Molecular cloning and characterization of a cotton phosphoenolpyruvate carboxylase gene  

作　　者：Zhixin Qiao Jin-Yuan Liu 

机构地区：[1]Laboratory of Molecular Biology and Protein Science Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China

出　　处：《Progress in Natural Science:Materials International》2008年第5期539-545,共7页自然科学进展·国际材料（英文版）

基　　金：This work was supported by the State Key Basic Research and Development Plan of China(2004CB 117303)；the Hi—Tech Research and Developm ent Program of China (2002AA207006）； the National Natural Science Foundation of China(30170080，39770078).

摘　　要：Phosphoenolpyruvate carboxylase （PEPC） plays diverse physiological functions during plant development. In this study, a new phos- phoenolpyruvate carboxylase gene GhPEPC2 is isolated from cotton （Gossypium hirsutum cv, zhongrnian 35） by RACE-PCR, The cloned cDNA of GhPEPC2 is 3364 bp in length, and has an open reading frame of 2913 bp, encoding for 971 putative amino acids with a calculated molecular mass of 110,6 kD and pI of 5,56. The deduced amino acid sequence of GhPEPC2 shares high similarity with other reported plant PEPCs, Southern blot analysis indicates that the cotton PEPC exists as a small gene family and the GhPEPC2 might have two copies in the cotton genome, The semi-quantitative RT-PCR reveals that GhPEPC2 constitutively expresses in all the tissues of cot- ton and accumulated highly in roots, flowers and embryos but relatively low in stems and fibers, In addition, the recombinant GhPEPC2 has been purified by expressing it in Escherichia coli and the catalytic properties of it were also investigated. The results showed that GhPEPC2 is a typical C3 PEPC with a higher Km （83,6 p.M） and lower Vmax （8,0 p.mol min^-1 mg^-1） compared with the C3 PEPCs previously reported.Phosphoenolpyruvate carboxylase (PEPC) plays diverse physiological functions during plant development. In this study, a new phos- phoenolpyruvate carboxylase gene GhPEPC2 is isolated from cotton (Gossypium hirsutum cv. zhongmian 35) by RACE-PCR. The cloned cDNA of GhPEPC2 is 3364 bp in length, and has an open reading frame of 2913 bp, encoding for 971 putative amino acids with a calculated molecular mass of 110.6 kD and pI of 5.56. The deduced amino acid sequence of GhPEPC2 shares high similarity with other reported plant PEPCs. Southern blot analysis indicates that the cotton PEPC exists as a small gene family and the GhPEPC2 might have two copies in the cotton genome. The semi-quantitative RT-PCR reveals that GhPEPC2 constitutively expresses in all the tissues of cot- ton and accumulated highly in roots, owers and embryos but relatively low in stems and bers. In addition, the recombinant GhPEPC2 has been puri ed by expressing it in Escherichia coli and the catalytic properties of it were also investigated. The results showed that GhPEPC2 is a typical C3 PEPC with a higher Km (83.6 lM) and lower Vmax (8.0 lmol min 1 mg 1) compared with the C3 PEPCs pre- viously reported.

关 键 词：Phosphoenolpyruvate carboxylase Recombinant protein Southern blot COTTON 

分 类 号：S562[农业科学—作物学]