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我国斯里兰卡木薯花叶病毒的分子特征及致病性分析
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作者 王国芬 李超萍 +5 位作者 时涛 吴会杰 蔡吉苗 李博勋 陆翠梅 黄贵修 《热带作物学报》 CSCD 北大核心 2024年第1期187-196,共10页
为进一步探究我国斯里兰卡木薯花叶病毒(Sri Lankan cassava mosaic virus,SLCMV)的分子特征及致病性。以感染SLCMV的木薯叶片基因组DNA为模板,PCR扩增获得DNA-A和DNA-B基因组全长,通过生物信息软件分析比较其核酸及氨基酸序列特征;构... 为进一步探究我国斯里兰卡木薯花叶病毒(Sri Lankan cassava mosaic virus,SLCMV)的分子特征及致病性。以感染SLCMV的木薯叶片基因组DNA为模板,PCR扩增获得DNA-A和DNA-B基因组全长,通过生物信息软件分析比较其核酸及氨基酸序列特征;构建了强、弱致病力分离物(SLCMV-Colombo和SLCMV-DG1922)的侵染性克隆,分别将2种致病力分离物的DNA-A和DNA-B组分进行重组,接种烟草(Nicotianatabacum),比较2种分离物的致病性差异。结果显示:我国SLCMV为“旧世界”双组份菜豆花叶病毒,编码包括AV2基因在内的8个开放阅读框(ORFs);具有双生病毒典型的共同序列(CR)、重复序列、TATABox和TAATATT↓AC茎环结构,Rep蛋白羧基末端有7个氨基酸缺失;我国SLCMV的基因组、编码和非编码区与柬埔寨、泰国和越南分离物的相似性在97.0%~100.0%之间,与印度和斯里兰卡早期的分离物相似性为86.5%~98.6%,DNA-B组份与印度木薯花叶病毒株系(ICMV)编码区序列相似性为95.0%~97.6%,与其他9个非洲木薯花叶病毒株系序列之间的序列相似性均低于80.0%。侵染性克隆接种结果证实,强致病力分离物SLCMV-Col的DNA-A(Col-A)组份在烟草叶片表现典型花叶症状中起主要作用,而我国分离物的DNA-B(DG1922-B)能协同Col-A对烟草产生强致病性。本研究分析了我国SLCMV的全基因组结构,建立的侵染性克隆及其技术为进一步解析SLCMV致病性机理提供重要的研究基础。 展开更多
关键词 斯里兰卡木薯花叶病毒 分子特征 侵染性克隆 致病性
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Molecular Cloning and Expression of RSSG58 Gene in Rice Sperm Cells 被引量:3
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作者 苗琛 苟小平 +4 位作者 兰利琼 鲍锦库 徐莺 王胜华 陈放 《Acta Botanica Sinica》 CSCD 2003年第2期234-241,共8页
Myosins, a large family of structurally diverse mechanoenzymes, which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force, play an important role in male reproductive proce... Myosins, a large family of structurally diverse mechanoenzymes, which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force, play an important role in male reproductive processes. In this study we report the rice ( Oryza sativa L.) RSSG58 gene, which was cloned from the cDNA library of rice sperm cells by using sperm cell mainly expression subtractive clone as probe. This gene encodes a putative 66.7 W polypeptide, which shows similarity to the myosin heavy chain of Arabidopsis thaliana, and consists of 579 amino acids with an isoelectric point (pI) of 4.885. RSSG58, which is a member of a divergent gene family, generates transcripts of 2 278 bp and 2 437 bp that differ only in their polyadenylation sites. Southern hybridization showed that RSSG58 has only one copy in rice genome and RSSG58 transcripts are most abundant in sperm cells, with two distinct signals. The RT-PCR analysis indicated that the transcriptions of the RSSG58 gene were various in the different development stages and tissues. The greatest accumulation of RSSG58 mRNA was detected in sperm cells, while weaker expression was detected in leaves, microspore mother cells, unicellular microspore pollen stage, two-cell stage pollens, mature pollens and pollinated ovaries. These results suggest that RSSG58 is especially abundantly expressed in rice sperm cells. 展开更多
关键词 molecular cloning RSSG58 gene sperm cell EXPRESSION RICE
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莴笋红叶基因图位克隆及分子标记开发
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作者 李春 何振 +7 位作者 刘小俊 梁根云 李艺凡 杨楠 蔡鹏 李跃建 房超 刘独臣 《四川农业大学学报》 CSCD 北大核心 2024年第3期523-528,共6页
