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《Chinese Traditional and Herbal Drugs》 2013-01
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Gene cloning, features of sequence, and analysis on temporal and spatial expression of Rehmannia glutinosa f. hueichingensis 3-ketoacyl CoA-thiolase

ZHOU Yan-qing 1,2,ZHANG Yong-hua 1,2,ZHANG Yu 1,2,CHEN Yan-mei 1,2,BAI Yan-yan 1,2,WEI Hai-fang 1,2,DUAN Hong-ying 1,2,ZHOU Chun-e 1,2 1.College of Life Sciences,Henan Normal University,Xinxiang 453007,China 2.Engineering Technology Research Center of Nursing and Utilization of Genuine Chinese Medicinal Materials,University and Colleges of Henan Province,Xinxiang 453007,China  
Objective To clone and analyze the full-length cDNA of Rehmannia glutinosa f.hueichingensis 3-ketoacyl CoA-thiolase(RghKAT) gene and to provide the candidate genes and theoretical basis for the molecular breeding of R.glutinosa f.hueichingensis.Methods The full-length cDNA sequence of RghKAT gene was amplified by quantitative RT-PCR and RACE techniques with degenerated primers being designed based on the conserved domain of ARGOS base sequences from other plants.The nucleotide and amino acid sequences were compared by bioinformatics technology.The temporal and spatial expression levels in 10 tissues of regenerated plantlets at two stages were detected by quantitative RT-PCR.Results The full-length of RghKAT gene was 1 713 bp,including an open reading frame(1 395 bp) encoding a 464-amino acid protein.Homology comparison and phylogenetic analysis revealed that RghKAT shared the high nucleotide sequence identity to those of Vitis vinefera(84%),Solanum lycopersicum(82%),Populus trichocarpa(82%),Arabidopsis thaliana(79%),and Triticum aestivum(73%),respectively.Meanwhile,the amino acid sequence coded by RghKAT gene shared the high identity to those of Petunia x hybrida(88%),V.vinefera(88%),Cucumis satiuus(86%),A.thaliana (87%),and T.aestivum(78%).The phylogenetic tree of RghKAT was consistent with the evolutionary relationship among the species.The physicochemical properties of RghKAT indicated that it was a slightly alkaline and stable protein,and the secondary structure was made of α-helixes,random coil,β-sheets,and β-turns.There was a putative signal peptide composed of 70 amino acid residues in the N-terminus,and the three-dimensional structure of RghKAT protein showed a typical characteristic sequence of thiolases.The quantitative RT-PCR assay demonstrated that the RghKAT mRNA could be detected in all 10 tissues examined at seedling and blooming stages with the highest expression level in the petals at blooming stage and the lowest one in the young leaves at seedling stage.Conclusion The full-length cDNA of RghKAT is cloned successfully,with thestructral properties of KAT gene and the typical characteristic sequence of product thiolases,which has the highest expression level in the petals of blooming stage.
【Fund】: 河南省基础与前沿技术研究计划项目(092300410009);; 河南省教育厅自然科学研究计划项目(2011A180022);; 河南省高校道地药材保育与利用研究工程技术中心开放课题基金项目(20100911);; 河南师范大学第三届学生创新创业论坛暨大学生创新性实验计划项目
【CateGory Index】: S567.234
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