新型杀虫晶体蛋白Cry7Ab4空间结构的计算机解析

采用一级结构分析,二级结构预测及同源比对建模的方法,模拟出新型杀虫晶体蛋白Cry7Ab4的空间结构,并进行动力学优化.结合其杀大猿叶甲活性数据与蛋白结构比对分析,发现Cry7Ab4与Cry7Aa1的活性差异主要归因于α3,α4上疏水性氨基酸的不同.     

苏云金芽孢杆菌毒素Cry7Aa1三维结构模型

运用生物信息学软件和网络数据库资源,采取同源建模的方法以毒素蛋白Cry8Ea1为模板预测了苏云金芽孢杆菌毒素Cry7Aa1的三级结构并分析了相关的生物学功能.结果表明,Cry7Aa1为3个结构域组成的近似球状蛋白,Cry7Aa1在氨基酸序列上与Cry8Ea1有40%的同源性,在三维结构_ECry7Aa1与Cry8Ea1有高度的相似性,它们结构域Ⅰ几乎完全相同,在结构域Ⅱ之间和结构域Ⅲ之间存在较小差异.Cry7Aa1与Cry8Ea1的表面电势分布不同.2种毒素具有相似的毒杀机理.模型经Ramachandran图和Verify 3D检验具有合理性.     

杀虫晶体蛋白对小菜蛾杀虫活性的识别预测

考察对小菜蛾具有杀虫活性和无杀虫活性的晶体蛋白二肽组成的差异.结果发现,对小菜蛾具有杀虫活性的晶体蛋白中,SG,LS,SN,NI,GS,SV,SL,TV,EF,NQ二肽分数较高;而对小菜蛾无杀虫活性的晶体蛋白中,AN,LQ,DA,AQ,TF,SY,EI,LE,PT,RA二肽分数较高,总体分数上存在差异最大的是S*型二肽.在此基础上,发展一种识别杀虫晶体蛋白(ICP)对小菜蛾杀虫活性的统计学方法,通过对两组共27条杀虫晶体蛋白进行有无杀虫活性的初步识别,该方法识别平均正确率分别是71.4%和100%.     

桃蚜关键抗性基因挖掘及抗蚜Cry蛋白预测

为了挖掘桃蚜(Myzus persicae)的关键抗性基因并构建抗性调控网络,通过加权基因共表达网络分析(WGCNA)和差异表达基因分析(DEGs)对桃蚜抗性研究的GEO数据库进行分析,筛选出2 426个枢纽基因和2 263个差异表达基因探针.将关键抗性基因在String数据库中进行蛋白质相互作用(PPI)分析,获得154个桃蚜关键抗性基因,并绘制桃蚜的抗性调控网络.结果表明:acpp基因的上调表达可能是大多数Cry蛋白不能有效杀死桃蚜的原因之一;参考抗蚜Cry1Cb2蛋白和11个靶标蛋白的对接规则,通过同源建模和分子对接等生物信息学技术,预测了10个新的Cry蛋白可能对桃蚜具有杀虫活性.     

黑线仓鼠Cry1、Cry2克隆及生物信息学分析

隐花色素(Cryptochromes,包括Cry1和Cry2)编码黄素腺嘌呤二核苷酸结合蛋白,是昼夜节律核心振荡器复合物的关键组分,是各种生理功能的重要调节因子.本实验以黑线仓鼠(Cricetulus barabensis)为材料,通过设计特异性引物,RT-PCR技术克隆得到黑线仓鼠Cry1 cDNA序列1822 bp(GeneBank登录号:MK796241)、Cry2 cDNA序列1945 bp(GeneBank登录号:MK796242).运用生物信息学对Cry1、Cry2核酸序列以及蛋白质序列进行分析,构建系统进化树,结果表明:测得的Cry1 cDNA包括一个完整的开放阅读框1764 bp,部分5′端非翻译区和部分3′非翻译区,编码587个氨基酸,A+T含量略高于G+C含量;测得的Cry2的cDNA包括一个完整的开放阅读框1794 bp,部分5′端非翻译区和部分3′非翻译区,编码597个氨基酸,G+C含量远高于A+T含量,提示其Tm值较高,热稳定性强. CRY1、CRY2理论蛋白分子量为66469.86 Da、67427.08 Da,等电点(pI)为8.40、8.82,均为不稳定蛋白. CRY1、CRY2蛋白跨膜区和疏水性分析和信号肽分析等均表明,整体疏水性不强,为亲水性蛋白;没有信号肽,不属于分泌蛋白,与其作为转录抑制因子相符合.功能结构域预测表明,二者均含有两个高度保守的功能结构域:DNA光解酶及FAD结合域.同源性比对及系统进化树分析显示,黑线仓鼠与中国仓鼠亲缘关系最近,与灵长类目及鸡形目亲缘关系较远,这与传统的分类物种分类方式相一致,证实本实验测得的Cry1、Cry2基因序列正确,体现了Cry1、Cry2进化的保守性.本实验为探究昼夜节律基因Cry1、Cry2在动物繁殖调控中的作用奠定了基础,具有重要的理论意义.     