【目的】研究莴笋叶色变异的遗传基础,加速莴笋新品种育种进程,提高育种效率。【方法】通过构建红叶和绿叶莴笋杂交群体,分析遗传规律,利用混池分组分析法(BSA-seq)和图位克隆法精细定位并克隆了莴笋红叶基因Lactuca sativa Red Leaf 1(... 【目的】研究莴笋叶色变异的遗传基础,加速莴笋新品种育种进程,提高育种效率。【方法】通过构建红叶和绿叶莴笋杂交群体,分析遗传规律,利用混池分组分析法(BSA-seq)和图位克隆法精细定位并克隆了莴笋红叶基因Lactuca sativa Red Leaf 1(LsRL1),根据变异位点设计了分子标记用于红叶莴笋分子标记辅助育种。【结果】遗传分析结果显示F2分离群体中红叶个体与绿叶个体的分离比为3∶1,说明研究中莴笋叶色表型受一个基因控制,且红叶相对于绿叶为显性性状。混池分组分析法(BSA-seq)将莴笋叶色基因LsRL1初步定位在莴笋第5染色体336.00 Mb到339.64 Mb的范围内。利用图位克隆的方法进一步将LsRL1基因的范围缩小至2个Indel分子标记ls06和ls12之间的85.17 kb区间内,这一区间内包含2个基因LOC111892298和LOC111892911。亲本间差异位点分析将LsRL1的候选基因确定为LOC111892911,该基因在拟南芥中的同源基因编码一个bHLH转录因子,参与花青素合成途径的调控。【结论】基于LsRL1基因在2个亲本间的插入缺失变异,我们开发了一个可用于红叶莴笋分子标记辅助育种的Indel标记,以加速红叶莴笋的育种进程。 展开更多
关键词 莴笋 红叶 图位克隆 分子标记
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西方蜜蜂AmAGO1蛋白的分子特性、时空表达谱及抗体制备
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作者 叶亚萍 刘治滩 +7 位作者 李琪明 臧贺 冯佩林 王宁 王杰 黄枳腱 陈大福 郭睿 《昆虫学报》 CAS CSCD 北大核心 2024年第1期18-28,共11页
【目的】Argonaute(AGO)家族是一类在进化上高度保守的蛋白家族,其在真核生物中主要参与形成RNA诱导沉默复合体(RNA-induced silencing complex,RISC),进而通过沉默基因表达参与诸多生物学过程。蜜蜂AGO蛋白的相关研究迄今仍然缺失。本... 【目的】Argonaute(AGO)家族是一类在进化上高度保守的蛋白家族,其在真核生物中主要参与形成RNA诱导沉默复合体(RNA-induced silencing complex,RISC),进而通过沉默基因表达参与诸多生物学过程。蜜蜂AGO蛋白的相关研究迄今仍然缺失。本研究拟通过预测西方蜜蜂Apis mellifera的AGO1(AmAGO1)理化性质和分子特性,解析AmAGO1在西方蜜蜂中的时空表达谱,并制备AmAGO1的多克隆抗体,为深入开展AmAGO1的功能与机制研究提供参考和基础。【方法】PCR扩增西方蜜蜂AmAGO1的编码序列(coding sequence,CDS);通过生物信息学预测AmAGO1蛋白的理化性质和分子特性;利用RT-qPCR检测AmAGO1在西方蜜蜂工蜂卵、3日龄幼虫、7日龄预蛹、8日龄预蛹、12日龄蛹以及1,2,6,12,15和18日龄成虫以及工蜂成虫触角、咽下腺、脑、表皮、中肠、脂肪体和毒腺中的表达量;构建原核表达质粒后诱导表达AmAGO1融合蛋白,并鉴定其表达形式;制备AmAGO1多克隆抗体,利用ELISA,Western blot和免疫沉淀(immunoprecipitation,IP)分别检测抗体效价、灵敏度和特异性。【结果】成功克隆到西方蜜蜂AmAGO1的CDS;AmAGO1含928个氨基酸,分子式为C4624H7332N1316O1325S51,分子量约为104.2 kD,等电点为9.31,平均亲水系数为-0.2965,含86个磷酸化位点及典型的PAZ结构域和PIWI结构域,但不存在典型的信号肽;AGO1在智人Homo sapiens、斑马鱼Danio rerio、黑腹果蝇Drosophila melanogaster、家蚕Bombyx mori、西方蜜蜂、东方蜜蜂A.cerana和欧洲熊蜂Bombus terrestris中具有较高的氨基酸序列一致性;西方蜜蜂和东方蜜蜂的AGO1聚为一支,同源性最高。AmAGO1在西方蜜蜂工蜂卵、幼虫、预蛹、蛹和成虫中均有表达但表达量存在差异,3日龄幼虫和7日龄预蛹中AmAGO1的表达量显著低于卵中的表达量,而8日龄预蛹和12日龄蛹中AmAGO1的表达量显著高于卵中的表达量;AmAGO1在1,2,6,12,15和18日龄工蜂体内均有表达但表达量也存在差异,其中1日龄成虫体内AmAGO1的表达量显著高于其他日龄成虫体内的表达量;AmAGO1在工蜂成虫触角、毒腺、脑、中肠、咽下腺、脂肪体和表皮中均有表达但表达量存在差异,触角中AmAGO1的表达量与咽下腺中的相差无几,但二者均显著高于毒腺、脑、中肠、脂肪体和表皮中AmAGO1的表达量;AmAGO1融合蛋白的表达形式为包涵体;制备的AmAGO1多克隆抗体具有较高的效价、灵敏度和特异性。【结论】AmAGO1可能是亲水性胞内蛋白,含有典型的PAZ结构域和PIWI结构域;AmAGO1在西方蜜蜂工蜂不同组织和不同发育阶段发挥潜在的重要功能;成功制备出高效价、高灵敏度和强特异性的AGO1多克隆抗体。 展开更多
关键词 西方蜜蜂 AmAGO1 克隆 分子特性 时空表达谱 原核表达 多克隆抗体
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大黄鱼FcRs基因的克隆及其在免疫应答中的初步功能分析
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作者 杨玉生 程安怡 +2 位作者 张亚萌 崔正伟 陈新华 《应用海洋学学报》 CAS CSCD 北大核心 2024年第1期160-170,共11页
Fc受体(Fc Receptors, FcRs)是一类与免疫球蛋白的Fc段特异性结合,介导多种免疫反应的受体分子。本研究克隆得到了大黄鱼(Larimichthys crocea)6个FcRs基因,分别是LcFcγRⅠL、LcFcγRⅡ、LcFcγRⅡaL、LcFcγRⅢ、LcFcRL5和LcFcRLA,其... Fc受体(Fc Receptors, FcRs)是一类与免疫球蛋白的Fc段特异性结合,介导多种免疫反应的受体分子。本研究克隆得到了大黄鱼(Larimichthys crocea)6个FcRs基因,分别是LcFcγRⅠL、LcFcγRⅡ、LcFcγRⅡaL、LcFcγRⅢ、LcFcRL5和LcFcRLA,其开放阅读框(ORF)大小分别为1 070、2 486、1 145、 1 955、4 125、1 659 bp。大黄鱼的6个LcFcRs都具有一个信号肽和2~8个免疫球蛋白(Ig)结构域,LcFcγRⅡ、LcFcγRⅢ和LcFcRL5还包含一个跨膜区。实时荧光定量PCR结果显示LcFcγRⅠL、LcFcγRⅡ、LcFcγRⅡaL和LcFcγRⅢ主要在鳃或肠等黏膜组织中表达,而LcFcRL5和LcFcRLA主要在脾和头肾等系统免疫组织中表达;革兰氏阴性细菌细胞壁脂多糖(LPS)刺激后,这6个LcFcRs基因头肾和脾脏中的表达在不同时间出现了显著上调;双链RNA病毒类似物Poly(I:C)刺激后,6个LcFcRs在头肾和脾脏中的表达出现不同程度的上调,而LcFcRL5在脾脏中的表达显著上调,在头肾中则明显下调,提示大黄鱼FcRs可能在LPS和Poly(I:C)诱导的免疫反应中起着重要但又不同的作用。 展开更多
关键词 海洋生物学 大黄鱼 FcRs 免疫球蛋白 分子克隆
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水稻低温发芽力的研究进展与展望
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作者 周旭 杨梯丰 +1 位作者 刘祖培 周炼 《中国稻米》 北大核心 2024年第5期41-48,共8页
