根据日本晴cab4基因序列(GenBank:AK104499.1)设计引物,用RT-PCR的方法从籼稻9311中克隆了叶绿素a/b结合蛋白基因的全长cDNA,命名为cab-9311(cab gene from 9311)。insilico分析表明:cab-9311与cab4基因同源性为99%,编码的蛋白含有244...根据日本晴cab4基因序列(GenBank:AK104499.1)设计引物,用RT-PCR的方法从籼稻9311中克隆了叶绿素a/b结合蛋白基因的全长cDNA,命名为cab-9311(cab gene from 9311)。insilico分析表明:cab-9311与cab4基因同源性为99%,编码的蛋白含有244个氨基酸,与cab4基因编码的蛋白同源性为98%。蛋白分子质量为26.9kD,理论等电点为6.52。第54位～第216位氨基酸是一个典型的叶绿素a/b结合蛋白功能域(chlorophyll a/bbinding domain)。跨膜分析和蛋白质三级预测显示,该蛋白在C端有一个典型的跨膜区。亚细胞定位分析表明该蛋白定位于叶绿体,是一个叶绿体内囊体膜上的锚定蛋白。展开更多
Bovine coronavirus(BCoV)poses a significant threat to the global cattle industry,causing both respiratory and gastrointestinal infections in cattle populations.This necessitates the development of efficacious vaccines...Bovine coronavirus(BCoV)poses a significant threat to the global cattle industry,causing both respiratory and gastrointestinal infections in cattle populations.This necessitates the development of efficacious vaccines.While several inactivated and live BCoV vaccines exist,they are predominantly limited to calves.The immunization of adult cattle is imperative for BCoV infection control,as it curtails viral transmission to calves and ameliorates the impact of enteric and respiratory ailments across all age groups within the herd.This study presents an in silico methodology for devising a multiepitope vaccine targeting BCoV.The spike glycoprotein(S)and nucleocapsid(N)proteins,which are integral elements of the BCoV structure,play pivotal roles in the viral infection cycle and immune response.We constructed a remarkably effective multiepitope vaccine candidate specifically designed to combat the BCoV population.Using immunoinformatics technology,B-cell and T-cell epitopes were predicted and linked together using linkers and adjuvants to efficiently trigger both cellular and humoral immune responses in cattle.The in silico construct was characterized,and assessment of its physicochemical properties revealed the formation of a stable vaccine construct.After 3D modeling of the vaccine construct,molecular docking revealed a stable interaction with the bovine receptor bTLR4.Moreover,the viability of the vaccine’s high expression and simple purification was demonstrated by codon optimization and in silico cloning expression into the pET28a(+)vector.By applying immunoinformatics approaches,researchers aim to better understand the immune response to bovine coronavirus,discover potential targets for intervention,and facilitate the development of diagnostic tools and vaccines to mitigate the impact of this virus on cattle health and the livestock industry.We anticipate that the design will be useful as a preventive treatment for BCoV sickness in cattle,opening the door for further laboratory studies.展开更多
Phospholipase D （PLD, EC 3.1.4.4） plays an important role in adaptive response of postharvest fruit to environment. In this study, a novel cDNA of PLDα was isolated with the strategy of in silico cloning in combina...Phospholipase D （PLD, EC 3.1.4.4） plays an important role in adaptive response of postharvest fruit to environment. In this study, a novel cDNA of PLDα was isolated with the strategy of in silico cloning in combination with RT-PCR from peach （Prunus persica L. cv. Jiubao）. The obtained PLDα gene contained a complete open reading frame encoding a 92- kDa protein of 810 amino acid residues, which possessed the characteristic C2 domain and two catalytic HKD motifs. The alignment analysis of the deduced peach PLDa protein with other known PLDα family proteins indicated that peach PLDα was conserved and highly homologous with strawberry PLDα. Semi-quantitative RT-PCR and Northern blot analysis indicated PLDα mRNA in peach fruits could be induced by low temperature. This work provided a scientific basis for further investigating the mechanism of postharvest fruit adaptation to low temperature.展开更多
[ Objective ] This study aimed to verify the feasibility of in silico cloning of functional candidate genes in tea. [ Method ] Theobroma cacao caffeine syn- thase gene BCS1 was used as a probe to search the establishe...[ Objective ] This study aimed to verify the feasibility of in silico cloning of functional candidate genes in tea. [ Method ] Theobroma cacao caffeine syn- thase gene BCS1 was used as a probe to search the established tea EST database using BLAST; 26 tea ESTs highly homologous to BCS1 were obtained, which were assembled using CAP （contig assembly program） of BioEdit software; subsequently, two EST configs harboring ORF were obtained, which were named TCSnewl and TCSnew2, respectively. Nucleotide sequences and deduced amino acid sequences of theses two genes were compared with those of cDNA of tea caffeine synthase gene TCS in the GenBank database that was cloned with experimental biological method. A phylogenetic tree was constructed for homalogous analysis of the deduced amino acid sequences of theses three genes. [ Result] in silico cloning of functional candidate genes in tea using a homologous gene of distantly related species as a probe is a feasible technical means. [ Conclusion] This study provided the basis for in silico cloning of other functional genes in tea.展开更多