苏云金杆菌杀虫晶体蛋白的crylC基因

目前,有8种crylC类基因已被克隆,其主要分布在杀虫亚种和鲇泽亚种.本文对杀虫晶体蛋白CrylC的结构与功能、作用方式、抗性问题及基因改造等方面作一综述,还就存在的问题与前景进行探讨.     

苏云金芽孢杆菌杀虫剂的研究进展

综述了苏云金芽孢杆菌的毒性作用,它的杀虫晶体蛋白基因及基因产物的特性,将杀虫晶体蛋白基因转移到植物上获得抗虫植物以及如何防止昆虫对该杀虫剂产生抗性的问题.     

苏云金杆菌Ⅰ型抗癌晶体蛋白的分子模建

通过同源建模,得到Ⅰ型抗癌晶体蛋白Parasporin-1的初始三维结构经分子动力学方法优化后,利用Ramachandran Plot检测和结构匹配等方法对模型进行评价.结果显示,结构模型中的键长、键角及二面角的分布合理,与模板蛋白的主链α碳原子的均方根差值为0.537 592, 在合理范围之内.此外,通过分析Parasporin-1结构域Ⅰ中的β-loop-β结构片段,推测出第2个蛋白酶处理激活位点的位置在两个芳香族疏水性氨基酸F167与Y168之间.     

黑带蚜蝇触角感器扫描电镜观察

触角是昆虫进行觅食、求偶和躲避天敌等行为的重要感觉器官,具有嗅觉和触觉功能.基于扫描电子显微镜对不同性别黑带蚜蝇(Episyrphus balteatus De Geer, 1776)成虫的触角及触角感器进行了观察拍照,分析了触角感器的种类、数量、形态结构以及分布特点.主要研究结果如下：1)黑带蚜蝇的触角由柄节、梗节、鞭节3部分组成;2)触角感器包括8类13种,毛形感器(sensilla trichodea, ST)具3种亚型(STⅠ,STⅡ,STⅢ)、锥形感器(sensilla basiconica, SB)具两种亚型(SBⅠ,SBⅡ)、刺形感器(sensilla chaetica, SC)具两种亚型(SCⅠ,SCⅡ)、腔锥形感器(sensilla coeloclnica, CO)具两种亚型(COⅠ,COⅡ)、节间感器(sensilla intersegment, SIn)、棒状感器(sensilla clavate, Cl)、柱形感器(sensilla cylindrical, Cy)和B9hm氏鬃毛(b9hm bristle, BB);3)不同性别黑带蚜蝇成虫触角感器存在明显差异...     

二化螟的水稻种群和茭白种群对杀虫剂和Cry蛋白的敏感性比较

采用微量点滴法测定了二化螟的水稻和茭白种群幼虫对杀虫剂辛硫磷、毒死蜱、氯虫苯甲酰胺、杀虫单和阿维菌素的敏感性,采用人工饲料胃毒法测定了二化螟对Cry蛋白(Cry1Ac、Cry2Aa、Cry1Ca)的敏感性,目的是为二化螟科学防治和抗性治理提供理论基础。结果表明:二化螟两寄主种群对阿维菌素和氯虫苯甲酰胺的敏感性差异显著,但对其他3种药剂及Cry蛋白的敏感性差异不显著。水稻种群和茭白种群对5种杀虫剂的敏感性由高到低均为阿维菌素>氯虫苯甲酰胺>辛硫磷>毒死蜱>杀虫单;对3种Cry蛋白的敏感性由高到低为Cry1Ca>Cry1Ac>Cry2Aa。因此,针对不同寄主田的二化螟种群应采用不同类型的药剂,尽量不使用单一药剂,可考虑采用药效最好的阿维菌素与其他几种药剂进行混配施用。     