低温萌发力是水稻适应极端天气、特别是水稻直播生产不可或缺的一个重要性状。本文综述了近年来关于水稻低温萌发力的鉴定方法、生理机制、分子克隆和遗传育种等方面的研究进展,提出了研究趋势与展望。在水稻低温萌发力鉴定方面,多采用1... 低温萌发力是水稻适应极端天气、特别是水稻直播生产不可或缺的一个重要性状。本文综述了近年来关于水稻低温萌发力的鉴定方法、生理机制、分子克隆和遗传育种等方面的研究进展,提出了研究趋势与展望。在水稻低温萌发力鉴定方面,多采用13℃~15℃试验温度、低温萌发第10 d的萌发率为评价指标。利用图位克隆和全基因组关联分析(GWAS)等技术手段已鉴定的水稻低温萌发力相关QTL超过100个,但实现目的基因克隆的仅4个,关于分子机制方面的研究极少。当前水稻低温萌发力强的品种主要是从育成品种中筛选而来。水稻低温萌发生理机理和分子机理的研究将有助于提高低温萌发鉴定及其育种应用。 展开更多
关键词 水稻 低温萌发 鉴定方法 分子克隆 遗传育种
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黄颡鱼硒蛋白基因的克隆与分析
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作者 刘光辉 余岸艮 +3 位作者 何杨 郭雨诗 柯江 罗智 《华中农业大学学报》 CAS CSCD 北大核心 2024年第1期185-193,共9页
为探讨黄颡鱼硒蛋白selenow2a、selenop2和selenot2基因之间的关系,采用3′/5′RACE PCR克隆得到3个基因的cDNA全长,分别为891、1998和1432bp,其中ORF长度分别为288、828和600bp,编码95、275和219个氨基酸。在线工具SECISerach3对3个基... 为探讨黄颡鱼硒蛋白selenow2a、selenop2和selenot2基因之间的关系,采用3′/5′RACE PCR克隆得到3个基因的cDNA全长,分别为891、1998和1432bp,其中ORF长度分别为288、828和600bp,编码95、275和219个氨基酸。在线工具SECISerach3对3个基因的cDNA序列分析结果显示,它们都含有可以编码硒代半胱氨酸的终止密码子,以及在3′非编码区存在SECIS元件。通过氨基酸序列比对和系统发育树分析,发现sele-now2a、selenop2和selenot2基因预测得到的氨基酸序列与斑马鱼(Danio rerio)氨基酸相似性分别为82.24%、66.19%和79.45%,而与斑点叉尾鮰(Ictalurus punetaus)的氨基酸相似性分别为94.74%、68.50%和90.95%,在发育树上则显示为树杈相接近。采用实时荧光定量PCR检测3个硒蛋白基因的mRNA在黄颡鱼心脏、肝脏、肌肉、脑、肠、脾脏、精巢和卵巢组织中的表达,结果显示其mRNA表达水平呈现组织特异性。表明3个基因拥有硒蛋白家族的特征,但在组织表达上具有特异性。 展开更多
关键词 硒蛋白 基因克隆 分子特征 黄颡鱼 组织表达
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天麻PCR‑层析试纸条快速可视化检测方法的建立与评价
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作者 马秋贺 马玉贺 +9 位作者 刘悦 李涛 刘昂 徐子强 柴金军 王艳茹 高丽君 夏薇 李明成 曲永梅 《上海中医药杂志》 CSCD 2024年第3期79-85,共7页
目的通过聚合酶链式反应(PCR)与层析试纸条结合的方法,实现天麻快速可视化真伪鉴别,并对其检测效果进行评价。方法采用一步法提取天麻及其伪品基因组DNA,应用NCBI数据库设计天麻特异性引物。采用DNA分子克隆技术制备天麻阳性质粒,作为... 目的通过聚合酶链式反应(PCR)与层析试纸条结合的方法,实现天麻快速可视化真伪鉴别,并对其检测效果进行评价。方法采用一步法提取天麻及其伪品基因组DNA,应用NCBI数据库设计天麻特异性引物。采用DNA分子克隆技术制备天麻阳性质粒,作为天麻阳性对照品。建立PCR-层析试纸条法鉴定天麻真伪,摸索最优实验条件,并进行方法学评价。结果①样品DNA提取的纯度符合要求,最优的PCR反应引物浓度为1μmol/L,循环为29次。②分子克隆的天麻阳性质粒序列与天麻DNA分子标记特异性指纹区片段序列同源性为98%,可作为天麻PCR-层析试纸条法的阳性对照品。③PCR-层析试纸条法的方法学评价结果显示:天麻对照药材在试纸条上出现两个条带,伪品和阴性对照品出现1个条带,与琼脂糖凝胶电泳法结果一致,特异性良好;PCR-层析试纸条法较琼脂糖凝胶电泳法的灵敏度高100倍,天麻DNA浓度为10-1 mg/L时,试纸条仍有模糊条带;在第3、6、9、12个月采用PCR-层析试纸条法进行检测,检测结果与预期一致,稳定性良好;混合样品验证显示,PCR-层析试纸条法的最低检测限为10%,而琼脂糖凝胶电泳法的最低检测限为50%。④采用PCR-层析试纸条法对15份市售天麻样品进行检测,鉴别出3个伪品,与琼脂糖凝胶电泳法结果一致。结论所构建的PCR-层析试纸条检测方法特异性强、灵敏度高、操作简便快速,可在短时间内实现鉴定结果的可视化,检测结果准确、稳定,为道地药材天麻的真伪鉴别提供了新方法。 展开更多
关键词 天麻 聚合酶链式反应 试纸条 分子克隆 可视化鉴定 中药研究
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泸宁鸡MYOZ2基因的分子特征及其在不同日龄腿肌中的表达
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作者 王孜 陈楚雯 +3 位作者 侯绍云 陈兆贤 张开武 李志雄 《西南民族大学学报(自然科学版)》 CAS 2024年第3期276-282,共7页
旨在研究雄性泸宁鸡MYOZ2基因的分子特性及其在不同组织的表达谱和不同日龄腿肌中的表达水平,探讨该基因对泸宁鸡生长发育的影响.通过RT⁃PCR技术获得MYOZ2基因的CDS序列并进行生物信息学分析,利用qPCR技术检测MYOZ2基因在泸宁鸡的心、... 旨在研究雄性泸宁鸡MYOZ2基因的分子特性及其在不同组织的表达谱和不同日龄腿肌中的表达水平,探讨该基因对泸宁鸡生长发育的影响.通过RT⁃PCR技术获得MYOZ2基因的CDS序列并进行生物信息学分析,利用qPCR技术检测MYOZ2基因在泸宁鸡的心、肝、脾、肺、肾、胸肌与腿肌组织中的表达差异情况,以及在不同日龄腿肌中的表达差异情况.结果表明,泸宁鸡MYOZ2基因开放阅读框(ORF)长度为792 bp,可以编码263个氨基酸;泸宁鸡MYOZ2蛋白质主要存在于细胞核内,其二级结构中无规卷曲所占比例最高;与其他物种如原鸡、火鸡、珍珠鸡、蜂鸟、雨燕、麻雀、中国仓鼠、牛、羊、猪及人的核苷酸序列进行对比,与原鸡同源性最高,为100%;与人、中国仓鼠、牛、羊、猪等物种的亲缘性较低,说明MYOZ2基因在同种属间进化较为保守;经组织表达谱的分析,MYOZ2在心脏中的表达量显著高于除脾以外的组织(P<0.05);在20、36、51、94 d日龄腿肌组织中都有表达,其中MYOZ2基因在20 d中的表达量最高,显著高于51 d和94 d日龄腿肌(P<0.05).此研究为进一步了解MYOZ2基因在泸宁鸡公鸡生长发育等生理功能调控中的作用提供理论依据. 展开更多
关键词 泸宁鸡 MYOZ2 克隆 分子特征 表达
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贵州白山羊肌细胞生成素基因克隆与生物信息学分析
