苏云金杆菌辅助蛋白基因p19和orf1-orf2对杀虫晶体蛋白CytlAa表达的影响

为检测苏云金杆菌辅助蛋白P19和0RFl—0RF2对杀虫晶体蛋白CytlAa表达的影响,构建了5个重组表达质粒。5个质粒都含有cytlAa基因,但pT1只含有cytlAa基因,p他同时含有p19基因,pt3同时含有orf1-orf2串联基因,pT4同时含有p19基因和p20基因,pT5同时含有orf1-orf2串联基因和p20基因。将这5个表达质粒和质粒pWF45电转化到苏云金杆菌晶体缺陷型4Q7中,分别获得转化菌株Bt—T1、Bt—T2、Bt—T3、Bt—T4、Bt—T5和Bt—WF45。SDS—PAGE结果显示,菌株Bt—T1、Bt—T2和Bt—T3只产生少量的27kD cytlAa蛋白,而且部分降解为大约24kD的蛋白。而Bt—T4和Bt—T5能产生大量的cytlAa蛋白,但Bt—T4和Bt—T5的cytlAa蛋白产量都明显少于Bt—WF45。电镜观察和生物测定结果表明Bt—T4和Bt—T5与Bt—WF45的晶体大小和杀蚊毒力没有显性差异。研究表明p19和ORF1—ORF2对CytlAa。蛋白的合成显示可能有抑制作用。     

苏云金杆菌以色列亚种杀蚊蛋白Cyt1Aa在离中不粘柄菌中的表达

在蚊幼虫生活水域里的离中不粘柄菌(Asticcacaulis excentricus,Ae)中已成功表达苏云金芽孢杆菌以色列亚种(Bacillus thuringiensis subsp.israelensis,Bti)杀蚊蛋白基因cry11Aa的基础上,将另一Bti杀蚊蛋白基因cyt1Aa转化入Ae中表达。构建并转化了分别单独含有cyt1Aa基因、及同时含有cry11Aa基因的表达质粒pSODCyt20和pSODCryCyt20,蛋白免疫杂交检测相应的Ae重组子分别表达产生了Cyt1Aa和Cry11Aa蛋白。为了探究Ae(pSODCryCyt20)重组子不能表达cyt1Aa的原因,提取了重组子总RNA、并与同是革兰氏染色阴性的大肠杆菌的总RNA比较,结果显示两者RNA系统显著不同,推测Ae中多个外源基因的表达,可能要求每个基因必需一个启动子。     

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.     

苏云金芽孢杆菌以色列亚种cyt1 Aa基因在球形芽孢杆菌中表达

将编码cyt1Aa基因和 p2 0蛋白基因的DNA片段分别克隆连接于两个不同的穿梭载体 pBU 4和pMK 3上 ,构建了重组质粒 pBA 30和 pMA 6,通过电击法 ,将重组质粒分别转化 B .s野生株2 2 97,获得了转化菌株Bs 97 30和Bs 97 6。SDS PAGE和Westernblot分析证实了cyt1Aa基因在转化菌株Bs 97 30中获得了表达 ,而在转化菌株Bs 97 6中未检测到cyt1Aa基因表达的蛋白。转化菌株Bs 97 30中 ,cyt1Aa基因与B .s二元毒素基因同步于菌体生长的对数期起始表达 ,并持续至芽孢形成。生测结果表明 ,转化菌株Bs 97 30中cyt1Aa基因的表达并未明显增强其对敏感和抗性致倦库蚊幼虫的毒力。其原因可能是弱毒性的 cyt1Aa蛋白在转化菌株中的表达量不高。     

苏云金芽孢杆菌分子伴侣串联基因p19-p29的克隆和表达载体的构建

根据已知序列设计一对PCR引物(ORF5S,ORF3N),可从cry2Aa或cry2Ac操纵子中扩增出包含串联分子伴侣基因p19p29的DNA片段,预期大小分别为16kb和20kb。对150株苏云金芽孢杆菌菌株进行PCR检测,从26株中获得了大小为16kb的扩增片段,但未获得20kb的片段。这表明cry2Aa型操纵子p19p29基因存在较广泛,而cry2Ac型较罕见。将来自Y2菌株的16kb片段回收,通过一系列亚克隆,最终构建成一个含有p19p29串联基因的Bt表达载体,为进一步研究p19p29串联基因的功能奠定了基础。     

