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Expression, purification, and subcellular localization of phospholipase C in Dunaliella salina

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Journal of Oceanology and Limnology 2019年第4期37卷 1363-1371页

作者： CONG Yuting WANG Yuan YUE Jinrong XING Zhenyu GAO Xiangnan chai xiaojie Key Laboratory of Hydrobiology in Liaoning Province College of Fisheries and Life Science Dalian Ocean University

Plants possess effective mechanisms to respond quickly to the external environment. Rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs after a stimulus. The PLC in Dunaliella salina plays important roles in growth and stress responses. However, the molecular basis of PLC action in D. salina remains little understood. To gain insight into the potential biological functions of this enzyme, we cloned a phospholipase C gene from D. salina in a previous study, named DsPLC (GenBank No. KF573428). Here, we present the prokaryotic expression, purification, and characterization of the DsPLC gene. The entire coding region of DsPLC was inserted into an expression vector pET32a, and the DsPLC gene was successfully expressed in Escherichia coli. The DsPLC protein was purified and identified using a polyclonal antibody and western blotting. Expressing DsPLC fused with a green fluorescent protein (GFP) in onion showed that DsPLC-GFP was localized to the intracellular membrane. Quantitative real-time PCR analysis revealed that the relative expression of the DsPLC gene was induced significantly by 3.0-mol/L NaCl at 4 h. Our results support the importance of PLC enzymes in plant defense signaling. This study provides a basis for further functional studies of the DsPLC gene and for additional analysis of the potential roles of PLC enzymes in response to abiotic stress.

关键词： Dunaliella salina DsPLC gene prokaryotic expression subcellular localization salt stress

Cloning of Ds MAPK Gene and Construction of Antisense Expression Vector

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Agricultural Biotechnology 2018年第4期7卷 8-11,24页

作者： Jinrong YUE Yuting CONG Xiangnan GAO Zhenyu XING Wenjing YUE xiaojie chai Dalian Ocean University/Key Laboratory of Maricuhure&Stock Enhancement in North China's Sea Ministry of Agricuhure/Key Laboratory of Marine Bio-Resources Restoration and Habitat Reparation in Liaoning ProvinceDalian 116023China

In order to investigate the role of MAPK gene in adaptation of Dunaliella salina to hypersaline environment, the Ds MAPK gene of D. salina was amplified by PCR. After inverted insertion of the open reading flame ofDs MAPK gene into downstream sequence of 35S promoter of plant expression vector, an antisense expression vector of Ds MAPK gene was successfully constructed and introduced into D. salina cells by LiAc/PEG-mediated method. The expression of Ds MAPK gene in transgenic D. salina was analyzed by semi-quantitative RT-PCR method. The results showed that the expression of Ds MAPK gene in D. salina was significantly inhibited at the transcriptional level. The study laid the foundation for further identification of the function of Ds MAPK gene.

关键词： Dunaliella salina Ds MAPK Antisense expression vector Semi-quantitative RT-PCR

Functional Analysis of Dunaliella salina Calmodulin Kinase Gene

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Agricultural Biotechnology 2020年第2期9卷 10-13,20页

作者： Zhenyu XING Mingfang WANG Xiangnan GAO Weiwei XU Yuting CONG xiaojie chai Key Laboratory of Hydrobiology in Liaoning Provincial Universities Dalian Ocean UniversityDalian 116023China

[Objectives] This study was conducted to investigate the function of Dunaliella salina calmodulin kinase(CaM K) gene.[Methods] The sense and antisense gene fragments(223 bp) and spacer sequence(129 bp) of *** calmodulin kinase gene were cloned and inserted into the downstream part of the35 S promoter of the eukaryotic expression vector pM *** siRNA expression system of CaM K gene was successfully *** p CaM K-RNAi expression vector was transformed into *** cells by the LiA c/PEG-mediated method,giving transgenic *** expression of CaM K gene was then analyzed by real-time fluorescence quantitative PCR.[Results]The expression of CaM K gene in the transgenic *** was significantly reduced,by 70% compared with the control group,suggesting that the expression of CaM K gene was significantly *** examination of the growth status of *** showed that *** cell division and proliferation were also *** is proved that CaM K gene has a positive regulation effect on the division and proliferation of *** cells.[Conclusions] The study provides important information for further elucidating the function and action mechanism of *** calmodulin kinase gene.