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作者 宋兴超 孟金柱 +2 位作者 赵园园 吴震洋 安清明 《中国畜牧兽医》 CAS CSCD 北大核心 2024年第3期926-935,共10页
【目的】克隆贵州白山羊肌细胞生成素(myogenin)基因并对其结构特性进行生物信息学分析,为进一步探索该基因调控山羊肌肉发育的分子机制提供重要参考依据。【方法】以贵州白山羊血液基因组DNA为模板,设计4对特异性引物,运用PCR扩增、San... 【目的】克隆贵州白山羊肌细胞生成素(myogenin)基因并对其结构特性进行生物信息学分析,为进一步探索该基因调控山羊肌肉发育的分子机制提供重要参考依据。【方法】以贵州白山羊血液基因组DNA为模板,设计4对特异性引物,运用PCR扩增、Sanger直接测序及序列拼接方法获得贵州白山羊myogenin基因完整编码区核苷酸序列,应用BioEdit 7.0、NetPhos 3.1、Conserved Domains Search、SMART、Motif Scan等生物信息学软件分别分析该基因核苷酸与氨基酸序列的组成、编码蛋白磷酸化位点、关键结构域和功能模体等结构特性,通过DNAStar Lasergene、DNAMAN 8.0和Mega 5.0软件分别进行15个偶蹄目物种myogenin基因编码氨基酸序列相似性比对及系统进化树构建。【结果】贵州白山羊myogenin基因序列全长2424 bp(获得GenBank登录号:HZ53289),由3个外显子、2个内含子、部分5′-UTR和3′-UTR构成,长度分别为471、82、122、765、589、135和260 bp,编码区长度为675 bp,共编码224个氨基酸。贵州白山羊myogenin蛋白第1—86位氨基酸为生肌决定因子(myogenic determining factors,MyoD)家族特有碱性结构域(Basic domain),第81—139位氨基酸为螺旋-环-螺旋(helix-loop-helix,H-L-H)结构域,第160—170位氨基酸为低复杂度结构域,存在21个磷酸化位点和核定位信号蛋白等功能模体结构。氨基酸序列比对表明,6个半胱氨酸残基、1个核定位信号和1个低复杂度结构域在15个偶蹄目物种完全保守。myogenin基因编码氨基酸序列相似性与系统进化分析均显示,贵州白山羊与波尔山羊、湖羊和马可波罗盘羊的亲缘关系最近。【结论】本研究成功克隆得到贵州白山羊myogenin基因全长序列,大小为2424 bp,编码224个氨基酸,myogenin蛋白包含Basic domain和H-L-H结构域。该结果可为myogenin基因调控山羊肌肉发育的分子机制研究提供理论参考。 展开更多
关键词 贵州白山羊 myogenin基因 克隆 分子系统进化 生物信息学
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Molecular Cloning and Sequence Analysis ofBoPGIP2 Gene from Brassica oleracea L. var. alboglabra
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作者 张弢 《Agricultural Science & Technology》 CAS 2009年第4期91-95,104,共6页
PGIP gene was obtained from Brassica oleracea L. var. alboglabra, named BoPGIP2. The full length of BoPGIP2 gene is 1 102 bp and the exon is 993 bp which encodes a protein of 330 amino acids with a predicted molecular... PGIP gene was obtained from Brassica oleracea L. var. alboglabra, named BoPGIP2. The full length of BoPGIP2 gene is 1 102 bp and the exon is 993 bp which encodes a protein of 330 amino acids with a predicted molecular mass of 37.1 kDa, interrupted by one intron of 95 bp in, length. Sequence analysis revealed that it has five potential N-giycosylation sites, two protein kinase C phosphrylation sites, five casin kinase Ⅱ phosphrylation sites and four N-myristoylation sites. The amino acids sequences alignment confirmed that ^145 LRR stucture was highly conserved in all aligned PGIP sequences. 展开更多
关键词 Brassica oleracea L. vat. alboglabra BoPGIP2 molecular cloning Sequence analysis
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Molecular Characterization, Expression Patterns and Binding Properties of Two Pheromone-Binding Proteins from the Oriental Fruit Moth, Grapholita molesta(Busck) 被引量:10
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作者 SONG Yue-qin DONG Jun-feng +1 位作者 QIAO Hui-li WU Jun-xiang 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2014年第12期2709-2720,共12页
Insect pheromone-binding proteins (PBPs) play important roles in transporting hydrophobic pheromone components across the sensillum lymph to the surface of olfactory receptors (ORs). However, the PBPs of the orien... Insect pheromone-binding proteins (PBPs) play important roles in transporting hydrophobic pheromone components across the sensillum lymph to the surface of olfactory receptors (ORs). However, the PBPs of the oriental fruit moth, Grapholita molesta, an important destructive pest of stone fruits