Transgenic tobacco plants expressing synthetic Cry1Ac and Cry1Ie genes are more toxic to cotton bollworm than those containing one gene

Transgenic tobacco plants carrying Cry1Ac, Cry1Ie or both genes were obtained. In the leaves of transgenic plants carrying both genes, the contents of Cry1Ac and Cry1Ie proteins were 0.173% and 0.131% of the total proteins, respectively. Cry1Ac protein content was 0.182% and Cry1Ie protein con- tent was 0.124% of the total proteins in the leaves of transgenic plants containing only one Bt gene. Fresh leaves of transgenic tobacco and wild-type plants were used for the insect bioassay against wild-type and Cry1Ac-resistant cotton bollworm (Helicoverpa armigera). The bioassay results showed that transgenic plants carrying both genes were significantly more toxic to wild-type and Cry1Ac-resistant cotton bollworm than those carrying Cry1Ac or Cry1Ie alone. This study indicates that the higher toxicity of transgenic tobacco plants carrying both genes is caused by the cooperative function of both Bt proteins, thus providing a potential way to delay the development of insect resis- tance to transgenic crops.     

The role of β18-β19 loop structure in insecticidal activity of Cry1Ac toxin from Bacillus thuringiensis

The β18-β19 loop in domain Ⅲ of Cry1Ac toxin is unique among Bacillus thuringlensis Cry proteins. In this study, the role of the loop structure in insecticidal activity of Cry1Ac toxin was investigated. Alanine scanning mutations within the loop were initially generated and most mutants were over-expressed and reduced toxicity at different degrees, except mutant N546A that showed almost 2 times enhanced toxicity against Helicoverpa armigera larvae. Further mutagenic analysis of N546 revealed that a charged amino acid in this position would cause very unfavorable influence on insecticidal activity. In addition, the deletion of N546 led to protein instability because of destruction of the loop integrity. Besides, mutant W544F was much more toxic than W544Y, indicating that hydrophobic nature of the position was important for maintaining the stability and activity of Cry1Ac protein. These findings are the first biological evidence for a structural function of β18-β19 loop in insecticidal activity of the Cry1Ac toxin.     

Transgenic tobacco plants expressing synthetic Cry1Ac and Cry1Ie genes are more toxic to cotton bollworm than those containing one gene

Transgenic tobacco plants carrying CrylAc, Crylle or both genes were obtained. In the leaves of transgenic plants carrying both genes, the contents of CrylAc and Crylle proteins were 0.173% and 0.131% of the total proteins, respectively. CrylAc protein content was 0.182 % and Cry1 le protein content was 0.124% of the total proteins in the leaves of transgenic plants containing only one Bt gene. Fresh leaves of transgenic tobacco and wild-type plants were used for the insect bioassay against wild-type and Cry1Ac-resistant cotton bollworm （Helicoverpa armigera）. The bioassay results showed that transgenic plants carrying both genes were significantly more toxic to wild-type and CrylAc-resistant cotton bollworm than those carrying CrylAc or Crylle alone. This study indicates that the higher toxicity of transgenic tobacco plants carrying both genes is caused by the cooperative function of both Bt proteins, thus providing a potential way to delay the development of insect resistance to transgenic crops.     

Agrobacterium tumefaciens-mediated Transformation of CryⅠA(b) gene to Trichoderma harzianum

In this study, Cry ⅠA(b) gene was successfully transferred into the biocontrol fungus Trichoderma harzianum with an efficiency of 60-180 transformants per 10^6 spores by using Agrobacterium tumefaciens-mediated transformation. Putative transformants were analyzed to test the presence of Cry ⅠA(b) gene by Southern blot. Most transformants contained a single T-DNA copy. RT-PCR analysis showed that the Cry ⅠA(b) gene was transcribed. Antifungal activities and insecticidal activities of the transformants were examined. There was no obvious difference in antifungal activities between the transformants and their wild strains. The modified mortalities of the transformants T1 and T2 were 69.57% and 91.30%, respectively. The tranformation system mediated by A. tumefaciens proved to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability of transformants.     

