Introduction of Bacillus thuringiensis(Bt)gene does not reduce potassium use efficiency of Bt transgenic cotton(Gossypium hirsutum L.)

Background:Potassium(K)deficiency has become a common field production problem following the widespread adoption of Bacillus thuringiensis(Bt)transgenic cotton(Gossypium hirsutum L.)worldwide.The purpose of this study was to clarify whether the introduction of Bt gene directly reduces the K-use efficiency of cotton to induce K deficiency.Results:The cotton variety,Jihe 321(wild type,WT)and its two Bt(Cry1Ac)-transgenic overexpression lines(OE-29317,OE-29312)were studied in field with low soil-test K+(47.8 mg·kg^(−1)).In the field with low soil-test K+,only OE-29317 had less biomass and K+accumulation than the WT at some growth stages.Both Bt lines produced similar or even greater seed cotton yield than WT in the field.When the Bt gene(~70%)in OE-29317 and OE-29312 plants was silenced by virus-induced gene silencing(VIGS),the VIGS-Bt plants did not produce more biomass than VIGSgreen fluorescent protein(control)plants.Conclusions:The introduction of Bt gene did not necessarily hinder the K use efficiency of the cotton lines under this study.

Identification of similar transcriptional regulatory mechanisms in multiple cry genes in Bacillus thuringiensis HD12

Bacillus thuringiensis subspecies morrisoni strain HD12, whose genome harbors multiple insecticidal protein-encoding genes, includes eight cry genes, as indicated by genome sequencing. This strain produces crystals that are toxic to lepidopteran species. These crystal inclusions were purified by sucrose gradients and sodium dodecyl sulphate-polyacrylamide gel electrophoresis （SDS-PAGE）, followed by liquid chromatography-mass spectrometry, and found to contain five proteins （Cry1Da, Cry1Ae, Cry1Bb, Cry1Fb, and CrylJa）. The transcriptional activities of the cry1Da, cry1Ae, cry1Bb, cry1Fb, and cry11Ja promoters indicated that transcription of crylDa is controlled by SigE; however, the other four cry genes were found to be controlled by both SigE and SigK. The activities of the crylJa and crylFb promoters were the strongest among the five genes studied. These promoters were therefore used to direct the expression of the Cry1Ac- and Cry2Ab-encoding genes concurrently in a single strain. Our findings indicate that promoters with the same transcriptional profile may be used to direct the expression of different cry genes in one strain. Our results are expected to be valuable for the construction of strains with efficient expression of multiple cry genes in order to overcome current limitations associated with the application of B. thuringiensis-based insecticides.

Geographical Distribution of Bacillus thuringiensis Strains and vip3 Genes in Different Climatic Zones in China

Vegetative Insecticidal Proteins(VIPs), a large family of insecticidal proteins, are produced from Bacillus thuringiensis(Bt) during the vegetative growth stage. VIPs represent the second generation of bio-insecticides that confer a wider insecticidal spectrum and have stronger activity. This work compared the geographical distribution of Bt strains and their vip3 genes in different climatic zones in China, the tropical(Hainan Province), subtropical(Guangxi Province) and temperate zones(Heilongjiang Province). A total of 156 Bt strains were isolated from 841 soil samples in Hainan Province tropical region, 356 Bt strains from 1 420 soil samples in Guangxi Province and 167 Bt strains from 1 010 soil samples in different geographical regions in Heilongjiang Province. Twenty-two out of 156 strains from tropical Hainan Province and two out of 356 from subtropical Guangxi Province were found to express vip3 genes, while vip3 genes were not expressed from temperate zone in Heilongjiang Province. Restriction Fragment Length Polymorphism(RFLP) was used to identify different types of vip3 genes that were within the same family and three fulllength vip3 genes were isolated. The genes cloned from Bacillus thuringiensis strain SL3 expressed in the transformed E. coli BL21 strain. Through SDS-PAGE, 88.6 ku insecticidal protein was expressed. The bioassays used two-instar larva of Lepidoptera insects(Spodoptera exigua and Agrotis ipsilon) were performed. The results of the bioassays showed that the protein strongly inhibited the body weight increasement on Spodoptera exigua and Agrotis ipsilon in a standard bioassay. Taken together, the results indicated that the distribution of Bt strains and vip3 genes had regional preference. Tropical and subtropical regions were the rich resources of Bt strains and vip3 genes compared with temperate region. These results would undoubtedly facilitate the studies of insecticidal proteins and expand the list of the pest-killing candidates to make fully use of the extremely rich microbial resources. The new vip3 genes isolated in the current study might also help resolve the emerging insecticidal resistance problems.