worldwide, are not well characterized. In this study, two new putative PBP genes, GmolPBP2 and GmolPBP3, were identiifed from G. molesta antennae. The deduced amino-acid sequences of these two putative PBP genes are characteristic of the odorant binding protein family, containing six conserved cysteine residues. The genomic DNA sequence of each gene contained two introns. However, the lengths and positions of the introns differed. RT-PCR analyses revealed that the two GmolPBP genes are only expressed in the antennae of female and male moths. Quantitative real-time PCR indicated that the transcription levels of GmolPBP2 are far greater than those of GmolPBP3 in both female and male antennae. GmolPBP3 showed higher transcription levels in female antennae than in male antennae, while GmolPBP2 showed similar transcription levels in both female and male antennae. The transcript levels of both genes were signiifcantly different in premating and post-coitum individuals, implying that mating affects the process of sex pheromone reception. To better understand the functions, two GmolPBPs were expressed in Escherichia coli, and the ligand binding assays were conducted. Results showed that GmolPBP2 has strong binding afifnities to two sex pheromone components, E8-12:Ac and Z8-12:Ac, as well as weaker binding afifnities to Z8-12:OH and 12:OH. GmolPBP2 also bound some ordinary odor molecules. However, the afifnity of GmolPBP3 to both sex pheromones and ordinary odor molecules was very weak. These results show that GmolPBP2 plays the main role in pheromone discrimination and recognition in the oriental fruit moth. 展开更多
关键词 Grapholita molesta pheromone-binding proteins molecular cloning mRNA expression prokaryotic expression lfuorescence competitive binding assays
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Molecular cloning and expression analysis of Crustin-like gene from Chinese shrimp Fenneropenaeus chinensis 被引量:2
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作者 LIU Fengsong LI Fuhua +4 位作者 XIANG Jianhai DONG Bo LIU Yichen ZHANG Xiaojun ZHANG Liusuo 《Acta Oceanologica Sinica》 SCIE CAS CSCD 2008年第2期81-92,共12页
A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fennero- penaeus chinensis by 3 ′and 5′ RACE. The full-length cDNA of Crustin-like gene contains a 3... A new member of antimicrobial protein genes of the Crustin family was cloned from haemocytes of the Chinese shrimp Fennero- penaeus chinensis by 3 ′and 5′ RACE. The full-length cDNA of Crustin-like gene contains a 390 bp open reading frame, encoding 130 amino acids. The deduced peptide contains a putative signal peptide of 17 amino acids and mature peptide of 113 amino acids. The molecular mass of the deduced mature peptide is 12. 3 ku. It is highly cationic with a theoretical isoelectric point of 8.5. The deduced amino acids sequence of this Crustin showed high homology with those of Penaeus ( Litopenaeus ) setferus. Northern blotting showed that the cloned Crustin gene was mainly expressed in haemocytes, gill, intestine, and RNA in situ hybridization indicated that the Crustin gene was constitutively expressed exclusively in haemocytes of these tissues. Capillary elec- trophoresis RT-PCR analysis showed that Crustin was up-regulated dramatically from 12 to 48 h after a brief decrease of mRNA during first 6 h in response to microbe infection. The level of Crustin mRNA began to restore at 72 h post-challenge. This indicated that Crustin gene might play an important role when shrimps are infected by bacterial pathogen. 展开更多
