The toxicity test and hypothetical model of Bacillus thuringiensis Cry1Aa helix4

Development of targeted biological agents against agricultural insect pests is of prime importance for the elaboration and implementation of integrated pest management strategies that are environment-friendly, respectful of bio-diversity and safer to human health through reduced use of chemical pesticides. A major goal to understand how Bt toxins work is to elucidate the functions of their three domains. Domains II and III are involved in binding specificity and structural integrity, but the function of Domain I remains poorly understood. Using a Manduca sexta BBMV (brush border membrane vesicles) system, we analyzed its responses to Cry1Aa 15 single-point mutations with altered Domain I helix 4 residues. Light scattering assay showed that toxicity was almost lost in 3 mutants, and we observed significantly reduced toxicity in other 7 mutants. However, 5 mutants retained wild-type toxicity. Using computer software, we simulated the three-dimensional structures of helix 4. Both experimental and bioinform     

The toxicity test and hypothetical model ofBacillus thuringiensis Cry1Aa helix4

Development of targeted biological agents against agricultural insect pests is of prime importance for the elaboration and implementation of integrated pest management strategies that are environment-friendly, respectful of bio-diversity and safer to human health through reduced use of chemical pesticides. A major goal to understand how Bt toxins work is to elucidate the functions of their three domains. Domains II and III are involved in binding specificity and structural integrity, but the function of Domain I remains poorly understood. Using a Manduca sexta BBMV (brush border membrane vesicles) system, we analyzed its responses to Cry1Aa 15 single-point mutations with altered Domain I helix 4 residues. Light scattering assay showed that toxicity was almost lost in 3 mutants, and we observed significantly reduced toxicity in other 7 mutants. However, 5 mutants retained wild-type toxicity. Using computer software, we simulated the three-dimensional structures of helix 4. Both experimental and bioinformatic analysis showed that residues in Cry1Aa Domain I helix 4 were involved in the formation of ion channels that is critical for its insect toxicity.     

用cDNA差式分析法克隆受GA抑制的豌豆基因

采用新兴的cDNA差式分析法(representational difference analysis,RDA)比较了经过赤霉素(GA)处理和未经过处理G2豌豆基因表达的差异,发现该豌豆细胞内有数个被GA特异性抑制的mRNA.克隆其中分子量较大的片段PGAS1～3(PeaGA suppressed cDNA 1～3),经Northern杂交证实这些基因只在未经GA处理的豌豆细胞内表达.比较现行核DNA(或 cDNA)减法及差式显示(Differential display)等技术,认为本方法不仅操作简单、易于推广,而且重复性好,假阳性少,是分子克隆技术的重要进步.     

粪产碱菌nif H,nif D和部分nif K的克隆、定位及序列分析

提取粪产碱菌(Alcaligenes faecalis)总DNA,经限制性内切酶酶切和琼脂糖凝胶电泳,以含肺炎克氏杆菌(Klebsiella pneumoniae)nif H和nif H-D基因的DNA片段为探针进行Southern杂交,筛选出与nif HDK同源的4.6kb片段,克隆到pBluesript SK~+载体上,构建了重组质粒pBZl.经亚克隆、酶切、DNA序列分析后发现,粪产碱菌具有与其它固氮菌相似的结构特征,其nif HDK共用1个启动子,具有上游激活序列UAS,RNA聚合酶σ⁵⁴因子识别序列、1个A-T富集区和SD序列.nifH和nif D的阅读框架分别为888和1476bp,GC含量各为61.6%和60.2%.nifH-nif D和nif D-nif K的基因间隔区长度分别为101和105bp,各存在1个7bp的反向重复和1个SD序列.由阅读框架(ORF)推导的铁蛋白和钼铁蛋白α亚基的氨基酸序列与其它固氮菌相比有较高的同源性,高度保守的氨基酸残基所处的位置也很相似.同源性比较说明,粪产碱菌与棕色固氮菌(Azotobacter vinelandii)同源性最高.     

蜘蛛杀虫肽在大肠杆菌中的表达及其活性分析

以Ｔ７为启动子构建了蜘蛛杀虫肽基因的表达质粒ｐＴＨＩ１９,转化大肠杆菌ＢＡＬ３１,在其对数生长期加入１ｍｍｏｌ／Ｌ ＩＰＴＧ能诱导杀虫肽的产生,表达产物占菌体总蛋白的１３．７５％,并且表达的杀虫肽具有功能活性。     

外源钙调蛋白对植物细胞分裂增殖作用的研究

外源钙调蛋白（Ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）对胡萝卜悬浮细胞增殖具明显促进作用,不同浓度ＣａＭ的促进程度不同,７ｕｇ／ｍｌ时促进作用最大。ＣａＭ抑制剂ＴＦＰ（Ｔｒｉｆｌｕｏｐｅｒ－ａｚｉｎｅ）则明显抑制该悬浮细胞的增殖,ＴＦＰ浓度越高则抑制作用越强。另外,外源ＣａＭ可以加快珍珠梅花粉第二次有丝分裂,改变生殖细胞有丝分裂各期花粉管的比例,说明外源ＣａＭ对植物体细胞和性细胞的增殖和分裂均有促进作用。     

北京大学蛋白质工程及植物基因工程国家重点实验室

北京大学蛋白质工程及植物基因工程国家重点实验室１概况北京大学蛋白质工程及植物基因工程国家重点实验室筹建于１９８７年,１９９０年通过验收,由生物化学家张龙翔教授任学术委员会主任,生物物理学家顾孝诚教授任实验室主任。１９９４年４月换届时,经国家教委批准,...     

蜘蛛杀虫肽基因的合成及其在植物中表达质粒的构建

近年来用生物制剂防治害虫,虽然可以避免环境污染,但效果往往不稳定,而用基因工程方法,将抗虫基因导入植物的基因组中,让植物自身产生抗虫物质,将是一种理想的途径［１,２］。抗虫的植物基因工程主要是利用苏云金杆菌的内毒素蛋白,转化此基因的烟草和番茄显示了对虫的抗性［３—６］。澳大利亚Ｄｅａｋｉｎ大学从一种蜘蛛毒液中分离纯化到一种只有３７个氨基酸的小肽,体外实验发现其能杀死多种对农业生产有害的昆虫,但对哺乳动物没有毒害作用［７］。我们根据此肽的氨基酸序列,采用植物偏爱的密码子,人工合成并克隆了此肽的基因…     

豌豆核糖体小亚基蛋白S21的cDNA克隆、序列分析及在G2豌豆中的表达研究

以G2豌豆幼苗为材料,构建了滴度为6.5×106pfu的cDNA文库,用同源序列筛选法从该文库中得到一个全长465bp的cDNA。杂交分析认为它是一完整的cDNA序列。DNA序列分析表明,它拥有一个282bp的开放读码框,编码94个氨基酸。计算机同源序列比较发现,它可能编码豌豆核糖体小亚基蛋白S21(RS21-PEA),因为该序列与已知的玉米、水稻S21在氨基酸全序列水平上有32%～35%的同源性,并含有与RNA结合的特征结构域。进化树分析显示S21蛋白可在一定程度上反映生物的进化及遗传变异趋势。