苏云金芽孢杆菌双组份信号转导系统的生物信息学分析

苏云金芽孢杆菌(Bacillus thuringiensis)能产生杀虫晶体蛋白等多种活性成分,是目前应用最广泛的微生物杀虫剂。本文采用生物信息学方法,系统分析了由本实验室完成全基因组测序的苏云金芽孢杆菌YBT-1520、CT-43和BMB171 3个菌株的双组分信号转导系统(Two-componentsignal transduction system,TCS)的分布、结构及功能,并初步构建了部分TCS的调控网络关系图。本研究旨在为深入研究苏云金芽孢杆菌的生长、代谢以及毒力因子的表达与调控,全面了解伴孢晶体的形成机制开辟新的研究方向。     

Microcalorimetric investigation on the growth model and the protein yield of Bacillus thuringiensis

A novel microcalorimetric technique based on the bacterial heat output was applied to evaluate the special growth model, the protein expression and the generation time of Bacillus thuringiensis for the first time. The thermogenic curves of the aerobic metabolism of B. thuringiensis strains YBT-833, YBT-1520 and YBT-833-2-1 were determined by using an LKB-2277 BioActivity Monitor. The analysis of the thermogenic curves indicated both the mutant strain and the wild-type strains followed the same linear growth model during sporulation. The metabolism heat output revealed heat output was correlated to the yield of the insecticidal crystal proteins (ICPs) very well, the more protein product, and the less heat output. Based on the data acquired, we proposed that this method could be a useful tool in monitoring the fermentation of B. thuringiensis.     

iTRAQ analysis reveals the effect of gabD and sucA gene knockouts on lysine metabolism and crystal protein formation in Bacillus thuringiensis

Lysine metabolism plays an important role in the formation of the insecticidal crystal proteins of Bacillus thuringiensis (Bt). The genes lam, gabD and sucA encode three key enzymes of the lysine metabolic pathway in Bt4.0718. The lam gene mainly affects the cell growth at stable period, negligibly affected sporulation and insecticidal crystal protein (ICP) production. While, the deletion mutant strains of the gabD and sucA genes showed that the growth, sporulation and crystal protein formation were inhibited, cells became slender, and insecticidal activity was significantly reduced. iTRAQ proteomics and qRT-PCR used to analyse the differentially expressed protein (DEP) between the two mutant strains and the wild type strain. The functions of DEPs were visualized and statistically classified, which affect bacterial growth and metabolism by regulating biological metabolism pathways: the major carbon metabolism pathways, amino acid metabolism, oxidative phosphorylation pathways, nucleic acid metabolism, fatty acid synthesis and peptidoglycan synthesis. The gabD and sucA genes in lysine metabolic pathway are closely related to the sporulation and crystal proteins formation. The effects of DEPs and functional genes on basic cellular metabolic pathways were studied to provide new strategies for the construction of highly virulent insecticidal strains, the targeted transformation of functional genes.     

转Bt基因作物释放杀虫晶体蛋白对土壤生态安全的影响

随着转Bt基因抗虫作物的大面积推广种植,其环境安全性问题日益引起关注。转Bt基因作物在生长期内持续不断地向环境释放杀虫晶体蛋白,这些杀虫晶体蛋白积累一旦超过了昆虫的消耗及环境因子对其的钝化,就可能对非靶标昆虫或土壤微生物造成伤害。转Bt基因作物向土壤中释放杀虫晶体蛋白的途径主要有3种:根系分泌、花粉飘落和秸秆还田。释放到土壤中的Bt杀虫晶体蛋白能够迅速被土壤活性颗粒吸附,1～3 h就能达到吸附平衡。吸附态Bt杀虫晶体蛋白不易被土壤微生物或酶降解,导致杀虫活性持续时间显著延长。土壤微生物种群变化是衡量Bt作物对土壤生态影响的重要指标。研究表明,Bt作物根系分泌物或Bt生物体降解释放的杀虫晶体蛋白对于土壤蚯蚓、线虫、原生动物、细菌和真菌没有毒性,但可使丛枝菌根真菌(AMF)菌丝长度减小,不能形成侵染单元。Bt杀虫晶体蛋白对土壤酶活性的影响程度依这类蛋白的导入方式或Bt作物生育期的不同而呈现差异。土壤中Bt Cry1Ab蛋白能被部分后茬作物吸收,但不同的商品试剂盒检测结果存在差异。文章综述了Bt杀虫晶体蛋白在土壤中释放、吸附、残留特性及其对土壤动物、土壤微生物、土壤酶活性和后茬作物的影响,旨在为转Bt基因作物释放杀虫晶体蛋白的土壤生态安全评价提供参考依据。     