关键词 Crustin-like gene Fenneropenaeius chinensis molecular cloning expression analysis microbe challenge
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Infectivity Analysis of Cloned Genomic DNA of P1 Agent in vitro 被引量:3
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作者 WEN Li-bin HE Kong-wang YANG Han-chun 《畜牧兽医学报》 CAS CSCD 北大核心 2009年第S1期43-46,共4页
The ultrastructure of porcine kidney(PK)-15 cells was examined after lipofectamine-aided transfection of the molecular clone of the P1 agent.PK-15 cells transfected with the tandem dimer of the P1molecular DNA clone h... The ultrastructure of porcine kidney(PK)-15 cells was examined after lipofectamine-aided transfection of the molecular clone of the P1 agent.PK-15 cells transfected with the tandem dimer of the P1molecular DNA clone had numbers of intracytoplasmic inclusions,and a few cells had intranuclear inclusions.Intracytoplasmic inclusions were round to oval and 0.1-0.3μm in diameter,and intranuclear inclusions,which were more electron dense,were of two general types:the first were round and small(0.1μm approximately)and the second were hexagonal and larger(0.4-0.8μm in diameter).Cells transfected with the tandem dimer of the P1 molecular DNA clone tested positive for P1 DNA at passage 5.This is the first report that the P1 molecular clone has infectivity in vitro and it will provide fundamental materials for further study of the biological characterization of P1. 展开更多
关键词 P1 AGENT molecular clone in vitro infectivity ANALYSIS
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Molecular cloning of human heat shock protein 27 and study of its protective effects on oxidative damage in rat cardiomyocte H9c2 被引量:3
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作者 Li Liu Xiaojin Zhang +3 位作者 Surong Jiang Xiang Gao Guoxain Ding Yunlin Cheng 《Journal of Nanjing Medical University》 2005年第4期187-190,共4页
Objective: To clone human cardiac heat shock protein 27 (HSP27) gene and to determine the effects of HSP27 on the oxidative stress in rat cardiomyocyte cell line H9c2. Methods: Full length of HSP27 cDNA which got ... Objective: To clone human cardiac heat shock protein 27 (HSP27) gene and to determine the effects of HSP27 on the oxidative stress in rat cardiomyocyte cell line H9c2. Methods: Full length of HSP27 cDNA which got by RT-PCR was constructed into pCDNA3.1^+ . The recombinant was transfected into rat cardiomyocyte cell line H9c2 and the stable trahsfection cell line was selected by G418. Then we observe the effects of HSP27 over-expression on LDH release and apoptosis induced H2O2 in H9c2. Results: ①pCDNA3.1^+/HSP27 provided a sound expression of HSP27 in both 293T and H9c2. ②LDH releasing induced by 0, 100,250,500, 1000 μmol/L H2O2 in HSP27 over-expression group and wild type group were 0.396±0.017 vs. 0.390±0.01)9 (p 〉0.05), 0.437±0. 014 vs. 0.416±0.015 (P〈0.05), 0.471±0.018 vs. 0.417±0.009 (P 〈0.001), 0.505±0.030 vs. 0.657± 0.022(P 〈0.001), 0.547 ±0.027 and 0.661 ± 0.011( P 〈 0. 001 ), respectively. ③Apoptosis induced by 150 μmol/L H2O2 in HSP27 over-expression group and wild type group were (10.693± 1.122)% vs. (4.027 ± 1.628)%( P 〈0.01). Conclusion: We cloned and constructed human cardiac HSP27 gene successfully, and over-expression of human HSP27 could inhibit oxidative damage significantly in H9c2. 展开更多
