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Quantitative Screening of Secretory Protein Genes in <i>Candidatus</i>Liberibacter Asiaticus

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American Journal of Plant Sciences 2018年第12期9卷 2408-2419页

作者： Binbin Li Yi Yang Zhiwen Luo Zhixin Liu naitong yu Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences Haikou China Institute of Tropical Agriculture and Forestry Hainan University Haikou China Environment and Plant Protection Institute Chinese Academy of Tropical Agricultural Sciences Haikou China Tropical Fruit Trees Institute Hainan Academy of Agricultural Sciences Haikou China

Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The disease is caused by Candidatus Liberibacter spp., which is vectored by the psyllids Diaphorina citri Kuwayama and Trioza erytreae. Secretory proteins are important in bacterial pathogenesis and structure components. Some of them are expressed at a high level. To obtain the highly-expressed secretory protein genes (SPGs) for antiserum preparation, six candidate SPGs were chosen from Candidatus Liberibacter asiaticus by bioinformatic analysis and were further tested by qPCR and RT-qPCR methods, respectively. The result showed that two SPGs, 408 and pap (both are Flp pilus assembly protein genes), have relative high amounts of DNA and RNA transcripts of early HLB-infected green orange leaves. The 408 and pap genes were further constructed into the plant expression vector pCAMBIA1300 (GV1300: GFP) and expressed in tobacco leaf epidermal cells for subcellular localization analysis. The transient expression results indicated that the 408 protein is located in the nuclei and cytoplasm of tobacco leaf cells. However, the pap protein is located in the cytoplasm of tobacco leaf cells, which may help the pathogen invade into plant cells. This research is an important foundation for the preparation of the antiserum against Candidatus Liberibacter asiaticus and the early detection of HLB disease.

关键词： Candidatus Liberibacter Asiaticus Secretory Protein DNA Amount RNA Transcription Subcellular Localization

Antiserum Preparation and Specific Detection of Banana RING-E3 Ubiquitin-Protein Ligase

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American Journal of Plant Sciences 2022年第3期13卷 371-379页

作者： naitong yu Qin Zhou Hongwei yuan Shuli Xian Jianhua Wang Yi Yang Zhixin Liu Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences Haikou China Hainan Academy of Tropical Agricultural Resource Haikou China Hainan Key Laboratory of Tropical Microbe Resources Haikou China Environment and Plant Protection Institute Chinese Academy of Tropical Agricultural Sciences Haikou China

According to the data of banana transcriptome sequencing, an E3 ubiquitin-protein ligase gene was cloned by RT-PCR method using the cDNA sample of banana leaves. The complete ORF of E3 ubiquitin-protein ligase is 681 bp long and its encoded protein showed 100% sequence identity to homologue RING-H2 finger protein (XP_009407047.1) of Musa_acuminata. Bioinformatic analysis indicated that E3 ubiquitin-protein ligase contains the Ring finger domain in C terminus and eight cross-brace motifs are found in the domain. The target gene was digested by EcoR V and EcoR I, and was inserted into prokaryotic expression vector pET-32a of the same digestions to obtain the plasmid pET32a-E3 ubiquitin-protein ligase. The recombinant plasmid was introduced into Escherichia coli strain BL21 (DE3), and induced at 25°C with 0.4 mmol/L IPTG for 6 hours. The soluble fusion protein was expressed and high purity fusion protein was obtained by Ni2+-NTA agarose affinity chromatography purification. The fusion protein was injected into mice 3 times to prepare the antiserum. Western blot analysis showed a specific protein band was detected in total protein sample of banana leaves, but not for the samples of wild-type Nicotiana benthamiana (N.B.) and wild-type Arabidopsis thaliana (A.T.), implying the antiserum was specific to banana E3 ubiquitin-protein ligase.

