The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vect...The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.展开更多
[Objective] The research aimed to induce the expression of FMDV structural protein VP1 in E.coli and purify the protein,then detect the activity.[Method] The fragment coding VP1 was amplified by PCR and doubly digeste...[Objective] The research aimed to induce the expression of FMDV structural protein VP1 in E.coli and purify the protein,then detect the activity.[Method] The fragment coding VP1 was amplified by PCR and doubly digested with BamH Ⅰ and XhoⅠ,then cloned into expression vector pGEX-4T-1 and pPROExHTb respectively to get recombinant plasmid pGEX-4T-1-VP1 and pPROExHTb-VP1.The recombinant plasmid pGEX-4T-1-VP1 and pPROExHTb-VP1 was transformed into E.coli BL21(DE3)and induced by IPTG,fusion protein was identifie...展开更多
Heat stress transcription factors (Hsfs) are the central regulators of defense response to heat stress. We identified a total of 25 rice Hsf genes by genome-wide analysis of rice (Oryza sativa L.) genome, including th...Heat stress transcription factors (Hsfs) are the central regulators of defense response to heat stress. We identified a total of 25 rice Hsf genes by genome-wide analysis of rice (Oryza sativa L.) genome, including the subspecies of O. japonica and O. indica. Proteins encoded by OsHsfs were divided into three classes according to their structures. Digital Northern analysis showed that OsHsfs were expressed constitutively. The expressions of these OsHsfs in response to heat stress and oxidative stress differed among the members of the gene family. Promoter analysis identified a number of stress-related cis-elements in the promoter regions of these OsHsfs. No significant correlation, however, was found between the heat-shock responses of genes and their cis-elements. Overall, our results provide a foundation for future research of OsHsfs function.展开更多
Parotid secretory protein (PSP) secreted abundantly in saliva, whose function is related with the anti-bacterial effect. The PSP cDNA has been isolated from pig parotid glands by 3′ and 5′ rapid amplification of cDN...Parotid secretory protein (PSP) secreted abundantly in saliva, whose function is related with the anti-bacterial effect. The PSP cDNA has been isolated from pig parotid glands by 3′ and 5′ rapid amplification of cDNA end (RACE), based on the conserved signal peptide region among the known mammalian PSP. The result of homologous comparison shows that pig PSP and human PSP shares the high identity at the level of the primary, secondary and tertiary protein structure. A search for functionally significant protein motifs revealed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu- X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is important to its function. RT-PCR, Dot blot and Northern blot analysis demonstrated that PSP was strongly expressed in parotid glands, but not in other tissues.展开更多
【目的】挖掘参与油茶糖代谢及逆境响应的糖外排转运子(sugars will eventually be exported transporters,SWEETs)。【方法】利用生物信息学方法分析油茶SWEETs家族的基因结构、蛋白基序、染色体定位、共线性关系、启动子区顺式作用元...【目的】挖掘参与油茶糖代谢及逆境响应的糖外排转运子(sugars will eventually be exported transporters,SWEETs)。【方法】利用生物信息学方法分析油茶SWEETs家族的基因结构、蛋白基序、染色体定位、共线性关系、启动子区顺式作用元件及上游调控因子等,并利用RT-qPCR分析CoSWEETs在不同时期、不同组织及不同逆境胁迫下的基因表达情况。【结果】从油茶中鉴定得到14个CoSWEETs基因,不均匀分布于10条染色体上,不同成员间内含子-外显子数目存在差异。根据系统进化关系,14个CoSWEETs可分为 4个分支,均具有1-2个MtN3 保守结构域,同一分支具有相似的基因结构和基序。根据启动子顺式作用元件和上游转录因子预测的分析结果,CoSWEETs启动子中含有多个与生长发育、植物激素和应激相关的调节元件,其表达可能受到ERF、DOF、BBR-BPC、MYB等转录因子的调控。RT-qPCR分析表明大部分CoSWEETs成员在果实和根中高表达,在种子中的表达水平与发育时期相关,并根据低温、高盐和干旱等非生物胁迫下CoSWEETs的表达模式挖掘出CoSWEET1、CoSWEET2、CoSWEET17等响应油茶低温、干旱或高盐胁迫的基因。【结论】CoSWEET基因的表达受到多种激素及转录因子调控,并在油茶种子发育与逆境胁迫响应中发挥重要作用。展开更多
基金Supported by NSFC-Joint Personnel Training Fund of Henan Province(U1204327)Special Fund for Construction of Provincial Key Laboratory in Henan Province(122300413217)
文摘The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.