Biodiversity of <i>Bacillus thuringiensis</i>Strains and Their <i>Cry</i>Genes in Ecosystems of Kyrgyzstan

The present study aims to isolate the unknown and known serotypes of Bacilllus thuringiensis (Bt) from natural objects in Kyrgyzstan. A total of 83 Bt strains were isolated from natural substrates, of which 30% were taken from the soil and litter samples, 69.7% from dead insects and about 0.3% from slugs. Serological examination revealed that such subspecies as var. thuringiensis (H-1), var. alesti (H-3), var. sotto (H-4a4b) and var. entomocidus (H-6) predominated in the upper horizon of soils in all climatic zones. In the dead insects such species as subsp. thuringiensis, subsp. galleria, subsp. sotto, subsp. kurstaki, subsp. Aizawai and subsp. Entomocidus dominated. A set of Bt strains isolated from insects and soil samples, selected from different ecosystems in Kyrgyzstan was molecular taxonomically characterized using the pycA gene as marker for phylogenetic reconstruction. Within the Bacillus cereus sensu lato species complex, all Kyrgyz isolates were shown to belong to the B. cereus subspecies thuringiensis. Most isolates were assigned to the lineage Bt tolworthi, with two isolates each belonging to the lineages Bt kurstaki and Bt sotto. A high degree of cry gene diversity was demonstrated in the set of Bt isolates, with several gene copies simultaneously present in a single strain;a particularly conspicuous trait was the frequent combination of Lepidopteran-specific cryI with Dipteran-specific cryIV genes in the same Bt isolate.

Bacillus thuringiensis营养期杀虫蛋白Vip3与其转基因植物研究进展

苏云金芽孢杆菌(Bacillus thuringiensis,Bt)是目前应用最多的生物杀虫剂。它能够产生多种杀虫因子,其中,最主要的是杀虫晶体蛋白(Insecticidal Crystal Proteins,ICPs)和营养期杀虫蛋白(Vegetative insectici-dal protein,Vip)。当前,大部分商业化利用的转基因作物均为杀虫晶体蛋白类,随着这些转基因作物种植面积的扩大,害虫对这些较为单一的杀虫蛋白产生抗性已成为一个严峻的问题。Vip3是Vip杀虫蛋白中的一类,不形成蛋白晶体,和ICPs在进化上没有同源性;其对鳞翅目、鞘翅目和同翅目等害虫具有毒杀作用,抗虫谱较广。目前,已经把Vip3基因导入了水稻、玉米和棉花等多种作物中,为作物抗虫育种、延缓害虫产生抗性和减少作物产量损失等带来新的前景。

Identification and Distribution of Bacillus thuringiensis Isolates from Primeval Forests in Yunnan and Hainan Provinces and Northeast Region of China