鳞翅目昆虫中肠Bt杀虫蛋白受体研究进展

杀虫晶体蛋白(insecticidal crystal proteins,ICPs;含有Cry和Cyt 2大家族)和营养期杀虫蛋白(vegetative insecticidal proteins,Vips)等Bt杀虫蛋白可有效防治鳞翅目害虫,其中Cry应用最广泛。然而,一些地区的鳞翅目害虫已对Bt杀虫蛋白产生了抗性。目前,普遍认为鳞翅目昆虫中肠受体与Bt杀虫蛋白结合能力的改变是导致其对Bt杀虫蛋白产生抗性的最主要因素。在鳞翅目昆虫中,Cry受体是研究得最为透彻的Bt受体,已经被证实的有氨肽酶N、钙黏蛋白、碱性磷酸酶和ABC转运蛋白等。Vips杀虫蛋白类与鳞翅目昆虫中肠受体的结合方式与Cry杀虫蛋白相似,但结合位点与Cry杀虫蛋白不同。本文从结构特点、作用机制及不同鳞翅目昆虫间的表达差异等角度对以上4种鳞翅目昆虫中肠Bt受体进行了综述,并提出如下展望:(1)以棉铃虫或小菜蛾等鳞翅目昆虫为农业害虫模式生物进行深入研究,阐明其对Bt杀虫蛋白产生抗性的机制,为研究其他鳞翅目农业害虫对Bt杀虫蛋白产生抗性的机制提供理论借鉴;(2)鉴于在不同鳞翅目昆虫间,中肠Bt受体与Bt杀虫蛋白结合存在差异,且同一Bt杀虫蛋白与鳞翅目昆虫Bt受体并不专一性结合,Bt杀虫蛋白多基因组合策略是较为有效的田间鳞翅目昆虫防治策略,是今后一段时间内Bt杀虫蛋白应用的发展方向。     

晶胞粘连苏云金芽胞杆菌C15杀线虫毒力因子发掘与功能鉴定

苏云金芽胞杆菌（Bacillus thuringiensis,Bt）制剂作为一种高效的微生物杀虫剂,在植物病虫害防控领域有着广泛的应用。Bt制剂的主效成分为杀虫晶体和芽胞,其中,杀虫晶体的环境低持久性是Bt农药应用的重要限制因素之一。自然界中存在着一些Bt菌株,其产生的杀虫晶体位于芽胞外壁和芽胞衣之间,这种特殊的表型被称为晶胞粘连（spore-crystal association, SCA）表型。由于芽胞外壁对晶体的保护作用,SCA表型可以提升晶体抵抗不良环境因素的能力,是开发新型Bt生物囊杀虫剂的有效育种策略。本文选取对线虫具有强毒杀能力的 SCA菌株C15作为研究对象。获得了C15菌株的完整基因组序列,包括一个5 637 049 bp的环状染色体和8个不同大小的环形质粒（240 314 bp到3 188 bp）。C15基因编码了5个杀虫蛋白（Cry蛋白）基因：cry21-99、cry21-67、cry21-66、cry21-46和cry-N。在Bt无晶体突变株BMB171中异源表达cry21-99基因,发现其表达产物形成菱形晶体,且对秀丽隐杆线虫（Caenorhabditis elegans）和南方根结线虫（Meloidogyne incognita）均有毒杀活性。同时,还在全基因组范围内预测了Cry毒素以外的杀线虫毒力因子和次生代谢产物。此外,在C15基因组中预测了本团队已报道的苏云金芽胞杆菌幕虫亚种（B. thuringiensis serovar finitimus）菌株YBT-020 SCA表型决定因子的同源基因,缺失后突变体仍然保留稳定的SCA表型,说明C15菌株的SCA表型形成机制与YBT-020不同,该菌株代表了一种新的SCA表型形成机制。本研究为转基因作物防控线虫提供了新的遗传资源,也为研究SCA表型形成机制,开发新型高效Bt制剂提供了新线索。     

