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澳洲坚果MiMYB1基因克隆及生物信息学分析

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澳洲坚果MiMYB1基因克隆及生物信息学分析

2019年全国热带作物学术年会

作者：王文林肖海艳郑树芳谭秋锦覃振师黄锡云贺鹏汤秀华许鹏王立丰陈海生广西南亚热带农业科学研究所农业农村部橡胶树生物学与遗传资源利用重点实验室/省部共建国家重点实验室培育基地-海南省热带作物栽培生理学重点实验室/农业农村部儋州热带作物科学观测实验站/中国热带农业科学院橡胶研究所

【目的】克隆澳洲坚果MiMYB1基因,利用生物信息学对Mi MYB进行分析预测其结构和功能,为研究澳洲坚果R2R3-MYB家族转录因子成员在澳洲坚果产量形成中的作用机制提供理论参考。【方法】利用PCR技术从澳洲坚果品种"桂热一号"叶子中克隆MiM... 详细信息

【目的】克隆澳洲坚果MiMYB1基因,利用生物信息学对Mi MYB进行分析预测其结构和功能,为研究澳洲坚果R2R3-MYB家族转录因子成员在澳洲坚果产量形成中的作用机制提供理论参考。【方法】利用PCR技术从澳洲坚果品种"桂热一号"叶子中克隆MiMYB1基因,将其进行生物信息学分析预测。【结果】克隆获得的MiMYB1基因cDNA序列全长1205bp bp,开放阅读框（ORF）1059 bp,编码353个氨基酸。MiMYB1具有两个SANT保守结构域,属于R2R3-MYB家族,是无跨膜结构、无信号肽且定位细胞核的不稳定亲水蛋白。系统发育进化分析MiMYB1与荷花NnMYB39氨基酸序列亲缘关系最近,在拟南芥中,MiMYB1与AtMYB41亲缘关系最近,MiMYB1、AtMYB41、AtMYB74、AtMYB102聚类为S11亚族。【结论】克隆MiMYB1基因并分析其结构及功能,为深入阐明其在澳洲坚果的产量形成中的机制提供理论参考。

关键词： Macadamia Integrifolia MiMYB1 gene cloning Bioinformatics analysis

cloning and Expression Analysis of Two MADS-Box genes from Strawberry Fruit

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Cloning and Expression Analysis of Two MADS-Box Genes from S...

第七届世界草莓大会

作者： H.C.Zhou Z.R.Kang X.Zhao G.Li L.Li Zhengzhou Fruit Research Institute Chinese Academy of Agricultural Sciences(CAAS) College of Horticulture and Landscape Architecture Southwest University

A MADS-box protein gene named FaMADSl was isolated from fruit tissue of strawberry（Fragaria ×ananassa Duch.’Toyonoka’） by RT-PCR and *** result of the sequencing showed that the nucleotide sequence of FaMADSl gene has98%similarity with the Fv-MADS-9 gene sequences from the *** cDNA gene sequence is 1,167 bp in length with a 747 bp open reading frame encoding 249 amino acid proteins which displayed the structure of typical plant MADS-box proteins,and 100 bp in the 5’UTR,320 bp in the 3’UTR,and 30 bp in the poly（A）*** SqRT-PCR experiment showed that the FaMADSl gene is only expressed in fruit tissue,with no expression detected in flower *** expression levels in different stages of fruit development and ripening are from low in young flower receptacles to high as fruit receptacles begin turning red with brown achenes,and then expression is lower at the full red fruit *** MADS-box protein gene named FaMADS2 from strawberry fruit was simultaneously cloned by RT-PCR and3’*** cDNA sequence length is 870 bp with a 617 bp coding frame,254 bp in the 3’UTR,and 27 bp in the poly（A） *** nucleotide sequence of the FaMADS2 gene has 96%identity with the FcMADS2 gene *** expression pattern of FaMADSl and FaMADS2 genes is different in tissues and stages of berry ***2 expression appears to be high in flower organs,and shows two peaks of expression in the young flower receptacle and white receptacle *** information provides new clues to aid in understanding the ripening process in strawberry fruit.