关键词： Banana E3 Ubiquitin-Protein Ligase Antiserum Preparation Polyclonal Antibody

Bioactive Constituents from the Peels of Clausena lansium

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Bioactive Constituents from the Peels of Clausena lansium

罗非鱼罗湖病毒ORF10蛋白的亚细胞定位及组织表达分析

中国植物病理学会2017年学术年会

作者： Deng Huidong Luo Zhiwen Guo Lijun Fan Hongyan Hu Fuchu yu naitong Liu Zhixin Hua Min Institute of Tropical Fruit Trees Hainan Academy of Agricultural Science Key Laboratory of Tropical Fruit Tree Biology of Hainan Province Haikou Scientific Observation and Experiment Station of Tropical Fruits Ministry of Agriculture Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences

Clausena lansium Skeels（Wampee） is a tropical species of the Rutaceae family,whichis widely distributed in southern *** fruits of wampee have high medicinal and edible value,which can be used for digestive disorders,abdominal pain,colic,phlegm,cough,and *** order to discover the bioactive constituents with medicinal value in peels of *** in Hainan,one new monoterpenoid,nerol oxide-8-carboxylic acid,and one new flavonoid glycoside,claulansoside A,together with six known compounds,clausenamide,quercetin,isorhamnetin,dihydromyric,2″,3″-dihydroxyanisolactone and（E,E）-8-（7-hydroxy-3,7-dimethylocta-2,5-dienyloxy） psoralen,have been isolated from the peels of *** column *** structures were determined using a combination of 1 D,and 2 D NMR（HMQC,HMBC,COSY and NOESY） techniques,and HR-ESI-MS *** antimicrobial activities against Staphylococcus aureus of compounds 1-8 were measured using paper disc diffusion method,among which compounds 1 and 7 exhibited antibacterial activity against Staphylococcus aureus with the diameter of inhibition zones of 11.5 mm and 14.2 *** hypoglycemic activities of compounds 1-8 were evaluated in vitro by the inhibition of α-glucosidase method and compounds 3 and 6 showed α-glucosidase inhibitory activity in vitro.

关键词： Clausena lansium Monoterpenoid Flavonoid Antibacterial activity Hypoglycemic activity

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中国水产科学 2021年第7期28卷 896-902页

作者：陈中元王荣华刘志昕余乃通湖南文理学院生命与环境科学学院水生动物重要疫病分子免疫技术湖南省重点实验室常德市农业生物大分子研究中心中国热带农业科学院热带生物技术研究所农业农村部热带作物生物学与遗传资源利用重点实验室

为了研究罗非鱼罗湖病毒(tilapia lake virus,TiLV) ORF10蛋白的功能,从染病莫桑比克罗非鱼(Oreochromis niloticus)脾脏和肝脏组织中克隆获得TiLV ORF10基因编码序列,并成功构建了荧光定位载体pEGFP-ORFl0和重组表达载体 pET32a-ORF10... 详细信息

为了研究罗非鱼罗湖病毒(tilapia lake virus,TiLV) ORF10蛋白的功能,从染病莫桑比克罗非鱼(Oreochromis niloticus)脾脏和肝脏组织中克隆获得TiLV ORF10基因编码序列,并成功构建了荧光定位载体pEGFP-ORFl0和重组表达载体 pET32a-ORF10。将荧光定位载体 pEGFP-ORF10 转染鲤(Cyprinus carpio)上皮瘤细胞(Epithelioma papulosum cyprinid,EPC),经荧光显微镜观察,在EPC的细胞核中观察到绿色荧光信号,表明该蛋白定位于细胞核中。将重组载体pET32a-ORF10转化大肠杆菌BL21(DE3)进行目的融合蛋白表达,聚丙烯酰胺凝胶电泳(SDS-PAGE)检测表明,His-TiLV ORF10融合蛋白成功表达并主要存在于上清液中。随后,采用Ni-NTA亲和层析的方法纯化融合蛋白His-TiLV ORF10,以其为抗原多次免疫Balb/C小鼠,制备多克隆抗体。蛋白印迹法(WB)分析显示,制备的抗体可以特异性识别His-TiLV ORF10。利用蛋白印迹法进一步对感染TiLV莫桑比克罗非鱼的不同来源组织进行检测,结果显示,TiLV ORF10蛋白在染病莫桑比克罗非鱼肌肉、脾脏、肝脏和肾脏中的表达存在较大差异,其中以肝脏和肾脏组织表达量为最高,脾脏次之,肌肉中表达量最低。本研究为深入了解TiLV ORF10蛋白的功能和病毒侵染与致病等机理提供了重要基础。

关键词：莫桑比克罗非鱼罗湖病毒 ORF10 亚细胞定位组织表达

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海南番茄蕨叶型CMV分子检测及其序列分析

基因组学与应用生物学 2015年第8期34卷 1723-1728页

作者：刘锋李涛余乃通王健华刘志昕海南大学环境与植物保护学院海口570228 中国热带农业科学院热带生物技术研究所农业部热带作物生物学与遗传资源利用重点实验室海口571101 海南省植物保护总站海口570100