Ninety-two Bacillus thuringiensis isolates were screened from 683 soil samples collected from tropical and semitropical primeval forests in Yunnan and Hainan provinces of China. Several shapes of crystals, including bipyramidal, square, ovoid, spherical, and amorphous, were observed in the B. thuringiensis isolates. Twenty-six pairs of primers were used to identify 31 holotype cry genes at primary rank of the B. thuringiensis cry gene nomenclature system. The cry gene-types of 92 B. thuringiensis isolates and 33 B. thuringiensis isolates screened from Northeast region of China were identified by PCR-RFLP and SDS-PAGE methods. Fifty-eight isolates harbored cry1 genes, 32 isolates cry2 genes, 12 isolates cry8 genes, 3 isolates cry9 genes, 12 isolates cry11 genes, and 13 isolates cry30 genes. Of the tested isolates, 42 produced no reaction product with 26 pairs of primers and also exhibited no toxicity against 8 insect species tested. The isolate Z2-34 harbored a novel cry30 gene, exhibited insecticidal activity against Aedes albopictus of Dipterans. The accession number of the novel genes in this study is AY916046. Isolation and identification of B. thuringiensis and cry gene are important for investigating the diversity of B. thuringiensis resources and cloning new cry gene.

Polyhedrosis Virus in Bacillus thuringiensis

This study was conducted to build a recombinant strain with highly insecticidal activity and a wide host range by using the crylAc and p74 gene. Firstly, the p74 gene was amplified from the genosome ofAutographa californica multicapsid nucleopolyhedrovirus. The crylAc gene and the terminator gene of crylAc, named crylAct, were amplified from the plasmid of Bt 4.0718 strain. Three T vectors, named pTp74, pT1Ac, and pT1Act which held the aimed gene p74, cry1Ac, and crylAct, respectively, and two middle vectors, named pTp74Act and pTIAcp74 which held the aimed fusion gene p74-crylAct and cry lAc-p74, respectively, were built by using pMDI 8-T. Then pTiAcp74 and the shuttle plasmid were digested and linked and an expressing-vector pH1Acp74 was built. Finally, pH1Acp74 was transformed into the acrystalliferous strain XBU001 and the aimed recombinant strain XBU-H1Acp74 was obtained. The expression of Bt transformant XBU-H1Acp74 was analyzed by SDS-PAGE which showed XBU-H1Acp74 could produce 130 kDa CrylAc protein and 50 kDa P74 protein. The insecticidal activity of transformant against Spodoptera exigua was evaluated compared with the contrast strains HTX-42 （only crylAc gene was transformed into XBU001） after autolysis. The LCs0 of HTX-42 was higher than that of the XBU-H IAcp74＇s, which implied that P74 could increase the efficacy and range of Bt Cry toxins in insect control. The fusion gene of crylAc and p74 were constructed successfully which will be served as the foundation lbr constructing the fusion genes of Bt cry gene and other foreign genes.

A Novel Bacillus thuringiensis Cry57 Protein Domain Swap Influence on Insecticidal Activity

Bacillus thuringiensis （Bt） is widely used in insecticides. Bt is a gram positive sporulation bacterium belonging to Bacillaceae family. It produces different insecticidal proteins like Cry toxin, Vip toxin, β-exotoxin and chitinase, etc. Bt insecticidal crystal proteins （ICPs） are not homologous to other known Vip protein and then act against lepidopteran, dipteran, coleopteran and nematodeslarvae via a unique process. In this experiment, modern high-throughput sequencing technique and sequencing were used and the whole genome sequence of BtLTS290 was obtained. The results compared to the database of GenBank showed that there was a cry57 gene in the genome sequence of BtLTS290. A novel cry57 gene was cloned and named cry57Ab1 （accession number is KF638650） by International Nomenclature Committee of Bt Endotoxin. cry57Ab1 gene could be expressed with the molecular weight of 90 ku. Cry57Ab1 protein had no obvious activity against Spodoptera exigua and Helicoverpa armigera. And Cry57Ab1 protein had a slight insecticidal activity against Ostrinia furnacalis and Plutella xylostella. Furthermore, the domain Ⅱof Cry57Ab1 and Cry1Bb were exchanged by overlapping extension PCR. SDS-PAGE showed that the molecular weight of Cry57Ab/1Bb/57Ab was about 90 ku. The insecticidal activity of Cry57Ab1 protein and Cry57Ab/1Bb/57Ab recombinant protein were determined. The results showed that the insecticidal activity of the recombinant protein to Spodoptera exigua and Helicoverpa armigera was very low, and the corrected mortality was less than 10%. The insecticidal activities against Ostrinia furnacalis and Plutella xylostella were reduced. The corrected mortality of Ostrinia furnacalis was 4.4%, and the corrected mortality rate for Plutella xylostella was 6.7%. Domain Ⅱof cry toxin played a key role on affecting host specifcity.