关键词： strawberry(Fragaria×ananassa Duch.) MADS-box gene gene cloning semi-quantitative RT-PCR

Sequence analysis and expression of the calcium exchanger gene,MCAX-1,in mulberry（Morus alba L.）

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Sequence analysis and expression of the calcium exchanger ge...

现代分子植物育种与粮食安全研讨会

作者： Liu Zhaoyue1,2,Zhao Weiguo 1,2,Zhang Yinghua1,2,Tong Wei1,2,Fang Rongjun1,2,Li Long 1,2 * (1 College of biological and Environmental Engineering,Jiangsu University of Science and technology,Zhenjiang Jiangsu 212018,China 2 Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhenjiang Jiangsu 212018,China)

For the sake of keeping the balance of plant cells,Calcium,as a necessary nutrition element to plant growth development,can get into the cytoplast by calcium channel and out of it by Ca2+-ATPase and calcium exchanger to hold a low calcium ***,calcium exchanger is related to resisting adversity of plants,to make the calcium mediated signal system unblocked and regulate adaptability accurately and ***,people are more and more focusing on the study of the calcium exchanger *** full-length cDNA that is cloned through RACE and RT-PCR methods,named as MCAX-1,cDNA fragments of which has been foundby measuring the function comments of ESTs sequence in mulberry,is 1784bp by the sequence *** the research focuses on gene expressions and prokaryotic expressions of the calcium exchanger gene under low temperature,drought,and salt stress conditions to find out gene expression patterns and responses of MCAX-1 in different *** will help us provide the theoretical basis and strong applications to excavate excellent gene resources and resistant molecular breeding in mulberry.

关键词： Mulberry Calcium exchanger gene(MCAX-1) gene cloning Sequence analysis gene expression

Amplification and Bioinformatics Analysis of h-ns gene of Vibrio alginolyticus

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Asian Agricultural Research 2023年第4期15卷 29-33页

作者： Ying CHEN Shi WANG Liangchuan CHEN Haiyun FENG Junlin WANG Huanying PANG Na WANG Fisheries College Guangdong Ocean UniversityZhanjiang 524025China Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture&Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes Zhanjiang 524025China Chinese Academy of Inspection and Quarantine Beijing 100176China

[Objectives]To amplify the h-ns gene of Vibrio alginolyticus and analyze it by bioinformatics.[Methods]According to the h-ns gene sequence of *** HY9901,a pair of specific primers were designed and amplified by PCR.[Results]The h-ns gene was 408 bp in length and 135 amino acids were *** predicted theoretical protein molecular weight was about 14.98 kD,and the isoelectric point was *** subcellular localization,SignalP 5.0,TMHMM Server 2.0 and SoftBerry-Psite predictions showed that H-NS was located outside the cell membrane,and the protein was unstable and *** was no signal peptide cleavage site,no transmembrane region and no KEGG metabolic *** amino acid sequence contained three phosphorylation sites,one N-terminal myristoylation site and three microsomal C-terminal target signal *** MEGA 5.0,H-NS phylogenetic tree was constructed by ortho-connection *** results showed that H-NS of *** was closer to H-NS of Vibrio *** SWISS-MODEL,the three-dimensional structure model of H-NS subunit was simulated,which was similar to the crystal structure of Salmonella typhimurium H-NS1-83.[Conclusions]This study lays a foundation for exploring the regulation mechanism of *** H-NS protein on bacterial virulence in the future.