为了鉴定引起海南定安县番茄叶片呈蕨叶型或变窄成线条状,叶片表面凹凸不平、生长不规则等症状的病原物,对该病样的叶片提取总RNA,反转录成第一链c DNA,然后根据CMV RNA1保守序列设计2对特异引物,再利用常规PCR方法鉴定其病原是否为CMV... 详细信息

为了鉴定引起海南定安县番茄叶片呈蕨叶型或变窄成线条状,叶片表面凹凸不平、生长不规则等症状的病原物,对该病样的叶片提取总RNA,反转录成第一链c DNA,然后根据CMV RNA1保守序列设计2对特异引物,再利用常规PCR方法鉴定其病原是否为CMV。利用两对特异引物进行PCR方法扩增后,分别获得大小约为500 bp和700 bp的目的 DNA条带,经序列相似性比对,发现海南定安县番茄的病原物为CMV。构建系统进化树和同源性分析,进一步确定了引起番茄叶片蕨叶和生长不规则的CMV为重花叶株系(包括蕨叶症状),属CMV IB亚组。本研究鉴定了CMV是引起番茄叶片蕨叶和生长不规则等症状的重要病原物,通过序列比较和同源分析确定了该病原物为CMV的重花叶株系(包括蕨叶症状)。

关键词：番茄黄瓜花叶病毒 PCR 序列分析

香蕉束顶病毒海口分离物Rb蛋白的原核表达、纯化及多克隆抗血清制备

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热带作物学报 2010年第8期31卷 1239-1243页

作者：冯团诚余乃通刘志昕中国热带农业科学院热带生物技术研究所农业部热带作物生物技术重点开放实验室海南海口571101

应用PCR方法从感染香蕉束顶病毒的香蕉植株幼嫩假茎和叶片总DNA中克隆了Rb(Rb-binding-like protein)基因的编码区,将其插入原核表达载体pET-28b中构建重组质粒pET28b-Rb。转化重组质粒的*** BL21(DE3)进行IPTG诱导表达,SDS-PAGE分析结... 详细信息

应用PCR方法从感染香蕉束顶病毒的香蕉植株幼嫩假茎和叶片总DNA中克隆了Rb(Rb-binding-like protein)基因的编码区,将其插入原核表达载体pET-28b中构建重组质粒pET28b-Rb。转化重组质粒的*** BL21(DE3)进行IPTG诱导表达,SDS-PAGE分析结果表明,表达产物大小为22.6ku,且主要以包涵体形式稳定表达。目的蛋白经Ni2+-NTA琼脂糖亲和层析纯化后免疫家兔并获得抗血清。Western blot检测结果表明,抗血清与诱导表达的BBTV编码Rb蛋白发生特异性反应。间接ELISA法测定的抗血清效价大于1:250000。用抗血清对感病香蕉和健康香蕉进行检测,结果表明,抗血清对感病香蕉有很高特异性,最佳工作浓度为1:1500。

关键词：香蕉束顶病毒 Rb蛋白原核表达抗血清间接ELISA

柑橘黄龙病菌(Candidatus Liberibacter asiaticus)分泌蛋白基因DNA含量及其RNA表达量关系分析（英文）

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柑橘黄龙病菌(Candidatus Liberibacter asiaticus)分泌蛋白基因D...

中国植物病理学会第十一届全国会员代表大会暨2018年学术年会

作者：李宾宾 Yang Yi Liu Zhixin Wang Jianhua yu naitong Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences 海南大学 Environment and Plant Protection Institute Chinese Academy of Tropical Agricultural Sciences

Huanglongbing(HLB) is the most destructive disease of citrus *** disease is caused by Candidatus Iiberibacter spp.,which vectored by the psyllids Diaphorina citri Kuwayama and Trioza *** proteins are important in bacterial pathogenesis and structure *** of them express at high *** obtain at least one high-level expression of secretory protein genes(SPGs),ten candidated SPGs were chosen from the Candidatus Iiberibacter asiaticus by bioinformatic *** SPGs were tested by PCR,qPCR and RT-qPCR method,*** result showed that two SPGs,408 and pap,are highly *** 408 and pap genes were further constructed into the plant expression vector pCAMBIA 1300(GV1300:GFP) and expressed in tobacco leaf epidermal cells for subcellular localization *** transient expression results indicated that 408 protein is located in the nucleus and cytoplasm of tobacco leaf ***,pap protein was located in the radially arranged micro-fibrils of tobacco guard cells that may relate to help pathogen invade into plant *** this study,408 and pap secretory protein genes of Candidatus Iiberibacter asiaticus were obtained and the subcellular localization of these two genes in tobacco leaves was *** research laies an important foundation for the preparation of the antiserum against the Candidatus Iiberibacter asiaticus and the early detection and prevention of citrus HLB.