基金Supported by National Supporting Plan(2006BAD06A14)Transgenic Major Projects(2008ZX08011-004)~~
文摘[Objective] The research aimed to induce the expression of FMDV structural protein VP1 in E.coli and purify the protein,then detect the activity.[Method] The fragment coding VP1 was amplified by PCR and doubly digested with BamH Ⅰ and XhoⅠ,then cloned into expression vector pGEX-4T-1 and pPROExHTb respectively to get recombinant plasmid pGEX-4T-1-VP1 and pPROExHTb-VP1.The recombinant plasmid pGEX-4T-1-VP1 and pPROExHTb-VP1 was transformed into E.coli BL21(DE3)and induced by IPTG,fusion protein was identifie...
基金Project (No. 30471118) supported by the National Natural Science Foundation of China
文摘Heat stress transcription factors (Hsfs) are the central regulators of defense response to heat stress. We identified a total of 25 rice Hsf genes by genome-wide analysis of rice (Oryza sativa L.) genome, including the subspecies of O. japonica and O. indica. Proteins encoded by OsHsfs were divided into three classes according to their structures. Digital Northern analysis showed that OsHsfs were expressed constitutively. The expressions of these OsHsfs in response to heat stress and oxidative stress differed among the members of the gene family. Promoter analysis identified a number of stress-related cis-elements in the promoter regions of these OsHsfs. No significant correlation, however, was found between the heat-shock responses of genes and their cis-elements. Overall, our results provide a foundation for future research of OsHsfs function.
基金supported by the National Major Basic Research Development Program(Grant No.G20000161)Beijing Natural Science Foundation(Grant No.5030001).
文摘Parotid secretory protein (PSP) secreted abundantly in saliva, whose function is related with the anti-bacterial effect. The PSP cDNA has been isolated from pig parotid glands by 3′ and 5′ rapid amplification of cDNA end (RACE), based on the conserved signal peptide region among the known mammalian PSP. The result of homologous comparison shows that pig PSP and human PSP shares the high identity at the level of the primary, secondary and tertiary protein structure. A search for functionally significant protein motifs revealed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu- X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is important to its function. RT-PCR, Dot blot and Northern blot analysis demonstrated that PSP was strongly expressed in parotid glands, but not in other tissues.
文摘【目的】挖掘参与油茶糖代谢及逆境响应的糖外排转运子(sugars will eventually be exported transporters,SWEETs)。【方法】利用生物信息学方法分析油茶SWEETs家族的基因结构、蛋白基序、染色体定位、共线性关系、启动子区顺式作用元件及上游调控因子等,并利用RT-qPCR分析CoSWEETs在不同时期、不同组织及不同逆境胁迫下的基因表达情况。【结果】从油茶中鉴定得到14个CoSWEETs基因,不均匀分布于10条染色体上,不同成员间内含子-外显子数目存在差异。根据系统进化关系,14个CoSWEETs可分为 4个分支,均具有1-2个MtN3 保守结构域,同一分支具有相似的基因结构和基序。根据启动子顺式作用元件和上游转录因子预测的分析结果,CoSWEETs启动子中含有多个与生长发育、植物激素和应激相关的调节元件,其表达可能受到ERF、DOF、BBR-BPC、MYB等转录因子的调控。RT-qPCR分析表明大部分CoSWEETs成员在果实和根中高表达,在种子中的表达水平与发育时期相关,并根据低温、高盐和干旱等非生物胁迫下CoSWEETs的表达模式挖掘出CoSWEET1、CoSWEET2、CoSWEET17等响应油茶低温、干旱或高盐胁迫的基因。【结论】CoSWEET基因的表达受到多种激素及转录因子调控,并在油茶种子发育与逆境胁迫响应中发挥重要作用。