关键词： Vibrio alginolyticus gene cloning H-NS Bioinformatics analysis

Cloing and Bioinformatics Analysis of ndk gene from Vibrio alginolyticus

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Asian Agricultural Research 2023年第7期15卷 30-34页

作者： Yujia ZHANG Shi WANG Jian ZHONG Weijie ZHANG Xing XIAO Zhiqing WEI Huanying PANG Na WANG College of Fisheries Guangdong Ocean UniversityZhanjiang 524025China Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture/Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes Zhanjiang 524025China Zhanjiang Customs Zhanjiang 524022China Chinese Academy of Inspection and Quarantine Beijing 100176China

[Objectives]The paper was to clone and analyze bioinformatics of ndk gene from Vibrio alginolyticus.[Methods]A pair of specific primers was designed based on the ndk gene sequence of *** *** full length of ndk gene was amplified by PCR and bioinformatics analysis was *** 5.0 software was used to construct NDK phylogenetic tree by neighbor-joining ***-MODEL program was used to obtain the three-dimensional structural model of single subunit from NDK protein.[Results]The ndk gene,molecular structural formula C702H1094N192O214S7,was 426 bp in total,encoding 141 amino acids,with the theoretical molecular weight of 15.87199 kD and the theoretical pI value of *** prediction results of protein subcellular localization,SignalP 5.0,TMHMM Server 2.0 and SoftBerry-Psite showed that NDK mainly existed in the cytoplasm,and the protein was unstable and *** was neither signal peptide cleavage site,nor transmembrane region and KEGG metabolic *** amino acid sequence had two protein kinase C phosphorylation sites,a casein kinaseⅡphosphorylation site,a N-myristoylation site,three microbody C-terminal target signal sites,and a nucleoside diphosphate kinase active *** analysis showed that the NDK of *** had high homology with that of ***,with a similarity of 98.58%.Analysis of the structural functional domain revealed that the protein had one NDK structural functional *** prediction results of secondary structure showed that theα-helix,random coil,β-sheet and extended strand accounted for 53.19%,28.37%,7.09%and 11.35%,*** of NDK protein via STRING database demonstrated that the proteins interacting with NDK protein were NrdA,NrdB,GmK,CmK,TmK,PyrG,PyrH,RelA,FolE and SpoT.[Conclusions]The study plays a positive role in the prevention and control of vibriosis and the improvement of the current aquaculture environment.

关键词： Vibrio alginolyticus gene cloning ndk Bioinformatics analysis

牡丹种子油体发育特点及其OLEs基因的表达特性研究

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牡丹种子油体发育特点及其OLEs基因的表达特性研究

作者：李志远扬州大学

学位级别：硕士

牡丹(Paeonia suffruticosa Andr.)属芍药科芍药属灌木,是近些年新兴的木本油料作物,因其籽油中含有大量不饱和脂肪酸而倍受关注。目前,关于牡丹籽油的研究主要集中于籽油的提取、脂肪酸成分的分析及脂肪酸的生物合成等方面,而关于脂肪... 详细信息