Evaluation of Cuban Bacillus thuringiensis(Berliner,1911)(Bacillales:Bacillacea)isolates with larvicidal activity against Aedes aegypti(Linnaeus,1762)(Diptera:Culicidae)

Objective:To evaluate 11 Cuban native Bacillus(B.)thuringiensis isolates in order to select one with the best larvicidal activity against Aedes(Ae.)aegypti and low cytotoxicity.Methods:The cry and cyt genes of the isolates(A21,A51,L95,L910,M29,R84,R85,R87,R89,U81 and X48)were amplified by PCR.The influence of organic matter and NaCl on the larvicidal activity was tested by bioassays.Cytotoxicity was assayed on peritoneal macrophages of BALB/c mice.Results:The cyt1(Aa,Ab,Ba),cyt2,cry4aA,cry4Ba,cry11(Aa,Ba,Bb)and cry10 genes were identified in all native Cuban isolates.The larvicidal activity(LC_(90))of seven isolates was affected by the presence of organic matter in the water,while A21,A51,L910,R84,U81 and X48 had better LC_(50),LC_(90),LC_(95) than the 266/29-Ⅶ-98 control strain.The LC_(50) of two isolates was affected by the presence of NaCl and A21,A51,R85 isolate had better larvicidal activity than the 266/29-Ⅶ-98 control strain.In terms of toxicity against macrophages,the extracts of nine isolates were less cytotoxic than the control strains.Conclusions:Native isolate A21 had the main virulence factors against Ae.aegypti larvae,displayed a good larvicidal activity in presence of different factors related with Ae.aegypti breeding sites,and had low citotoxicity against macrophages.These results can contribute to the improvement of existing biological control strategies and the development of new biolarvicides.

产黑色素B.thuringiensis重组菌株的构建及其培养条件的优化

将嗜麦芽假单胞菌中产生黑色素的基因 (melgene)克隆到穿梭载体 pHT3 10 1中 ,并将它处于表达系统cry3A的控制下 ,构建得到重组质粒 pHTAM .将此重组质粒用电脉冲的方法转入苏云金芽胞杆菌受体菌株BMB171中 ,得到重组菌株RSA .研究结果表明 ,处于cry3A控制下mel基因在BMB171菌株中得到了成功的表达 .本研究进一步采用红外光谱扫描法检测RSA所产黑色素的性质 。

B.thuringiensis穿梭载体的构建及cry1C基因的表达

以pUC19为母体，克隆了Btken－Ag（B.thuringiensissubsp.kenyaeAg）的复制起始区（～1．6kb）和pUC4K的aphⅠ基因，构建成穿梭载体pHV－1。pHV－1在E.coli中经100个世代，质粒保持率在80％以上；5在Bti4Q8（B．thuringiensissubsp.israelensis4Q8）中经40个世代，质粒保持率在80％以上。将B．licheniformis热稳定α－淀粉酶基因启动子控制下的Bt9510（B．thuringiensis9510）的crylC结构基因插入pHV－1，构建成重组质粒pHV－crylC，电激转化导入Bit4Q8。光学显微镜下观察，Bit4Q8无晶体产生；而Bit4Q8（pHV－crylC）则出现典型的菱形晶体，其晶体略小于Bt9510。生物测定结果表明表达的Cry1C蛋白对甜菜夜蛾具有一定的毒杀活性。

Expression of Bacillus Thuringiensis δ-Endotoxin Gene With Recombinant Baculovirus in Insect Cell

Insect baculovirus(IBV) is a biological insecticide in agriculture and forestry, butits slow speed of killing pests and narrow range of host make its application quitelimited. Hence, polyhedrin (ocu) gene of IBV was chosen for construction of

EXPRESSION OF THE Bacillus thuringiensis SUBSP, KURSTAKI HD-1 δ-ENDOTOXIN GENE IN Escherichia coli

In 1987, Vaeck and Fischhoff reported the transformation of the B. thuringiensis (abbreviated to B. t. ) δ-endotoxin gene into tobacco and tomato respectively, and obtained the transgenic plants. Since then, more attention has been paid to plant resistance to insect attack by plant genetic engineering.