苏云金芽胞杆菌营养期杀虫蛋白基因的克隆及表达分析

选择本实验室分离的苏云金芽胞杆菌李氏亚种 (subsp. Leesis) 菌株YBT833、鲇泽亚种(subsp.Aizawai) 菌株YBT-1416和库斯塔克亚种(subsp. Kurstaki)菌株YBT1535为出发菌株,以营养期杀虫蛋白基因PCR扩增的特异片段为探针,进行总DNA酶切片段的Southern杂交定位。结果显示3株菌株的营养期杀虫蛋白基因,均位于经XbaI完全消化的4～5kb大小的DNA 片段上。将该区域DNA片段回收后克隆到pUC19载体,建立了3个较基因组文库小的亚基因组文库。通过菌落原位杂交筛选和酶切鉴定分别得到3个相应的营养期杀虫蛋白基因vip83、vip14和vip15,并对其测序。DNA序列比较发现基因vip83与已知营养期杀虫蛋白基因存在5个差异碱基。将vip83、vip14基因亚克隆到苏云金芽胞杆菌大肠杆菌穿梭载体pHT315, 分别得到重组质粒pBMB8901和pBMB8902。将它们电转化到vip^-的B.t.受体菌BMB171和4Q7,获得了相应的工程菌BMB8901-171,BMB8902-171,BMB8901-4Q7和BMB8902-4Q7。SDS-PAGE电泳检测均有88kD大小的蛋白表达。生物测定结果亦表明了,营养期杀虫蛋白Vip83和Vip14对鳞翅目棉铃虫、小菜蛾和甜菜夜蛾的三龄幼虫均有一定的杀虫活性;其中对小菜蛾的毒力最高,LC₅₀值分别为28.6,31.6,45.4和37.6μL/mL。该结果为构建高效广谱工程菌提供了实际材料和理论依据。      

Proteome analysis of metabolically engineered Escherichia coli producing Poly(3-hydroxybutyrate)

Recombinant Escherichia coli strains harboring heterologous polyhydroxyalkanoate (PHA) biosynthesis genes were shown to accumulate unusually large amounts of PHA. In the present study, integrated cellular responses of metabolically engineered E. coli to the accumulation of poly(3-hydroxybutyrate) (PHB) in the early stationary phase were analyzed at the protein level by two-dimensional gel electrophoresis. Out of 20 proteins showing altered expression levels with the accumulation of PHB, 13 proteins were identified with the aid of mass spectrometry. Three heat shock proteins, GroEL, GroES, and DnaK, were significantly up-regulated in PHB-accumulating cells. Proteins which play essential roles in protein biosynthesis were unfavorably influenced by the accumulation of PHB. Cellular demand for the large amount of acetyl coenzyme A and NADPH for the PHB biosynthesis resulted in the increased synthesis of two enzymes of the glycolytic pathway and one enzyme of the Entner-Doudoroff pathway. The expression of the yfiD gene encoding a 14.3-kDa protein, which is known to be produced at low pH, was greatly induced with the accumulation of PHB. Therefore, it could be concluded that the accumulation of PHB in E. coli acted as a stress on the cells, which reduced the cells' ability to synthesize proteins and induced the expression of various protective proteins.     

Molecular approaches towards development of novel Bacillus thuringiensis biopesticides

Bacillus thuringiensis (Bt) has been used for control of lepidopteran, dipteran and coleopteran insects for over three decades. Novel Bt strains harbouring new types of insecticidal genes are being discovered worldwide. Recombinant strains with enhanced toxicity and broadened insecticidal spectrum have been constructed. To increase the field persistence of insecticidal crystal proteins (ICPs), alternative modes of their delivery in Pseudomonas sp. and endophytes have been developed. ICPs have been modified by site-directed mutagenesis to improve their insecticidal efficacy. Higher yields of ICPs have been achieved by use of strong expression promoters and other regulatory elements. Gene-disabling of the sporulation-specific protease has led to yield enhancement of ICPs. Interestingly, Bt toxins have been found to act synergistically with some other pesticidal agents. Optimization of fermentation conditions is an essential requirement for cost-effective commercial production of Bt biopesticides. The environmental impact of deployment of genetically engineered biopesticides has been assessed. Recombinant Bt strains that do not carry any non-Bt DNA, endophytes, encapsulation in killed bacteria (such as Pseudomonas) and asporogenous Bt strains are ecologically safe approaches. Efficient resistance management strategies require judicious use of Bt transgenic plants in conjunction with refugia and Bt biopesticides in an Integrated Pest Management (IPM) program. This revised version was published online in November 2006 with corrections to the Cover Date.     