牡丹(Paeonia suffruticosa Andr.)属芍药科芍药属灌木,是近些年新兴的木本油料作物,因其籽油中含有大量不饱和脂肪酸而倍受关注。目前,关于牡丹籽油的研究主要集中于籽油的提取、脂肪酸成分的分析及脂肪酸的生物合成等方面,而关于脂肪酸的储存报道较少。油体是植物储存油脂的细胞器,它的正常发育是脂肪酸积累的前提。为了明确牡丹种子油体发育特点及其油体膜蛋白基因(OLFs)的表达特性,我们以油用牡丹'凤丹'(Paeoniaostii)为材料,一方面从油体入手,观察油体形态、分析理化特性、分析与脂肪酸积累的相关性、鉴定油体蛋白种类;另一方面,从油体蛋白入手,克隆OLEs序列、检测表达模式、观察亚细胞定位、构建超表达载体并对烟草进行遗传转化,主要研究结果如下:(1)牡丹种子出油率在种子成熟过程中呈先升高后降低的趋势,在花后100天时出油率最高,达到27%;牡丹籽油主要包含5种脂肪酸:棕榈酸、硬脂酸、油酸、亚油酸、α-亚麻酸,其中含量最高的是α-亚麻酸,最低的是硬脂酸。牡丹籽油的脂肪酸成分的积累趋势与种子出油率类似,各脂肪酸积累的峰值都出现于花后100天。(2)通过石蜡切片,发现牡丹种子油体的积累呈先增加后减少的趋势,油体从花后80天开始进入迅速积累阶段并于花后100天时达到最大积累量。通过超薄切片,发现牡丹种子油体数量在种子发育过程中不断增加,油体在细胞中变得越来越充实,但相对于其他时期,花后100天时出现了许多更小的油体。(3)牡丹种子油体粒度随着种子发育时期的变化呈现先减小后增大的趋势,在花后100天时其粒度达到最小值,而后略有增大。牡丹种子的出油率、脂肪酸含量与油体粒度均呈显著负相关,其中种子出油率与油体粒度的相关性达到-0.98。在种子成熟过程中,牡丹种子油体表面蛋白质所带的电荷与粒度趋势一致,同样在花后100天时Zeta电位达到最低,之后略有增加。通过对牡丹种子油体的稳定性研究,我们发现油体在NaCl溶液中的稳定性随着溶液浓度的升高而变差;通过对不同pH值下油体的形态进行观察,我们发现当pH值为5.0时,油体的结构被严重破坏,pH为9.0时的油体相对于中性条件下的油体显得更加稳定,而当pH值达到11.0时,油体在乳液中的运动变得非常剧烈,并出现许多超大油体。通过对不同温度下油体的形态观察发现,随着温度的升高,油体的结构并没有发生明显的变化。(4)牡丹种子发育前100天的油体蛋白相对集中,在17、22、37和55 kD处分别有4条明显的条带,杂带较少,但花后120天时油体蛋白组成发生了一定的变化,在37和55kD处均出现了其他条带。进一步通过MALDI-TOF-MS分析发现,牡丹种子前三个时期的油体蛋白主要以油体膜蛋白类为主,未鉴定出其他家族蛋白,但在花后120天时,发

关键词： Tree peony Oil body Oleosin gene cloning Expression characteristics

法克隆紫穗槐4-香豆酸:CoA连接酶全长cDNA(英文)

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Forestry Studies in China 2002年第1期4卷 13-17页

作者：刘文哲胡学军高晓蓉袁晓东刘哲范琦安利佳大连理工大学生物工程系大连116029 宝生物大连有限公司大连116600

An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) PCR. We designed 5′RACE primers based on 4CLA1 fragment which obtained from degenerate PCR. Inverse PCR and nested PCR enabled cloning of the remainder fragments of the gene included 5′ and 3′ end sequence. The ORF encodes a polypeptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins, in particular a postulated AMP binding site, catalytic domain, and conserved Cys residues.

关键词： gene cloning random amplification of cDNA end PCR Amorpha fruticosa 4 coumarate:CoA ligase lignin

阴道毛滴虫Fd基因真核表达质粒的构建（英文）

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阴道毛滴虫Fd基因真核表达质粒的构建（英文）

2005年寄生虫学国际学术研讨会暨中国动物学会寄生虫专...

作者： Xie Hui Wang Yajing Tie Chaonan Bi Shiliang Liu Peina (Department of Parasitology,West China School of Preclinical and Forensic Medicine Sciences, Sichuan University, Chengdu 610044, China Department of Obstetrics and Gynecology, West China Second Hospital, Sichuan University, Chengdu 610041, China)

＜正＞Aim To construct a eukaryotic cell expression recombinant plasmid containing ferredoxin gene of T. vaginalis. Methods Total DNA was extracted from T. vaginslis with Chelex-100 method and used as templates for PCR. The Fd gene was directionally cloned into plasmid pMD-18T simple vector, then subcloned into eukaryotic expression vector pcDNA3.1（+）.The transformants were screened and identified by PCR and restriction analysis. Results The size of amplified Fd gene was 306bp. The correct recombinant plasmid pcDNA3.1（+）-Fd was isolated and confirmed by PCR and restriction analysis. The DNA sequence of cloned gene was the same as the published sequence. Conclusion Recombinant plasmid are going to be used further study of function of the Fd.

关键词： T. vaginalis Ferredoxin Eukaryotic cell expression recombinant plasmid gene cloning