关键词： Candidatus Liberibacter asiaticus Secretory protein DNA amount RNA transcription Subcellular localization

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海南仙人掌matK基因的克隆和序列分析

基因组学与应用生物学 2016年第11期35卷 3135-3140页

作者：吴育鹏余乃通周启林王健华刘志昕文昌市农产品质量安全检验检测站文昌571300 中国热带农业科学院热带生物技术研究所农业部热带作物生物学与遗传资源利用重点实验室海口571101

2015年12月,为了分析采自海南海口城西仙人掌mat K基因的序列及同源关系,利用RNA plant Reagent试剂盒提取仙人掌叶总RNA,反转录成第一链c DNA。同时设计仙人掌成熟酶K基因(mat K)的1对保守引物,利用RT-PCR的方法扩增仙人掌成熟酶K基因... 详细信息

2015年12月,为了分析采自海南海口城西仙人掌mat K基因的序列及同源关系,利用RNA plant Reagent试剂盒提取仙人掌叶总RNA,反转录成第一链c DNA。同时设计仙人掌成熟酶K基因(mat K)的1对保守引物,利用RT-PCR的方法扩增仙人掌成熟酶K基因,所得序列经Blastn进行比对,发现与已报道的22个仙人掌mat K基因核苷酸序列一致;利用Blastn的Distance tree of results构建系统进化树,结果显示海南仙人掌mat K基因与其它报道的30个仙人掌mat K基因同源性最高,同源性在99%以上,属于同一组,而与Opuntia basilaris(Gen Bank accession ***786750.1)同源性最低,为96.6%。本研究利用RT-PCR方法获得了仙人掌成熟酶K基因序列,同时构建的系统进化树确定了该仙人掌成熟酶K基因的同源关系。本结果将为海南仙人掌的进化研究提供科学线索。

关键词：仙人掌 matK基因克隆序列分析

香蕉束顶病毒海口分离物核穿梭蛋白(NSP)的纯化及抗血清制备

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基因组学与应用生物学 2010年第2期29卷 375-378页

作者：余乃通冯团成王建华刘志昕中国热带农业科学院热带生物技术研究所农业部热带作物生物技术重点开放实验室海口571101 海南大学环境与植物保护学院海口570228

本研究以本实验室保存的含重组载体pDEST17-NSP的原核表达菌株*** BL21(DE3)为材料,于25℃、0.1mmol/L IPTG条件下诱导4h,集菌后超声波破碎,获得以包涵体形式表达的约20kD的融合蛋白。实验结果表明,将沉淀的融合蛋白溶于含6mol/L尿素的B... 详细信息

本研究以本实验室保存的含重组载体pDEST17-NSP的原核表达菌株*** BL21(DE3)为材料,于25℃、0.1mmol/L IPTG条件下诱导4h,集菌后超声波破碎,获得以包涵体形式表达的约20kD的融合蛋白。实验结果表明,将沉淀的融合蛋白溶于含6mol/L尿素的Binding Buffer中,再经Ni2+-NTA亲和层析纯化后,可获得高纯度的融合蛋白。将纯化融合蛋白经12%SDS-PAGE电泳,切胶回收目的带,液氮研磨并按1:1(W/V)混合佐剂,4次免疫家兔,获得BBTV病毒核穿梭蛋白的特异性抗血清。以融合蛋白作抗原,间接ELISA法测定其抗血清效价为1:5000。田间检测样品的最佳抗血清工作浓度为1:500。Western Blot鉴定结果表明抗血清能与目的蛋白特异性结合。本研究的结果将为下一步NSP基因转录调控和蛋白功能研究奠定一定基础。

关键词：香蕉束顶病毒核穿梭蛋白纯化抗血清制备