals (Cry8Ca2). The plasmid pSTK-3A was stable when strain 3A-HBF was grown in medium without antibiotics. The growth rate of 3A-HBF was not significantly different from that of the recipient strain, HBF-1. Strain 3A-HBF showed toxicity against two families of pests, Scarabaeidae and Chrysomelidae pests, which are susceptible to Cry8Ca (Anomala corpulenta) and Cry3Aa (Leptinotarsa decemlineata and Colaphellus bowringi). The 50% lethal concentrations of 3A-HBF against A. corpulenta, L. decemlineata and C. bowringi were 0.730 × 10⁸ c.f.u./g dry soil, 1.74 μg/ml and 1.15 μg/ml, respectively.     

Strategy for amplification and sequencing of insecticidal <Emphasis Type="Italic">cry1A</Emphasis> genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A feasible and fully described strategy, with a detailed list of primers, for amplifying, cloning and sequencing known and potentially novel cry1A genes harboured by a Bacillus thuringiensis strain was successfully established. Based on the analysis of conserved regions of the cry1A genes, the 1AF and 1UR oligonucleotide primers were designed to amplify the whole open reading frame of these genes. The PCR products obtained revealed the successful amplification of cry1A genes from 13 B. thuringiensis strains. These bacteria were previously known to harbour at least one cry1A gene. An Argentinean B. thuringiensis isolate INTA Mo1-12 was randomly chosen for cloning and sequencing of cry1A genes by using a primer set developed in this study. Both nucleotide and amino acid sequences similarity analysis revealed that cry1Aa and cry1Ac from B. thuringiensis INTA Mo1-12 are new natural variants, showing several differences with the other known cry1A subclasses. These genes were named by the B. thuringiensis Pesticidal Crystal Protein Nomenclature Committee as cry1Aa15 and cry1Ac21 respectively.     

二化螟钙黏蛋白CAD1的原核表达及与Cry1Ac、Cry2Aa蛋白的结合

【目的】二化螟是水稻的重要害虫之一,钙黏蛋白(cadherin,CAD)是一类重要的Bt杀虫蛋白受体,在获得二化螟钙黏蛋白基因(Cs CAD1)的基础上,明确Cs CAD1蛋白与Cry1Ac和Cry2Aa蛋白的结合能力。【方法】利用PCR技术克隆Cs CAD1基因片段,将构建的p ET-28a-(+)-Cs CAD1重组质粒转入原核表达菌株BL21(DE3)中,IPTG诱导表达。目的蛋白经Ni柱亲和纯化后SDS-PAGE电泳检测,利用western blot和ligand blot技术分析其与Cry1Ac和Cry2Aa蛋白的结合能力。【结果】重组载体可在表达菌株BL21中表达一个约44 ku的蛋白,原核表达载体构建成功。SDS-PAGE显示该蛋白条带单一,且纯度较好。Ni柱亲和层析纯化该目的蛋白后进行Ligand blot分析,结果显示Cs CAD1重组蛋白可以与Cry1Ac和Cry2Aa蛋白结合。【结论】Cs CAD1蛋白可以与Cry1Ac和Cry2Aa蛋白结合,是潜在的Cry蛋白受体,所得结果有助于阐明Cry1Ac和Cry2Aa蛋白对二化螟的作用机制。     

Novel cry gene from Paenibacillus lentimorbus strain Semadara inhibits ingestion and promotes insecticidal activity in Anomala cuprea larvae

A positive clone was selected from a library of total cell DNA of Paenibacillus lentimorbus strain Semadara that reacted with an antiserum that was raised against parasporal crystal proteins produced by this strain. The positive clone had a DNA insert containing two whole cry genes (cry43Aa1, cry43Ba1), one partial cry gene (cry43-like), and three smaller genes located upstream. Eight blocks that are conserved in the Cry proteins of Bacillus thuringiensis [Microbiol. Mol. Biol. Rev. 62 (1998) 775] were detected in their deduced amino acid sequences. The Escherichia coli transformant expressing cry43Aa1 caused inhibition of ingestion and 90% mortality in the first stadium larvae of Anomala cuprea. A low concentration of sporangia mixed with the transformant expressing cry43Aa1 easily infected the larvae of A. cuprea. The protein of approximately 150 kDa produced by the transformants expressing the cry genes reacted with antiserum specific for the parasporal crystal proteins. Southern hybridization analysis demonstrated that the cry genes were located on the chromosomal DNA of this strain, which possessed at least four cry genes.