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当前位置: 首页 > 文章 > 集胞藻PCC6803乙醇酸脱氢酶基因的克隆及其叶绿体定位分析 核农学报 2018 (7) 1330-1337
Position: Home > Articles > Cloning and Chloroplast-targeting Analysis of Glycolate Dehydrogenase From Synechocystis PCC6803 Journal of Nuclear Agricultural Sciences 2018 (7) 1330-1337

集胞藻PCC6803乙醇酸脱氢酶基因的克隆及其叶绿体定位分析

作  者：
胥华伟;侯典云
关键词：
光呼吸;集胞藻PCC6803;乙醇酸脱氢酶;叶绿体定位;瞬时表达
摘  要：
为明确集胞藻PCC6803乙醇酸脱氢酶与水稻Rubisco小亚基叶绿体转运肽(RCTP)的融合蛋白是否能准确定位到水稻叶绿体,本研究以特异引物从集胞藻PCC6803基因组DNA中扩增获得约1.5 kb的片段,将该片段与水稻Rubisco小亚基叶绿体转运肽连接,然后将融合片段连入瞬时表达载体P322-d1-e GFP,测序,获得融合基因RCTP-gdh,进一步构建了叶绿体定位的瞬时表达载体RCTPGDH-e GFP-d1并进行了叶绿体定位研究。结果表明,集胞藻PCC6803 GDH共编码492个氨基酸,含有FAD结合域、FAD关联的氧化酶活性(222~464 aa)以及乙醇酸氧化酶亚基(51~463 aa)。激光共聚焦显微镜分析表明,融合蛋白RCTP-GDH-e GFP可以准确定位到水稻叶绿体,但其转运效率低于RCTP-e GFP的转运效率,叶绿体和细胞质中的融合蛋白RCTP-GDH-e GFP都易于聚集而呈斑点状。本研究为进一步应用集胞藻gdh基因改造C3作物光呼吸代谢途径,抑制C3作物光呼吸速率,提高作物光合速率奠定了基础。
译  名：
Cloning and Chloroplast-targeting Analysis of Glycolate Dehydrogenase From Synechocystis PCC6803
作  者：
XU Huawei;HOU Dianyun;College of Agriculture,Henan University of Science and Technology;
关键词：
photorespiration;;Synechocystis sp. PCC6803;;glycolate dehydrogenase;;chloroplast-targeting;;transient expression
摘  要：
To investigate wether the fusion protein derived from glycolate dehydrogenase in Synechocystis PCC6803 and Rubisco small subunit chloroplast transit peptide in rice could target into rice chloroplasts perfectly,a fragment about1. 5 Kb was amplified using specific primers from Synechocystis sp. PCC 6803 genomic DNA,the fragment was fused to transit peptide of rice Rubisco small subunit( RCTP),the fusion fragment RCTP-gdh was then inserted into transient expression vector P322-d1-e GFP,the sequencing results showed that we have obtained the glycolate dehydrogenase gene. Then the recombinant chloroplast-targeting transient expression vector RCTP-GDH-e GFP-d1 was constructed and its subcellular localization was analyzed. The results showed that Synechocystis sp. PCC 6803 GDH encoded 492 amino acid residues and contained FAD binding domine,FAD-oxidase C,as well as glycolate oxidase subunit D domains.Subcellular localization analysis using confocal laser scanning microcopy( CLSM) showed that the fusion protein RCTPGDH-e GFP could be translocated into rice chloroplasts successfully,but the targeting efficiency was lower than RCTPe GFP and the fusion protein RCTP-GDH-e GFP tend to aggregate and form speckles in both cytoplasm and chloroplasts,which provide scientific reference to engineering the photorespiration pathway,inhibiting of photorespiration rate and increasing photosynthetic rate of C_3 crops by further using of gdh gene.

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