关键词： Dunaliella salina CaMK RNAi LiAc/PEG-mediated method Real-time fluorescence quantitative PCR

Subcellular Localization of Small GTP-binding Protein DsRab in Dunaliella salina

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Agricultural Biotechnology 2018年第3期7卷 77-80页

作者： Yuting CONG Jinrong YUE Zhenyu XING Xiangnan GAO Xiujuan LI xiaojie chai Dalian Ocean University Key Laboratory of Hydrobiology in Liaoning Province

With the total RNA of Dunaliella salina as a template,the cD NA sequence of D. salina small GTP-binding protein gene was amplified by RT-PCR technique,and cloned onto pM Dl8-T simple vector,the recon was subjected to PCR detection and restriction endonuclease analysis,and the total sequence of DNA was determined. The results showed that the cloned fragment was 612 bp,and shared 100% homology with reported D. salina DsRab gene（ GenB ank： JN989548）. The target gene fragment was inserted downstream of pM DCG 35 S promoter,constructing subcellular localization recombinant vector pM DCG-DsRab. The successfully constructed subcellular localization recombinant vector pM DCG-DsRab was transformed into Agrobacterium tumefaciens LBA4404,and positive single clones were screened and used for transinfection of onion epidemical cells by Agrobacterium-mediated method,and the instant expression of DsRab was observed under fluorescence microscope. The results showed that the fusion protein GFP-DsRab was successfully expressed in onion epidemical cells,and mainly distributed on cytomembrane. This study will provide reference for further illumination of the function and action mechanism of D. salina small GTP-binding protein DsRab.

关键词： Dunaliella salina Small GTP-binding proteins Subcellular localization Fusion protein

Cloning and Prokaryotic Expression of MAPKK kinase Gene from Dunaliella salina

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Cloning and Prokaryotic Expression of MAPKK kinase Gene from...

中国生物化学与分子生物学会2016年全国学术会议

作者： chai xiaojie Liu Yiqiong Cong Yuting Wang Yiyun Yue Jinrong Key Laboratory of Hydrobiology in Liaoning Province Dalian Ocean University

A MAPKK kinase gene was firstly cloned from Dunaliela salina by RT-PCR and RACE *** full length of c DNA for MAPKKK gene was 1 460 bp with a 27bp 5’UTR,a 554bp 3’UTR and a 879bp open reading frame coding 292 amino *... 详细信息

关键词： Dunaliella salina MAPKKK gene cloning bioinformatics prokaryotic expression western blotting

来源： cnki会议评论

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基于FPGA的SIFT特征点检测

高技术通讯 2012年第4期22卷 429-435页

作者：肖晗原魁何文浩柴晓杰中国科学院自动化研究所北京100190

为了在现场可编程门阵列（FPGA）中用硬件电路实现尺度不变特征转换（SIFT）特征点的实时检测，对原算法进行了改进，提出了一种基于面密度插值法和双重极值点约束的新方法。该方法显著提升了SIFT特征点的尺度不变性，同时有利于提高定... 详细信息