关键词 Heat Shock Protein 27 molecular Cloning TRANSFECTION Oxidafive Stress APOPTOSIS
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副溶血性弧菌不耐热溶血素的生物信息学分析与分子动力学模拟
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作者 张德福 于振兴 +4 位作者 张明 吕欣然 张永勤 张国清 励建荣 《食品与发酵工业》 CAS CSCD 北大核心 2024年第12期251-257,I0009-I0012,共11页
副溶血性弧菌是一种革兰氏阴性人畜共患致病菌,可引发人类胃肠炎等食源性疾病。副溶血性弧菌具有多种毒力因子,热不稳定溶血素(thermolabile hemolysin,TLH)即是其中之一。该研究对副溶血性弧菌的tlh基因进行了克隆及测序,对tlh基因编码... 副溶血性弧菌是一种革兰氏阴性人畜共患致病菌,可引发人类胃肠炎等食源性疾病。副溶血性弧菌具有多种毒力因子,热不稳定溶血素(thermolabile hemolysin,TLH)即是其中之一。该研究对副溶血性弧菌的tlh基因进行了克隆及测序,对tlh基因编码的TLH蛋白进行了生物信息学分析及结构和功能预测。结果表明,TLH蛋白由418个氨基酸组成,预测分子质量为47.36 kDa。在TLH蛋白中确定了几个保守的结构域和基序,包括SGNH水解酶结构域和GDSL基序;对TLH蛋白的三维结构进行了同源建模及验证,在此基础上通过分子动力学模拟分析其稳定性、与4-硝基苯基月桂酸酯(p-nitrophenylaurate,PNPL)的相互作用能力及结合底物对结构紧密度和残基灵活性的影响,揭示了催化三联体Ser153-His390-Asp393周边是一个重要的药物靶点活性口袋,具有高度保守的残基序列,并在底物结合后表现出残基灵活性差异。该研究分析了副溶血性弧菌TLH蛋白的性质和结构,可以为保障水产品的安全性、提高水产品原料安全评价水平提供理论支持。 展开更多
关键词 基因克隆 副溶血性弧菌 不耐热溶血素(tlh) 生物信息学分析 分子动力学模拟
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Molecular Cloning and Characterization of Three Novel Genes Related to Fatty Acid Degradation and Their Responses to Abiotic Stresses in Gossypium hirsutum L. 被引量:1
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作者 DONG Jia WEI Li-bin +1 位作者 HU Yan GUO Wang-zhen 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2013年第4期582-588,共7页
Fatty acid metabolism is responsible not only for oilseed metabolism but also for plant responses to abiotic stresses. In this study, three novel genes related to fatty acid degradation designated GhACX, Gh4CL, and Gh... Fatty acid metabolism is responsible not only for oilseed metabolism but also for plant responses to abiotic stresses. In this study, three novel genes related to fatty acid degradation designated GhACX, Gh4CL, and GhMFP, respectively, were isolated from Gossypium hirsutum acc. TM-1. The phylogenetic analysis revealed that amino acid sequences of GhACXand GhMFP have the highest homology with those from Vitis vinifera, and Gh4CL has a closer genetic relationship with that from Camellia sinensis. Tissue- and organ-specific analysis showed that the three genes expressed widely in all the tested tissues, including ovules and fiber at different developing stages, with expressed preferentially in some organs. Among them, GhACX showed the most abundant transcripts in seeds at 25 d post anthesis (DPA), however, GhMFP and Gh4CL have the strongest expression level in ovules on the day of anthesis. Based on real-time quantitative RT-PCR, the three genes were differentially regulated when induced under wounding, methyl jasmonate (MeJA), cold, and abscisic acid (ABA) treatments. The characterization and expression pattern of three novel fatty acid degradation related genes will aid both to understand the roles of fatty acid degradation related genes as precursor in stress stimuli and to elucidate the physiological function in cotton oilseed metabolism. 展开更多
关键词 genes related to fatty acid degradation molecular cloning expression analysis abiotic stress cotton( Gossypium hirsutum L.)