重组Bt伴胞晶体蛋白Cry5B对大熊猫西氏贝蛔虫的离体杀灭活性观察

大熊猫是中国特有的珍稀物种,西氏贝蛔虫是危害大熊猫最为严重的一种肠道寄生性线虫。针对目前长期药物驱虫导致的大熊猫西氏贝蛔虫耐药及生态污染问题,苏力菌(Bacillus thuringiensis,Bt)晶体蛋白Cry5B因其特异的线虫杀灭活性,是一种理想的新型抗蛔虫病药物候选。本研究拟通过原核表达方式产生Bt YBT-1518重组Cry5B蛋白,并评价其对大熊猫西氏贝蛔虫的离体杀灭活性。结果表明,Bt YBT-1518 Cry5B由1 246个氨基酸组成,分子量(MW)为139.889 kDa,包含Endotoxin_N、δ-Endotoxin_C、Endotoxin_C、Endotoxin_C2、Cry1Ac_D5结构域,与Bt PS86Q3晶体蛋白关系最近。重组Cry5B蛋白在IPTG浓度为1.4 mmol/L时表达量最大,表达于菌体上清液。离体杀灭活性实验表明,重组Cry5B蛋白的大熊猫西氏贝蛔虫抑杀效果具有显著的剂量依赖性,对肠道L4期幼虫第3天的ED₅₀值为14.5 μg/mL,第7天的ED₅₀值为0.16 μg/mL;但对成虫更敏感,仅作用2 d便可显著下降虫体活性,直至死亡,证实重组Cry5B蛋白对大熊猫蛔虫具有较强的毒杀作用。这些结果为后续论证Bt YBT-1518重组Cry5B蛋白可以作为一种新型、环保的抗大熊猫西氏贝蛔虫病药物提供了数据参考。     

苏云金杆菌营养期杀虫蛋白的研究

营养期杀虫蛋白 (vegetativeinsecticidalproteins ,VIPs)是苏云金杆菌 (Bacillusthuringiensis,Bt)在对数生长中期分泌的一类新型杀虫毒蛋白。VIPs主要分为VIP1、VIP2和VIP3三种。VIP1和VIP2构成二元毒素 ,对鞘翅目叶甲科的昆虫具有杀虫特异性 ;而VIP3对鳞翅目昆虫具有较广谱的杀虫活性。VIP1和VIP2的杀虫作用机理还不清楚 ;VIP3通过诱发细胞凋亡 ,最终导致昆虫死亡 ,这种作用机理与Bt杀虫晶体蛋白的作用机理完全不同 ,这为筛选新的杀虫活性物质提供了新的思路。vip基因现已被应用于转基因杀虫植物的构建 ,得到高效抗虫的多价转基因玉米。此外 ,VIPs嵌合蛋白的构建、vip及其融合基因导入其它许多宿主微生物等方面的研究也具有诱人的潜在应用前景。     

昆虫类钙粘蛋白与Bt Cry1A蛋白之间的相互作用

类钙粘蛋白(cadherin-likeprotein)位于昆虫中肠刷状缘膜囊泡(brushbordermembranevesicles,BBMV)上,是苏云金芽孢杆菌(Bacillusthuringiensis,Bt)产生的杀虫晶体蛋白(BtCry蛋白)的主要受体之一。它能够与BtCry蛋白结合,引起细胞膜的渗透性发生改变,促进BtCry蛋白对敏感昆虫的毒杀作用。类钙粘蛋白基因的突变还能导致敏感昆虫对BtCry蛋白产生抗性。因此,研究昆虫类钙粘蛋白与BtCry蛋白之间的相互作用,将有助于揭示BtCry蛋白杀虫作用机理。文章对昆虫类钙粘蛋白种类、结构特征、在昆虫体内的分布、及其与BtCry蛋白之间的相互作用等方面的研究现状进行详细论述。     

Characterization of melanin produced by a wild-type strain of Bacillus cereus

Bacillus cereus 58 (Bc58)is a UV-resistant wild type strain that has an ability to produce a sorrel pigment induced by L-tyrosine.The Fourier-transform infrared (FT-IR)spectra and chemical tests of its pigment are similar to that of the standard melanin (Sigma).A bioassay shows that the LC50 of a Bacillus thuringiensis (Bt)formulation added with the melanin of Bc58 and exposed to UV for 5 h is 16.1 μg/ml,which is similar to that of the Bt formulation without UV treatment,however,it is almost double that of the Bt formulation exposed to UV without the melanin of Bc58.The result of SDS-PAGE indicates that the melanin of Bc58 can protect the insecticidal crystal proteins from degradation.This suggests that it is an excellent UV protective agent for the insecticidal crystal proteins of the Bt formulation.     

Physiological response of non-Bt and Bt cotton to short-term drought stress

Drought stress triggered the accumulation of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) both in non-Bt and Bt cotton with simultaneous production of antioxidant enzymes. And there was no significant difference between non-Bt and Bt cotton under drought stress. In contrast to this, we observed a significant reduction of Bt toxin proteins under 72 h of drought stress in Bt cotton.     

