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作品数:2 被引量:6H指数:1
供职机构:大连海洋大学更多>>
发文基金:国家自然科学基金辽宁省教育厅高等学校科学研究项目更多>>
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Prokaryotic Expression and Purification of Ds MAPK from Dunaliella salina
2015年
Dunaliella salina is an important model organism for investigating the salt tolerance mechanism of plant. MAPK is the key gene in the molecular pathway of salt tolerance of plant. In this experiment, the open reading frame (ORF) of DsMAPK gene was amplified by PCR. The target fragment was cloned in pGS-21a, a prokaryotic expression vector with GST-tag. The recombinant plasmid pGS-21a- DsMAPK was then transformed into E. coil BL21 (DE3). The expression was induced with IPTG. Then the expression form of the recombinant protein was analyzed. The expression products were purified with GST-SefinoseTM Kit and identified with SDS-PAGE and Western blot. The results showed the recombinant expression vector pGS-21a-DsMAPK was constructed successfully, and the molecular weight of the expressed recombinant protein was as same as expected. Western blot analysis showed the purified recombinant protein could be identified specially by the anti- GST antibody and had a good immunological activity. The successful expression of DsMAPK will lay a basis for the further research on the role of DsMAPK in the salt tolerance mechanism at the protein level.
岳文静柴晓杰刘丽颖武天祥刘艺琼
关键词:PURIFICATION
杜氏盐藻磷脂酶C基因DsPLC的克隆及盐胁迫下的表达分析被引量:6
2014年
为了研究磷脂酶C基因的结构与功能,采用RT-PCR和RACE技术从盐藻中克隆出了一种磷脂酶C基因(GenBank Accession No.KF573428),命名为DsPLC,并对其进行了生物信息学分析和盐胁迫条件下的表达分析。结果表明,DsPLC的cDNA全长2 628 bp,开放阅读框1 782 bp,编码593个氨基酸。该蛋白质为亲水性稳定蛋白,没有信号肽,也没有跨膜区域,是一种非跨膜蛋白,存在多个潜在的磷酸化位点,二级结构的主要元件为无规卷曲和α-螺旋。进一步的进化分析结果表明,盐藻与衣藻、团藻的亲缘关系最近。实时荧光定量PCR结果显示,正常情况下DsPLC的表达量很低,当受到高盐胁迫时表达量急剧升高,且在胁迫4 h时表达量达到最高,是对照组的10倍左右,差异极显著(P<0.01),表明DsPLC可能在盐藻抵御外界盐胁迫的过程中起重要作用。这些研究结果为进一步阐明DsPLC的功能及其作用机制奠定了分子基础。
韩冬梅柴晓杰王逸云刘世才岳文静
关键词:杜氏盐藻磷脂酶C全长CDNA生物信息学分析实时荧光定量PCR
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