文摘Bovine coronavirus(BCoV)poses a significant threat to the global cattle industry,causing both respiratory and gastrointestinal infections in cattle populations.This necessitates the development of efficacious vaccines.While several inactivated and live BCoV vaccines exist,they are predominantly limited to calves.The immunization of adult cattle is imperative for BCoV infection control,as it curtails viral transmission to calves and ameliorates the impact of enteric and respiratory ailments across all age groups within the herd.This study presents an in silico methodology for devising a multiepitope vaccine targeting BCoV.The spike glycoprotein(S)and nucleocapsid(N)proteins,which are integral elements of the BCoV structure,play pivotal roles in the viral infection cycle and immune response.We constructed a remarkably effective multiepitope vaccine candidate specifically designed to combat the BCoV population.Using immunoinformatics technology,B-cell and T-cell epitopes were predicted and linked together using linkers and adjuvants to efficiently trigger both cellular and humoral immune responses in cattle.The in silico construct was characterized,and assessment of its physicochemical properties revealed the formation of a stable vaccine construct.After 3D modeling of the vaccine construct,molecular docking revealed a stable interaction with the bovine receptor bTLR4.Moreover,the viability of the vaccine’s high expression and simple purification was demonstrated by codon optimization and in silico cloning expression into the pET28a(+)vector.By applying immunoinformatics approaches,researchers aim to better understand the immune response to bovine coronavirus,discover potential targets for intervention,and facilitate the development of diagnostic tools and vaccines to mitigate the impact of this virus on cattle health and the livestock industry.We anticipate that the design will be useful as a preventive treatment for BCoV sickness in cattle,opening the door for further laboratory studies.
基金supported by China Post doctoral Science Foundation (20070420095)the National Natural Science Foundation of China (30571279,30871699,30901012)+1 种基金Innovative Foundation of Shanghai UniversitySystems Biology Research Foundation of Shanghai University
文摘Phospholipase D （PLD, EC 3.1.4.4） plays an important role in adaptive response of postharvest fruit to environment. In this study, a novel cDNA of PLDα was isolated with the strategy of in silico cloning in combination with RT-PCR from peach （Prunus persica L. cv. Jiubao）. The obtained PLDα gene contained a complete open reading frame encoding a 92- kDa protein of 810 amino acid residues, which possessed the characteristic C2 domain and two catalytic HKD motifs. The alignment analysis of the deduced peach PLDa protein with other known PLDα family proteins indicated that peach PLDα was conserved and highly homologous with strawberry PLDα. Semi-quantitative RT-PCR and Northern blot analysis indicated PLDα mRNA in peach fruits could be induced by low temperature. This work provided a scientific basis for further investigating the mechanism of postharvest fruit adaptation to low temperature.
基金Supported by National Science and Technology Support Program of China(2011BAD01B01)Youth Talent Innovation Fund of Fujian Academy of Agricultural Sciences(2011QC-2)Special Fund for"Double Hundred Plan"of Fujian Academy of Agricultural Sciences(sbmx1303-1)
文摘[ Objective ] This study aimed to verify the feasibility of in silico cloning of functional candidate genes in tea. [ Method ] Theobroma cacao caffeine syn- thase gene BCS1 was used as a probe to search the established tea EST database using BLAST; 26 tea ESTs highly homologous to BCS1 were obtained, which were assembled using CAP （contig assembly program） of BioEdit software; subsequently, two EST configs harboring ORF were obtained, which were named TCSnewl and TCSnew2, respectively. Nucleotide sequences and deduced amino acid sequences of theses two genes were compared with those of cDNA of tea caffeine synthase gene TCS in the GenBank database that was cloned with experimental biological method. A phylogenetic tree was constructed for homalogous analysis of the deduced amino acid sequences of theses three genes. [ Result] in silico cloning of functional candidate genes in tea using a homologous gene of distantly related species as a probe is a feasible technical means. [ Conclusion] This study provided the basis for in silico cloning of other functional genes in tea.
