Bhore Subhash Janardhan, Amelia Kassim, Wang Edina, Priyadharsini Sindhuja, Shah Farida Habib
Molecular Biology Division, Melaka Institute of Biotechnology, Lot 7, Melaka International Trade Centre City, 75450 Ayer Keroh, Melaka, Malaysia ; Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Bedong-Semeling Road, Bedong, 08100, Kedah, Malaysia.
Bioinformation. 2013;9(4):197-206. doi: 10.6026/97320630009197. Epub 2013 Feb 21.
The identification of genes and understanding of genes' expression and regulation in common bean (Phaseolus vulgaris L.) is necessary in order to strategize its improvement using genetic engineering techniques. Generation of expressed sequence tags (ESTs) is useful in rapid isolation, identification and characterization of the genes. To study the gene expression in P. vulgaris pods tissue, ESTs generation work was initiated. Early stage and late stage bean-pod-tissues cDNA libraries were constructed using CloneMiner cDNA library construction kit. In total, 5972 EST clones were isolated using random method of gene isolation. While processing ESTs, we found lycopene β-cyclase (PvLCY-β) and β-carotene hydroxylase (PvCHY-β) gene's cDNA. In carotenoid biosynthesis pathway, PvLCY-β catalyzes the production of carotene; and PvCHY-β is known to function as a catalyst in the production of lutein and zeaxanthin. To understand more about PvLCY-β and PvCHY-β, both strands of both cDNA clones were sequenced using M13 forward and reverse primers. Nucleotide and deduced protein sequences were analyzed and annotated using online bioinformatics tools. Results showed that PvLCY-β and PvCHY-β cDNAs are 1639 and 1107 bp in length, respectively. Analysis results showed that PvLCY-β and PvCHY-β gene's cDNA contains an open reading frame (ORF) that encodes for 502 and 305 amino acid residues, respectively. The deduced protein sequence analysis results also showed the presence of conserved domains needed for PvLCY-β and PvCHY-β functions. The phylogenetic analysis of both PvLCY-β and PvCHY-β proteins showed it's closeness with the LCY-β and CHY-β proteins from Glycine max, respectively. The nucleotide sequence of PvLCY-β and PvCHY-β gene's cDNA and it's annotation is reported in this paper.
为了利用基因工程技术制定普通菜豆（Phaseolus vulgaris L.）的改良策略，鉴定其基因并了解基因的表达和调控是必要的。表达序列标签（EST）的产生有助于快速分离、鉴定和表征基因。为了研究普通菜豆豆荚组织中的基因表达，启动了EST产生工作。使用CloneMiner cDNA文库构建试剂盒构建了早期和晚期菜豆荚组织cDNA文库。总共使用随机基因分离方法分离了5972个EST克隆。在处理EST时，我们发现了番茄红素β-环化酶（PvLCY-β）和β-胡萝卜素羟化酶（PvCHY-β）基因的cDNA。在类胡萝卜素生物合成途径中，PvLCY-β催化胡萝卜素的产生；已知PvCHY-β在叶黄素和玉米黄质的产生中起催化作用。为了更多地了解PvLCY-β和PvCHY-β，使用M13正向和反向引物对两个cDNA克隆的两条链进行了测序。使用在线生物信息学工具对核苷酸和推导的蛋白质序列进行了分析和注释。结果表明，PvLCY-β和PvCHY-β cDNA的长度分别为1639和1107 bp。分析结果表明，PvLCY-β和PvCHY-β基因的cDNA包含一个开放阅读框（ORF），分别编码502和305个氨基酸残基。推导的蛋白质序列分析结果还显示了PvLCY-β和PvCHY-β功能所需的保守结构域的存在。对PvLCY-β和PvCHY-β蛋白的系统发育分析表明，它们分别与来自大豆的LCY-β和CHY-β蛋白密切相关。本文报道了PvLCY-β和PvCHY-β基因cDNA的核苷酸序列及其注释。