苏云金杆菌辅助蛋白P20对杀虫晶体蛋白Cry1Ab表达的影响

苏云金杆菌(Bacillus thuringiensis, 简称Bt)杀虫晶体蛋白Cry1Ab因其C半端缺少了一段含4个半胱氨酸的氨基酸序列而导致蛋白的不稳定,报道苏云金杆菌辅助蛋白P20帮助Cry1Ab蛋白的表达及晶体的形成。利用穿梭载体pHT3101构建3个表达质粒,即pT1B、pP1B和pDP1B,3个质粒都含有cry1Ab基因,不同在于pT1B没有p20基因,pP1B含有p20全基因,而pDP1B不仅含有p20全基因,且在p20基因前插入cry1A?启动子。分别将这3个表达质粒经电转化到苏云金杆菌晶体缺陷型菌株CryB中,获得转化菌株T1B、P1B和DP1B。Western blot表明cry1Ab基因在这3株菌中均表达了130 kD的蛋白,部分降解为大约60 kD的蛋白。蛋白定量分析显示,3株菌130 kD蛋白量的比为1∶1.4∶1.5,降解后的60 kD蛋白量的 比为1∶1.1∶1.6,Cry1Ab蛋白总量的比为1∶1∶2∶1.6。镜检发现,Cry1Ab在3株菌中都形成典型的菱形晶体,其晶体大小为T1B Helicoverpa armigera）均具有明显的杀虫活性,三者的LC₅₀差异不显著。研究表明,P20对cry1Ab基因的表达和晶体形成均有帮助,P20表达量的多少可能是导致Cry1Ab蛋白最终产量有所不同的因素。     

苏云金杆菌辅助蛋白P19对杀虫晶体蛋白Cry11Aa表达的影响

苏云金杆菌以色列亚种的p19基因、cry11Aa基因和p20基因位于同一操纵子上,据推测辅助蛋白P19可能与Cry11Aa蛋白的晶体化相关。本研究利用穿梭载体pHT3101构建了两个重组质粒pHcy1和pHcy3,两质粒均携带cry11Aa基因,但后者完全缺失了cry11Aa基因上游的p19基因。将重组质粒电激转化至苏云金杆菌无晶体突变株4Q7中进行蛋白表达,SDS-PAGE结果表明在4Q7(pHcy1)和4Q7(pHcy3)中均能检测到正常表达的Cry11Aa蛋白,但单位体积培养液的Cry11Aa蛋白在辅助蛋白P19存在时的表达量明显高于其单独表达的表达量;透射电镜观察显示两菌株中的Cry11Aa蛋白形成了大小相近、形状相似的双梯形晶体;另外,生物测定结果表明重组菌株4Q7(pHcy1)和4Q7(pHcy3)对三龄致倦库蚊的杀虫活性没有显著性差异。该现象说明辅助蛋白P19的缺失对Cry11Aa蛋白的晶体形成和杀蚊活性没有影响,但P19作为分子伴侣在一定程度上帮助提高了Cry11Aa蛋白的表达水平。     

苏云金杆菌辅助蛋白P20对杀虫晶体蛋白CrylAb表达的影响

苏云金杆菌 (Bacillusthuringiensis,简称Bt)杀虫晶体蛋白Cry1Ab因其C 半端缺少了一段含 4个半胱氨酸的氨基酸序列而导致蛋白的不稳定 ,报道苏云金杆菌辅助蛋白P2 0帮助Cry1Ab蛋白的表达及晶体的形成。利用穿梭载体pHT310 1构建 3个表达质粒 ,即pT1B、pP1B和pDP1B ,3个质粒都含有cry1Ab基因 ,不同在于pT1B没有p2 0基因 ,pP1B含有p2 0全基因 ,而pDP1B不仅含有p2 0全基因 ,且在p2 0基因前插入cry1A(c)启动子。分别将这 3个表达质粒经电转化到苏云金杆菌晶体缺陷型菌株CryB中 ,获得转化菌株T1B、P1B和DP1B。Westernblot表明cry1Ab基因在这 3株菌中均表达了 130kD的蛋白 ,部分降解为大约 6 0kD的蛋白。蛋白定量分析显示 ,3株菌 130kD蛋白量的比为 1∶1.4∶1 5 ,降解后的 6 0kD蛋白量的比为 1∶1.1∶1.6 ,Cry1Ab蛋白总量的比为 1∶1∶2∶1 6。镜检发现 ,Cry1Ab在 3株菌中都形成典型的菱形晶体 ,其晶体大小为T1B 相似文献   