Construction of a new plant expression vector containing two insect resistant genes and its expression in transgenic tobacco plants

A new plant expression vector (pBS29K-BA) containing two insect resistant genes, a synthetic chimeric gene BtS29K encoding the activated insecticidal protein Cry1Ac and a gene API-BA encoding the arrowhead (Sagittaria sagittifolia L.) proteinase inhibitor (API) A and B, is constructed. Transgenic tobacco plants expressing these two genes are obtained through Agrobacterium-mediated transformation of tobacco leaf discs. The average expression levels of Cry1Ac and API-BA proteins in transgenic plants are of 3.2 μg and 4.9 μg per gram fresh leaf respectively. The results of insecticidal assay of transgenic plants indicate that the pBS29K-BA transformed plants are more resistant to insect damage than the plants expressing the Cry1Ac gene or API-BA gene alone.     

妇科腹腔镜术后抗感染341例临床分析

目的分析腹腔镜术后抗感染情况,探讨术后感染的有效防治措施.方法选取腹腔镜手术患者341例作为研究对象,腹腔镜子宫全切和次全切手术(A组)172例(污染类手术);腹腔镜下卵巢囊肿剥出术(B组)169例(清洁类手术),再依照术后预防性使用抗菌药物1,3,7 d和未使用抗菌药物分为Ⅰ~Ⅳ4个亚组,对比患者手术时间、术后退热时间、抗菌药物使用时间和术后感染发生率情况.结果 A组和B组手术时间、术后体温和肠功能恢复时间各亚组间差异均无统计学意义(P0.05);A组中使用抗菌药物的AⅠ~AⅢ3个亚组中AⅠ组抗菌药物平均使用时间明显短于AⅡ,AⅢ组,差异具有显著统计学意义(P0.01);AⅣ亚组术后退热时间明显长于AⅠ~AⅢ亚组,术后感染率明显高于AⅠ~AⅢ亚组,差异均具有统计学意义(P0.05);BⅠ~BⅣ4个亚组之间手术时间差异无统计学意义(P0.05);BⅣ亚组术后退热时间和术后感染率与BⅠ~BⅢ亚组相比差异均无统计学意义(P0.05).结论妇科腹腔镜手术中,对于清洁类手术患者术后不必给予抗菌药物,污染类手术延长术后抗菌药物给药时间与感染发生率之间无明显相关性,应根据患者实际情况尽量缩短给药时间.     

Banach空间的一些新的几何性质

对Ｂａｎａｃｈ空间引起了几种新的几何性质,它们是几何性质（Ｃ－Ｋ）的时偶性质。此外,我们得到了关于这些新的几何性质的一些重要而有用的结果。     

利多卡因和丙泊酚复合静脉麻醉在腹腔镜子宫切除术中的应用

目的通过综合评估不同剂量的丙泊酚和利多卡因复合应用于腹腔镜子宫切除术麻醉的可行性,并摸索适合的临床用药剂量。方法将60例择期行腹腔镜子宫切除手术患者,随机分为3组,给予不同剂量的丙泊酚和利多卡因维持麻醉。连续监测脑电双频指数（BIS）,血流动力学参数,苏醒情况。结果各组患者诱导后BIS值均明显降低,Ⅱ、Ⅲ组术中患者BIS值明显低于Ⅰ组。血液动力学参数变化Ⅰ组他时点心率（HR）低于阳时点,差异有统计学意义（P〈0．05）Ⅰ,Ⅱ,Ⅲ组T1、T2、T3、T4、T5、T6时点无刨平均动脉压（MAP）低于T0（P均〈0．05）。组间比较：Ⅱ组T2、T4时点MAP分别低于Ⅰ组相应时点,差异有统计学意义（P均〈0．05）。Ⅲ组患者自主呼吸恢复时间和唤之睁眼时间与Ⅰ、Ⅱ组相比明显增加,差异有统计学意义（P〈0．05）。Ⅰ组中有1例发生术中知晓,而Ⅲ、Ⅱ组无此情况。各组拔管时间、定向力恢复时间比较差异无统计学意义。结论丙泊酚和利多卡因复合静脉麻醉在腹腔镜子宫切除手术中应用具有可行性,推荐剂量为利多卡因1mg／（kg·h）加丙泊酚3mg／（kg·h）。     

杀鼠药毒性成份的GC/MS分析

本文利用毛细管色谱柱的ＧＣ／ＭＳ法，鉴定了１３种杀鼠药的有效成份，其中８种含有毒鼠强，２种含有氟乙酸，１种含有氟乙酰胺，１种含有氟乙酸和氟乙酰胺。结果经对照样品验证而进一步证实。