MOLECULAR CLONING AND EXPRESSION OF Bacillus thuringiensis subsp. galleriae INSECTICIDAL CRYSTAL PROTEIN GENES IN Escherichia coli

The location of the toxin gene of B. thuringiensis subsp. galleriae (H5ab) on the Mr-130Mdplasmid is determined by molecular cloning. Double digestion fragments (BamHⅠ and SalⅠ)and PstⅠ restriction fragments as well, from the 130 Md plasmid of B. thuringiensis subsp.galleriae, are ligated with the cloning vector pAT 153 respectively and transformed into E.coli strain HB 101. Out of 208 transformants, three colonies (FG2, FG9, FG19) give posi-tive hybridization reaction using the HD-1 delta-endotoxin gene as a probe. They are presum-ed to contain the delta-endotoxin gene of B. thuringiensis subsp. galleriae. Western bolt assaysindicate that Mr-130 kDal and 68 kDal, crystal proteins produced by clone FG 2 react withanticrystal protein antibody. The protein extracts of clone FG2 are lethal to Ostrinia furna-calis (Guenee). This is the first report with regard to the cloning and expression of the B. thuringiensissubsp. galleriae (H5ab) delta-endotoxin gene.

Acquisition of Insect-Resistant Transgenic Maize Harboring a Truncated cry1Ah Gene via Agrobacterium-Mediated Transformation

A novel insecticidal gene crylAh was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active CrylAh toxin has a toxicity level similar to that of the full-length CrylAh toxin. In this study, plant expression vector pMhGM harboring truncated crylAh gene was transformed into maize （Zea mays L.） immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions （madMARs） were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efficiency of 5% around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer （Ostriniafurnacalis）. These two events were further confirmed by molecular analysis. Southern blot suggested that a single copy of the crylAh gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene crylAh was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T l-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.

Construction of polyhedrin-positive recombinant viruswith expression of truncated δ-endotoxin fromBacillus thuringiensis in insect cell

Baculoviruses have been widely used as biological agents because of their specificpathogenicity for target insect and harmlessness to mammals, birds and plants as well asadvantages with persistence and epidemics as pestcides. A major drawback for morewide-spread use of these viruses is their low virulenee and slow speed of action, which re-stricted their use. No enhancement in pathogenicity of recombinant viruses was observedwith insertion of the Bacillus thuringiensis full-length endotoxin cryIA(c) and cryIA(b) geneinto the AcNOV (Autographa californica nuclear polyhedrosis virus) genome under the con-trol of polyhedrin gene promoter. The reason may be that protoxin, full-length genesproduct of recombinant virus in infected insect cells, which was short of insect gut alkalienvironment, cannot be degraded into active toxic polypeptide. Expression level of 3’

Cloning of Bt cry Genes by Rapid Screening of DNA Libraries with PCR-RFLP

Bacillus thuringiensis (Bt) strain C002 contains crylAa, cry2Ab, crylCa insecticidal crystal genes and an unkown gene cryX, among which crylCa is located in a 6 -9 kb EcoR I fragment of the chromosomal DNA. The total DNA and the plasmids DNA libraries of C002 were constructed in Bt-E. coli shuttle plasmid pHT315 by inserting 6 - 9 kb chromosomal and plasmid DNA fragments prepared respectively with EcoR I complete and 5au3A I partial digestion. On the basis of every 50 transformants pooled together from 5-10 tubes, the pools containing about 2 000 transformants from the plasmids DNA library and 400 transformants from the total DNA library were rapidly screened by PCR-RFLP. Clones containing crylAa, cryX, crylCa , and cry2Ab were isolated and named as pHT-1Aa, pHT-X, pHT-1Ca and pHT-2Ab respectively. Restriction analysis indicated that pHT-1Aa, pHT-1Ca and pHT-2Ab had the typical physical map of the homologous cry genes. Furthermore, each plasmid was transferred into Bt acrystalliferous strain cryB- by eletropora-tion. SDS-PAGE result showed that transformant of pHT-1Ca expressed 130 kDa protein and bioassay result proved its high toxicity against Spodotera exigua 1st instar larvae with 100% corrected motality.

