将去信号肽的耐酸性高温α-淀粉酶突变基因amyd克隆到大肠杆菌表达载体pET-30a上，实现重组质粒pET-amyd在大肠杆菌BL21（DE3）中的高效表达。经硫酸铵盐析、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶层析，重组酶AMYD...将去信号肽的耐酸性高温α-淀粉酶突变基因amyd克隆到大肠杆菌表达载体pET-30a上，实现重组质粒pET-amyd在大肠杆菌BL21（DE3）中的高效表达。经硫酸铵盐析、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶层析，重组酶AMYD的比活达到354．6U·mg^-1、纯化倍数为83．83，获得凝胶电泳条带单一的蛋白样品，经SDS-PAGE检测，AMYD酶分子量为63．5kDa。重组酶AMYD的最适温度80℃、最适反应pH值为4．5，在温度低于90℃、反应pH值4．0～6．5的条件下，酶活较稳定。展开更多
克隆并表达变形链球菌耐氟菌株耐酸相关基因ropA。以变形链球菌UA159的耐氟菌株全基因组为模版,PCR扩增ropA基因并与p MD-18T克隆载体连接构建重组克隆质粒,测序鉴定。Bam HⅠ、HindⅢ双酶切重组克隆质粒,回收目的基因片段并与p Pro EX ...克隆并表达变形链球菌耐氟菌株耐酸相关基因ropA。以变形链球菌UA159的耐氟菌株全基因组为模版,PCR扩增ropA基因并与p MD-18T克隆载体连接构建重组克隆质粒,测序鉴定。Bam HⅠ、HindⅢ双酶切重组克隆质粒,回收目的基因片段并与p Pro EX HTa表达载体通过粘性末端连接构建重组表达质粒,转化入大肠埃希菌感受态细胞DH5α,IPTG诱导,SDS-PAGE检测Rop A表达量。测序结果为变形链球菌UA159的耐氟菌株ropA基因碱基序列与亲代菌株UA159完全一致。SDS-PAGE结果显示IPTG成功诱导Rop A蛋白表达,并且随诱导时间的延长蛋白表达增多。变形链球菌UA159耐氟菌株的耐酸相关基因ropA未发生突变,说明变形链球菌耐氟菌株耐酸性增强不是由ropA碱基序列的改变导致。本研究成功诱导Rop A蛋白表达,为后续研究Rop A蛋白功能奠定了基础。展开更多
Objective: To knock out the entire Luxs gene of Streptococcus mutans(S.mutans) UA159 strain via homologous recombination and construct a Luxs-deleted mutant strain of S. mutans. To study the difference between the aci...Objective: To knock out the entire Luxs gene of Streptococcus mutans(S.mutans) UA159 strain via homologous recombination and construct a Luxs-deleted mutant strain of S. mutans. To study the difference between the acid resistance of S. mutans Ingbritt C international standard strain and the acid resistance of LuxS mutant strain. Methods: Two DNA fragments locating in the upper and downstream of Luxs gene were amplified and a erythromycin resistance gene of PJT10 between them were engineered into PUC19 plasmid for constructing the recombination plasmid pUCluxKO. Electrotransformation of S.mutans cells with pUCluxKO-mutant resulted in isolation of erythromycin resistant S. mutans transformants, which was identified by polymerase chain reaction, V.harveyi BB170 luminescence bioassay and sequencing analysis. Solutions of S. mutans standard strain and LuxS mutant strain with same density were made and cultured at pH 3.5 to 7.0 BHI liquid for the same period.Terminal growth situation was compared.Firstly acidized in pH 5.5 BHI liquid,the two strains were cultured at pH 3.0 BHI liquid. The acid tolerance responses of the two strains were compared.Results:Restriction endonuclease analyses showed that pUCluxKO-mutant vector had been successfully recombined. The Luxs-deleted status of S.mutans mutants was confirmed by PCR with primers which were specific for the genes of Luxs and Erythromycin resistance. S.mutans mutant can not induce bioluminescence, indiating the mutant had been successfully recombined. After twenty generations of culture, the constructed Chinese S.mutans mutants were confirmed to be stable. Significant difference of aciduricity was observed between S.mutans standard strain and LuxS mutant strain.The acid resistance of standard strain was stronger than that of LuxS mutant strain.The two strains both displayed the capability of acid tolerance responses. Conclusion:The S.mutans gene allelic exchange plasmid is constructed correctively and a Luxs-negative mutants of S.mutans is constructed, which can help to further study the role of Luxs in the pathogenesis of S.mutans. LuxS mutant strain is more sensitive to acid inactivation,but the capability of acid tolerance responses exist still.展开更多
We examined salt tolerance responsive genes in Pak-choi under salt stress and analyze their potential function. The LRNA differential display was used to screen the transcript derived fragments （TDFs） related to sal...We examined salt tolerance responsive genes in Pak-choi under salt stress and analyze their potential function. The LRNA differential display was used to screen the transcript derived fragments （TDFs） related to salinity tolerance in tolerant and Loderately tolerant Pak-choi germplasm. Seventy-eight primer combinations generated 101 differential eDNA fragments, which ere divided into 10 expression types. Seven cDNA sequences （GenBank accession Nos. DQ006915-DQ006921） obtained and ,~quenced were highly homologous to some known expression genes or the genes related to the signaling pathways in plants under ifferent abiotic stress.展开更多