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Molecular Cloning and Sequence Analysis of IGF-Ⅰfrom Triangular Bream (Megalobrama terminalis) 被引量:2
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作者 TONGFu-dan LIUHong-yun 《Agricultural Sciences in China》 CAS CSCD 2004年第9期700-706,共7页
The insulin-like growth factor Ⅰ(IGF-Ⅰ) gene of triangular bream (Megalobrama terminalis)(GenBank No.AY247412)(Tb)was cloned for the first time from liver by RT-PCR. Thenucleotide sequence analysis showed the Tb IGF... The insulin-like growth factor Ⅰ(IGF-Ⅰ) gene of triangular bream (Megalobrama terminalis)(GenBank No.AY247412)(Tb)was cloned for the first time from liver by RT-PCR. Thenucleotide sequence analysis showed the Tb IGF-ⅠcDNA consisted of 486 nucleotides andencoded 117 amino acids including B, C, A, D and E five domains. Analysis of E-domainindicated that cloned Tb IGF-Ⅰbelonged to IGF-ⅠEa-2 subtype. Identity analysis showedthe IGF-Ⅰnucleotide sequence shared 99.8% homology with bluntnose bream, 88.8% withgrass carp, 85.8% with common carp; the pre-IGF-Ⅰamine acid sequence shared 99.4% withbluntnose bream, 88.8% with grass carp, 85.4% homology with common carp. In the CyprinusCarpio, the higher homology of nucleotide sequence and amino acid sequence in IGF-Ⅰshowed that the closer relationship the fishes have. These results could provide basicdata for the research on Tb germplasm and the development and utilization of biologicalfeed additives. 展开更多
关键词 Triangular bream (Megalobrama terminalis) IGF-Ⅰ molecular cloning Sequence analysis
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Molecular Cloning and Nucleotide Sequence of the Attenuated Hog Cholera VirusLapinized Chinese Strain 被引量:1
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作者 Wang Jiafu Zhang Chuyu +3 位作者 Fu Liezhen Wang Ning Huang Qianhua Zhang Pengwei(College of Life Sciences, Wuhan University, Wuhan 430072, China) 《Wuhan University Journal of Natural Sciences》 CAS 1998年第4期499-503,共5页
The genomic sequence of the attenuated hog cholera virus Lapinized Chinese strain (HCLV) was determined from overlapping cDNA clones. The viral RNA of HCLV stain comprised 12 310 nucleotide (nt) including 374 nt and 2... The genomic sequence of the attenuated hog cholera virus Lapinized Chinese strain (HCLV) was determined from overlapping cDNA clones. The viral RNA of HCLV stain comprised 12 310 nucleotide (nt) including 374 nt and 239 nt at the 5′ and 3′-noncoding region, respectively. The complete genome sequence contained one large open reading frame which encoded an amino acid sequence of 3 898 residues with a calculated molecular weight of 437×103. Although there were mostly only small differences between the sequence of the HCLV strain and the published sequences of strains ALD, GPE?, Alfort and Brescia, there was one notable insertion of 12 nucleotides, TTTTCTTTTTTC in the 3′ non-coding region of HCLV strain. 展开更多
关键词 Key words hog cholera virus molecular cloning SEQUENCE
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Molecular cloning and characterization of human age-related NADH oxidase (arNOX) proteins as members of the TM9 superfamily of transmembrane proteins 被引量:2
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作者 Xiaoyu Tang Debby Parisi +2 位作者 Bradley Spicer Dorothy M. Morré D. James Morré 《Advances in Biological Chemistry》 2013年第2期187-197,共11页
Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is ... Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the TM9 family of transmembrane proteins. Peptide antisera raised to all five members of the human TM9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog TM9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited ar-NOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known TM9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the TM9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids1. 展开更多
关键词 AGE-RELATED NADH OXIDASE (arNOX) TM-9 SUPERFAMILY of TRANSMEMBRANE PROTEINS molecular Cloning HUMAN Serum Plasma and Body Fluids Saliva
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