热处理对转基因秸秆中重组蛋白和重组DNA的降解作用

随着转基因作物种植面积的不断扩大,如何高效处理转基因秸秆成为了一个重要的科学问题。未经处理的转基因秸秆中的重组蛋白和重组DNA可以在土壤中存在很长时间,并对土壤生物多样性产生潜在负面影响。因此寻找一个既节约成本又对环境无害的秸秆处理方法非常重要。高温处理是降解转基因秸秆重组蛋白和重组DNA的有效手段,但目前对处理温度和处理时间的选择还缺乏系统的研究。本研究通过试纸条、聚合酶链反应(PCR)等方法检测不同温度和时间处理的转基因作物秸秆中重组蛋白和重组DNA的水平。结果表明,转基因大豆、棉花、玉米、水稻的秸秆在50℃处理3 h后,其体内的抗除草剂蛋白草胺膦乙酰转移酶(phosphinothricin acetyltransferase, PAT)等重组蛋白基本降解;但相同温度下,重组DNA的降解则需要4 d时间;提高处理温度可以在一定程度上缩短重组蛋白和重组DNA降解所需的时间。50℃处理4 d的条件在实际生产中通过堆肥就能实现。本研究从分子生物学的角度为转基因秸秆处理提供了依据。

低温脂肪酶产生菌的筛选、表达及酶学性质分析

为筛选高产低温脂肪酶的菌株,并为脂肪酶的工业化开发提供生产资料。利用罗丹明B培养法从漠河县兴安落叶松林为主的林下土壤中分离出1株低温脂肪酶产生菌,对其形态学、生理生化特性进行鉴定,并结合16S rDNA基因序列,确定该菌株为苏云金芽孢杆菌(Bacillus thuringiensis)。以苏云金芽孢杆菌基因组为模板克隆脂肪酶基因Lip240,对Lip240进行异源表达以及酶学性质的分析。结果表明:该重组脂肪酶Lip240的最适反应温度为30℃,在4~30℃条件下处理6 h能维持60%以上的活性,是一种低温脂肪酶,最适pH为8.0。Ca^(2+)、Mn^(2+)、Fe^(2+)、Fe^(3+)和Cr^(3+)对脂肪酶Lip240的酶活有一定的促进作用。该酶具有较好的有机溶剂耐受性,SDS能够显著(P<0.05)抑制脂肪酶Lip240的酶活。该酶更偏好于水解中短链底物。该结论可为微生物资源开发利用和低温脂肪酶的工业应用提供一定的理论依据。

PEG介导BT基因转化大豆原生质体获转基因植株

通过ＰＥＧ法将Ｂ．Ｔ（ＢａｃｉｌａｓｔｈａｒｉｎｇｉｅｎｓｉｓＣｒｙＩＡｃ）毒蛋白基因导入到大豆主栽品种黑农３５，黑农３７，合丰２５和合丰３５的原生质体中，经３０ｍｌ／Ｌ潮霉素筛选，选择有抗性的愈伤组织进行分化，获得了三棵再生植株，移栽后全部成活，对移栽后植株的总ＤＮＡ进行ＰＣＲ分析，均显阳性。对ＰＣＲ阳性植株进行Ｓｏｕｔｈｅｒｎ杂交分析，证明Ｂ．Ｔ毒蛋白基因已整合到大豆细胞基因组中。