为了在现场可编程门阵列（FPGA）中用硬件电路实现尺度不变特征转换（SIFT）特征点的实时检测，对原算法进行了改进，提出了一种基于面密度插值法和双重极值点约束的新方法。该方法显著提升了SIFT特征点的尺度不变性，同时有利于提高定点数的计算精度。在此基础上，设计了一种高效率的硬件计算方案，将所有的计算步骤都实现在了流水线结构中，并在FPGA上完成了开发工作。与现有研究成果相比，这种高效的方案非常节约硬件资源，大大降低了硬件成本，同时又大幅度提高了计算精度。系统能够在采集图像的同时完成特征点的检测。对于360×288大小的图像，其理论最高处理能力为303帧／秒；由于受到摄像头的图像采集速度限制，目前的实际处理速度是25帧／秒。

关键词：尺度不变特征转换(SIFT) 现场可编程门阵列(FPGA) 特征点检测机器视觉硬件计算

VEGFR-2与抑制剂Sunitinib的分子对接及分子动力学研究

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化学学报 2012年第10期70卷 1232-1236页

作者：安康柴晓杰薛飞王媛张婷大连海洋大学、辽宁省海洋生物资源恢复与生境修复重点实验室大连116023

用分子对接方法研究了VEGFR-2和抑制剂Sunitinib的相互作用模式,并对其复合物进行了10 ns的分子动力学(Molecular Dynamics,MD)模拟.结果表明,抑制剂Sunitinib能与VEGFR-2中位于活性空腔的Glu885,Ile888,His1026,Asp1028,Asp1046五个氨基酸残基形成疏水作用;另外,VEGFR-2中His1026,Cys1024,Asp1046三个氨基酸残基能与Sunitinib形成三个作用强度不同的氢键.这些基团之间的相互作用是Sunitinib抑制VEGFR-2活性的关键因素.研究结果可为VEGFR-2抑制剂的结构改良、分子设计、合成提供理论参考,并有助于寻找活性更高、效果更好的抗肿瘤药物.

关键词： VEGFR-2 Sunitinib 分子对接分子动力学

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信息技术在事业单位档案管理中的应用探究

创新创业理论研究与实践 2019年第11期2卷 178-179页

作者：柴晓洁河南省医疗器械检验所

改革开放以来,经过几十年发展,中国进入信息化时代。信息技术在实际生活中优势巨大,不仅被用于高科技行业,也被用于企业和事业单位。而管理方面则是信息技术在企业或是事业单位中运用最广泛的领域。该文从事业单位档案传统管理方式出发... 详细信息

改革开放以来,经过几十年发展,中国进入信息化时代。信息技术在实际生活中优势巨大,不仅被用于高科技行业,也被用于企业和事业单位。而管理方面则是信息技术在企业或是事业单位中运用最广泛的领域。该文从事业单位档案传统管理方式出发,针对社会目前发展趋势以及事业单位档案管理存在的不足,对信息技术在事业单位档案管理中的应用进行探究。

关键词：档案管理技术优势技术运用

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轮状病毒VP7基因的克隆与表达

生物技术通报 2011年第3期27卷 160-162,174页

作者：柴晓杰王晓庆张婷代靖宇大连海洋大学生命科学与技术学院大连116023

应用聚合酶链式反应技术(PCR)扩增轮状病毒VP7基因,并将其克隆到pMD18-T simple载体上,对重组子进行PCR检测和限制性内切酶分析,并测定DNA全序列。结果显示,克隆片段全长为981 bp。将轮状病毒VP7基因定向的克隆到原核表达载体pET-32a启... 详细信息

应用聚合酶链式反应技术(PCR)扩增轮状病毒VP7基因,并将其克隆到pMD18-T simple载体上,对重组子进行PCR检测和限制性内切酶分析,并测定DNA全序列。结果显示,克隆片段全长为981 bp。将轮状病毒VP7基因定向的克隆到原核表达载体pET-32a启动子下游,构建原核表达载体pET-32aVP7。将质粒pET-32aVP7转化Transetta表达菌株进行诱导表达,裂解菌体细胞抽提蛋白质进行SDS-PAGE。结果表明,轮状病毒壳蛋白VP7基因在Transetta表达菌株内得到成功表达。

关键词：轮状病毒 PCR 克隆表达