对鳞翅目害虫高毒力的Bt cry1Aa基因的分离克隆及表达

Bt菌Ly30株是我国自行分离的对多种害虫具有高毒力的苏云金芽孢杆菌,经CAPS(cleaved amplified polymorphic sequences)系统鉴定,它含有cry1Aa基因。以全长基因PCR产物的粘端定向克隆的方法, 设计一对特异引物,分别引入NcoⅠ和BamHⅠ/NcoⅠ酶切位点。以Ly30质粒DNA为模板扩增cry1Aa全长基因,与表达载体Pkk233-2相应酶切产物连接,转化大肠杆菌,获得含有cry1Aa基因重组质粒pKKLy1Aa。完成了该基因的亚克隆和序列测定,结果表明,该基因的编码区为3 531 bp,编码蛋白分子量为133.2kD,含1.176个氨基酸,等电点Pi为4.99。该基因序列已在GenBank中登记注册,登录号为AF384211,并被国际Bt杀虫晶体蛋白基因命名委员会正式命名为cry1Aa12。对重组菌KKLy1Aa进行诱导表达研究。在0.6 mmol/L IPTG、37℃、8 h培养条件下,该基因获得高效表达,SDS-PAGE电泳检测到明显的133.2 kD蛋白带。室内生测结果表明,Cry1Aa蛋白对不同的小菜蛾品系均有较高的杀虫活性,其LC₅₀值分别为0.203 μg/mL和0.554 μg/mL。     

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。     

EFFECTS OF HELPER PROTEINS ENCODED BY p19 AND orf1-orf2 GENES ON Cyt1Aa PROTEIN EXPRESSION IN ACRYSTALLIFEROUS STRAIN OF BACILLUS THURINGIENSIS

A series of plasmids were constructed to examine the effects of p19 and orf1‐orf2 genes from Bacillus thuringiensis on Cyt1Aa synthesis and inclusion formation. The plasmids expressed the cyt1Aa gene along with either p19 or orf1‐orf2, or each of them coordinatively with p20 in the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. No effect on the expression of Cyt1Aa protein was found when P19 or Orf1‐Orf2 co‐expressed with Cyt1Aa. However, when including p20 gene, the constructs with p19 or orf1‐orf2 gene produced lower yield of Cyt1Aa proteins than without p19 or orf1‐orf2 gene. Electron microscopy observation and bioassay showed that P19 and Orf1‐Orf2 have no influence on the crystal size and toxicity of Cyt1Aa protein. It is presumed that P19 and Orf1‐Orf2 might have negative effects on Cyt1Aa synthesis in B. thuringiensis.     

Identification of a gene for Cyt1A-like hemolysin from Bacillus thuringiensis subsp. medellin and expression in a crystal-negative B. thuringiensis strain.