文摘将去信号肽的耐酸性高温α-淀粉酶突变基因amyd克隆到大肠杆菌表达载体pET-30a上，实现重组质粒pET-amyd在大肠杆菌BL21（DE3）中的高效表达。经硫酸铵盐析、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶层析，重组酶AMYD的比活达到354．6U·mg^-1、纯化倍数为83．83，获得凝胶电泳条带单一的蛋白样品，经SDS-PAGE检测，AMYD酶分子量为63．5kDa。重组酶AMYD的最适温度80℃、最适反应pH值为4．5，在温度低于90℃、反应pH值4．0～6．5的条件下，酶活较稳定。
文摘克隆并表达变形链球菌耐氟菌株耐酸相关基因ropA。以变形链球菌UA159的耐氟菌株全基因组为模版,PCR扩增ropA基因并与p MD-18T克隆载体连接构建重组克隆质粒,测序鉴定。Bam HⅠ、HindⅢ双酶切重组克隆质粒,回收目的基因片段并与p Pro EX HTa表达载体通过粘性末端连接构建重组表达质粒,转化入大肠埃希菌感受态细胞DH5α,IPTG诱导,SDS-PAGE检测Rop A表达量。测序结果为变形链球菌UA159的耐氟菌株ropA基因碱基序列与亲代菌株UA159完全一致。SDS-PAGE结果显示IPTG成功诱导Rop A蛋白表达,并且随诱导时间的延长蛋白表达增多。变形链球菌UA159耐氟菌株的耐酸相关基因ropA未发生突变,说明变形链球菌耐氟菌株耐酸性增强不是由ropA碱基序列的改变导致。本研究成功诱导Rop A蛋白表达,为后续研究Rop A蛋白功能奠定了基础。
文摘Objective: To knock out the entire Luxs gene of Streptococcus mutans(S.mutans) UA159 strain via homologous recombination and construct a Luxs-deleted mutant strain of S. mutans. To study the difference between the acid resistance of S. mutans Ingbritt C international standard strain and the acid resistance of LuxS mutant strain. Methods: Two DNA fragments locating in the upper and downstream of Luxs gene were amplified and a erythromycin resistance gene of PJT10 between them were engineered into PUC19 plasmid for constructing the recombination plasmid pUCluxKO. Electrotransformation of S.mutans cells with pUCluxKO-mutant resulted in isolation of erythromycin resistant S. mutans transformants, which was identified by polymerase chain reaction, V.harveyi BB170 luminescence bioassay and sequencing analysis. Solutions of S. mutans standard strain and LuxS mutant strain with same density were made and cultured at pH 3.5 to 7.0 BHI liquid for the same period.Terminal growth situation was compared.Firstly acidized in pH 5.5 BHI liquid,the two strains were cultured at pH 3.0 BHI liquid. The acid tolerance responses of the two strains were compared.Results:Restriction endonuclease analyses showed that pUCluxKO-mutant vector had been successfully recombined. The Luxs-deleted status of S.mutans mutants was confirmed by PCR with primers which were specific for the genes of Luxs and Erythromycin resistance. S.mutans mutant can not induce bioluminescence, indiating the mutant had been successfully recombined. After twenty generations of culture, the constructed Chinese S.mutans mutants were confirmed to be stable. Significant difference of aciduricity was observed between S.mutans standard strain and LuxS mutant strain.The acid resistance of standard strain was stronger than that of LuxS mutant strain.The two strains both displayed the capability of acid tolerance responses. Conclusion:The S.mutans gene allelic exchange plasmid is constructed correctively and a Luxs-negative mutants of S.mutans is constructed, which can help to further study the role of Luxs in the pathogenesis of S.mutans. LuxS mutant strain is more sensitive to acid inactivation,but the capability of acid tolerance responses exist still.
基金Project supported by the National "the Tenth Five-Year-Plan" Key Program (No. 2004BA525B08)China and the Key Laboratory of Vegetable Genetics and Physiology, Ministry of Agriculture, China
文摘We examined salt tolerance responsive genes in Pak-choi under salt stress and analyze their potential function. The LRNA differential display was used to screen the transcript derived fragments （TDFs） related to salinity tolerance in tolerant and Loderately tolerant Pak-choi germplasm. Seventy-eight primer combinations generated 101 differential eDNA fragments, which ere divided into 10 expression types. Seven cDNA sequences （GenBank accession Nos. DQ006915-DQ006921） obtained and ,~quenced were highly homologous to some known expression genes or the genes related to the signaling pathways in plants under ifferent abiotic stress.