Caloric restriction improves thermotolerance and reduces hyperthermia-induced cellular damage in old rats.

Adult-onset, long-term caloric restriction (CR) prolongs maximum life span in laboratory rodents. However, the effect of this intervention on an organism's ability to cope with a physical challenge has not been explored. We investigated the influence of CR and aging on stress tolerance in old rats exposed to an environmental heating protocol on two consecutive days. We hypothesized that CR would increase heat tolerance by reducing cellular stress and subsequent accrual of oxidative injury. All calorically restricted rats survived both heat exposures compared with only 50% of their control-fed counterparts. CR also decreased heat-induced radical generation, stress protein accumulation, and cellular injury in the liver. In addition, heat stress stimulated marked induction of the antioxidant enzymes manganese-containing superoxide dismutase and catalase, along with strong nuclear catalase expression in liver samples from rats subjected to CR. In contrast, stress-related induction of antioxidant enzymes was blunted, and nuclear catalase expression was unchanged from euthermic conditions in the control-fed group. These data suggest that CR reduces cellular injury and improves heat tolerance of old animals by lowering radical production and preserving cellular ability to adapt to stress through antioxidant enzyme induction and translocation of these proteins to the nucleus.     

苏云金杆菌杀虫晶体蛋白活性预测的支持向量机模型

藉均匀设计(UD)方法,构建了苏云金杆菌(Bt)杀虫晶体蛋白氨基酸组成特征与其杀虫活性之间关系的支持向量机(SVM)模型。当惩罚系数为0·01、epsilon值为0·2、gamma值为0·05、域值为0·5时,该模型对Bt杀虫晶体蛋白杀虫活性的预测平均准确率达73%。     

A proteomic analysis approach to study insecticidal crystal proteins from different strains of Bacillus thuringiensis

We obtained and compared a new cry2Ac6 gene from Bacillus wuhanensis 140, and Bacillus thuringiensis (Bt) subsp. kurstaki 4.0718 and B.t. kurstaki XL004 that share a similar genetic background but occupy different ecological niches. Using a proteomic approach and function-based activity profiling, we systemically identified the insecticidal crystal proteins (ICPs) from the three Bt species, which were found to be mainly distributed at pH 4–7 on two-dimensional electrophoresis (2DE) gels by PDQuest software. The proteins that exhibited a significant difference in expression were excised, digested in-gel and identified by MALDI-TOF-MS. Thirty-three differently expressed proteins were identified from the three Bt strains. The Cry2Ac6, Cry1Ab16, CryIG, CryH2, CryI, CryINA67-1 and CryI⁺ crystal protein mixture from B.t. wuhanensis 140, Cry1Ac, Cry2Aa and endotoxin delta1 from B.t. kurstaki 4.0718 were further analyzed by bioinformatics analysis. Two common proteins were founded in three strains, the heat shock proteins (HSP60) and the translation elongation factor Tu, which help with protein refolding and prevent protein degradation. The different enzymes of metabolism, including glutamate racemase, chemotaxis protein histidine kinase and related kinases pyruvate dehydrogenase complex E1orE3 were identified. Some protein spots could not be identified. The results indicate that each Bt strain has unique ICPs as well as some common proteins related to ICPs formation, and that the virulence of Bt strains is closely related to the expression of specific ICPs.     

Heat Stress in Wheat during Reproductive and Grain-Filling Phases

Ambient temperatures have increased since the beginning of the century and are predicted to continue rising under climate change. Such increases in temperature can cause heat stress: a severe threat to wheat production in many countries, particularly when it occurs during reproductive and grain-filling phases. Heat stress reduces plant photosynthetic capacity through metabolic limitations and oxidative damage to chloroplasts, with concomitant reductions in dry matter accumulation and grain yield. Genotypes expressing heat shock proteins are better able to withstand heat stress as they protect proteins from heat-induced damage. Heat tolerance can be improved by selecting and developing wheat genotypes with heat resistance. Wheat pre-breeding and breeding may be based on secondary traits like membrane stability, photosynthetic rate and grain weight under heat stress. Nonetheless, improvement in grain yield under heat stress implies selecting genotypes for grain size and rate of grain filling. Integrating physiological and biotechnological tools with conventional breeding techniques will help to develop wheat varieties with better grain yield under heat stress during reproductive and grain-filling phases. This review discusses the impact of heat stress during reproductive and grain-filling stages of wheat on grain yield and suggests strategies to improve heat stress tolerance in wheat.