A gene designated cyt1Ab1, encoding a 27,490-Da protein, was isolated from Bacillus thuringiensis subsp. medellin (H30 serotype) by using an oligonucleotide probe corresponding to the cyt1Aa1 gene. The sequence of the Cyt1Ab1 protein, as deduced from the sequence of the cyt1Ab1 gene, was 86% identical to that of the Cyt1Aa1 protein and 32% identical to that of the Cyt2Aa1 protein from B. thuringiensis subsp. kyushuensis. The cyt1Ab1 gene was flanked upstream by a p21 gene, in the same orientation, encoding a 21,370-Da protein that showed 84% similarity to the putative chaperone P20 protein from B. thuringiensis subsp. israelensis and downstream, on the opposite strand, by a sequence showing 85% identity to the IS240A insertion sequence. The cyt1Ab1 gene was expressed at a high level in a nontoxic strain of B. thuringiensis subsp. israelensis in which large inclusions of the Cyt1Ab1 protein were produced. Purified Cyt1Ab1 crystals were as hemolytic as those of the Cyt1Aa1 protein and were twice as hemolytic as those from the wild-type strain. Mosquitocidal activity toward Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae was assayed. The toxicity of the Cyt1Ab1 protein was slightly lower than that of the Cyt1Aa1 protein for all three mosquito species, and Cyt1Ab1 was 150, 300, and 800 times less active toward Culex, Anopheles, and Aedes larvae, respectively, than were the native crystals from B. thuringiensis subsp. medellin.     

Cytolytic toxin Cyt1Aa of Bacillus thuringiensis synergizes the mosquitocidal toxin Mtx1 of Bacillus sphaericus

Using the shuttle vector pBU4, the mosquitocidal toxin gene mtx1 from Bacillus sphaericus strain SSII-1 was introduced into an acrystalliferous strain of B. thuringiensis both individually and in combination with the accessory protein gene p20 and the cytolytic protein gene cyt1Aa from B. thuringiensis subsp. israelensis. Bioassay results indicated that the recombinants B-pMT4(Mtx1) and B-pMT9(Mtx1), both individually containing mtx1, had moderate toxicities to binary toxin susceptible and binary toxin resistant Culex quinquefasciatus larvae during the vegetative growth stage, but that their toxicities declined rapidly during the sporulation phase. The LC50 values were 2.5 and 4.8 mg/ml respectively, against 3-4 instar susceptible and resistant larvae for the final sporulated cultures of recombinants B-pMT9(Mtx1), and little toxicity was detected for B-pMT4(Mtx1). Meanwhile, the recombinant B-pMPX2(Mtx1+Cyt1Aa) expressing Mtx1, P20 alone, and Cyt1Aa in combination had stable toxicities during both the vegetative phase and the sporulation phase, with a LC50 ranging from 0.45-0.58 mg/ml. Furthermore, expression of Cyt1Aa appeared to enhance the activity of Mtx1 to target mosquito larvae, suggesting a synergism between Cyt1Aa and Mtx1 toxins.     

Cross-resistance spectra of Culex quinquefasciatus resistant to mosquitocidal toxins of Bacillus thuringiensis towards recombinant Escherichia coli expressing genes from B. thuringiensis ssp. israelensis

Sixteen Escherichia coli clones were assayed against susceptible and Bacillus thuringiensis-resistant Culex quinquefasciatus larvae. The clones expressed different combinations of four genes from Bacillus thuringiensis ssp. israelensis; three genes encoded mosquitocidal toxins (Cry11Aa, Cry4Aa and Cyt1Aa) and the fourth encoded an accessory protein (P20). The cross-resistance spectra of the mosquitoes were similar to the profiles for recombinant B. thuringiensis strains expressing B. thuringiensis toxin genes, but with varied toxicity levels. The toxicity of the recombinants towards resistant mosquito larvae was improved when p20 and cyt1Aa were expressed in combination with cry4Aa and/or cry11Aa. Recombinant pVE4-ADRC, expressing cry4Aa, cry11Aa, p20 and cyt1Aa, was the most active against the resistant Culex, and resistance levels did not exceed fourfold. These results indicate that B. thuringiensis ssp. israelensis genes expressed in a heterologous host such as E. coli can be effective against susceptible and B. thuringiensis-resistant larvae and suppress resistance.     

Cyt1A of Bacillus thuringiensis delays evolution of resistance to Cry11A in the mosquito Culex quinquefasciatus

Insecticides based on Bacillus thuringiensis subsp. israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported. Moreover, in contrast to B. thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B. thuringiensis subsp. israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations. Selection of Culex quinquefasciatus with mutants of B. thuringiensis subsp. israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance. This hypothesis, however, has not been tested experimentally. Here we report experiments in which separate C. quinquefasciatus populations were selected for 20 generations to recombinant strains of B. thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population. The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains. When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold. This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent. These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins.     

A new class of LINEs (ATLN-L) from Arabidopsis thaliana with extraordinary structural features.

The Arabidopsis thaliana genome has about 250 copies of LINEs (here called ATLNs). Of these, some, called ATLN-Ls, have an extra sequence of about 2 kb in the region downstream of two consecutive open reading frames, orf1 and orf2. Interestingly, the extra sequences in these ATLN-L members have another open reading frame, designated as orf3. Each member is flanked by direct repeats of a target site sequence, showing that ATLN-L members with the three open reading frames have retrotransposed as a unit. The ATLN-L members are also distinct from other ATLN members: orf1 terminates with TAA (or TAG) and is located in the same frame as orf2, and the ATG initiation codon of orf2 is not present in the proximal region. A sequence that may form a pseudoknot structure in ATLN-L mRNA was present in the proximal region of orf2, therefore the TAA (or TAG) termination codon of orf1 is assumed to be suppressed to produce an Orf1-Orf2 transframe protein during the translation of the ATLN-L mRNA. The region between orf2 and orf3 is several hundred bp long, suggesting that orf3 expression is independent of orfl-orf2. The amino acid sequences of the proteins Orf1 and Orf3 are highly homologous in their N-terminal half regions that have a retroviral zinc-finger motif for RNA binding. Orf3, however, has a leucine-zipper motif in addition to the zinc-finger motif. The C-terminal regions of the Orf1 and Orf3 proteins have poor homology, but seem to have nuclear localization signals, suggesting that these proteins are involved in the transfer of ATLN-L mRNA to nuclei. A phylogenetic tree shows that Orf3 proteins form a branch distinct from the branches of the Orf1 proteins encoded by ATLN-L members. This indicates that an ancestor element of ATLN-Ls has incorporated the orf1 frame carried by another ATLN member into its distal region to orf1-orf2 during evolution.     

Binding of Cyt1Aa and Cry11Aa toxins of Bacillus thuringiensis serovar israelensis to brush border membrane vesicles of Tipula paludosa (Diptera: Nematocera) and subsequent pore formation

Bacillus thuringiensis serovar israelensis (B. thuringiensis subsp. israelensis) produces four insecticidal crystal proteins (ICPs) (Cry4A, Cry4B, Cry11A, and Cyt1A). Toxicity of recombinant B. thuringiensis subsp. israelensis strains expressing only one of the toxins was determined with first instars of Tipula paludosa (Diptera: Nematocera). Cyt1A was the most toxic protein, whereas Cry4A, Cry4B, and Cry11A were virtually nontoxic. Synergistic effects were recorded when Cry4A and/or Cry4B was combined with Cyt1A but not with Cry11A. The binding and pore formation are key steps in the mode of action of B. thuringiensis subsp. israelensis ICPs. Binding and pore-forming activity of Cry11Aa, which is the most toxic protein against mosquitoes, and Cyt1Aa to brush border membrane vesicles (BBMVs) of T. paludosa were analyzed. Solubilization of Cry11Aa resulted in two fragments, with apparent molecular masses of 32 and 36 kDa. No binding of the 36-kDa fragment to T. paludosa BBMVs was detected, whereas the 32-kDa fragment bound to T. paludosa BBMVs. Only a partial reduction of binding of this fragment was observed in competition experiments, indicating a low specificity of the binding. In contrast to results for mosquitoes, the Cyt1Aa protein bound specifically to the BBMVs of T. paludosa, suggesting an insecticidal mechanism based on a receptor-mediated action, as described for Cry proteins. Cry11Aa and Cyt1Aa toxins were both able to produce pores in T. paludosa BBMVs. Protease treatment with trypsin and proteinase K, previously reported to activate Cry11Aa and Cyt1Aa toxins, respectively, had the opposite effect. A higher efficiency in pore formation was observed when Cyt1A was proteinase K treated, while the activity of trypsin-treated Cry11Aa was reduced. Results on binding and pore formation are consistent with results on ICP toxicity and synergistic effect with Cyt1Aa in T. paludosa.