绿色棉苯丙氨酸解氨酶基因GhPAL1的克隆和实时定量表达分析.pdf

分子植物育种，2012 年，第 10 卷，第 3 期，第 311-316 页 Molecular Plant Breeding, 2012, Vol.10, No.3, 311-316 研究报告 Research Report 绿色棉苯丙氨酸解氨酶基因 GhPAL1 的克隆和实时定量表达分析 赵伟涛 1 李艳军 2 张新宇 2 吴莉 1 孙杰 2* 1 石河子大学生命科学学院, 石河子, 832003; 2 石河子大学农学院, 新疆生产建设兵团绿洲生态农业重点实验室, 石河子, 832003 * 通讯作者, sunjiezh@ 摘 要 本研究以拟南芥( Arabidopsis thaliana )苯丙氨酸解氨酶(PAL)基因 cDNA 序列(AAC18870.1)为探针， 在 GenBank 中与棉花 EST 进行 tBLASTn 比对，将同源性高的 EST 序列拼接后获得一条棉花 PAL 全长 cDNA 基因序列，以此序列为模板设计引物，通过 RT-PCR 扩增从绿色棉纤维分离出了一个绿色棉 PAL 基 因全长 cDNA 序列，将该基因命名为 GhPAL1 (GenBank 登录号: JN032297)。序列分析表明，该 cDNA 全长 2 347 bp，含有一个编码 721 个氨基酸的开放阅读框。实时荧光定量 PCR 分析发现：该基因在绿色棉纤维中 的表达模式与白色棉纤维相似，但两者在同一时期的表达量差异显著，其在花后 6 d 绿色棉纤维细胞中的表 达量是同期白色棉的 6 倍以上，从该基因的表达特征推测 GhPAL1 基因可能在绿色棉纤维色素合成过程中 发挥重要作用。 关键词 绿色棉, 苯丙氨酸解氨酶, 实时荧光定量 PCR Cloning and Expression Analysis of a Phenylalanine Ammonia-lyase Gene GhPAL1 from Natural Green Cotton Fiber Zhao Weitao 1 Li Yanjun 2 Zhang Xinyu 2 Wu Li 1 Sun Jie 2* 1 College of Life Science, Shihezi University, Shihezi, 832003; 2 College of Agriculture, Shihezi University, The Key Oasis Eco-agriculture Laboratory of Xinjiang Production and Construction Group, Shihezi, 832003 * Corresponding author, sunjiezh@ DOI: 10.3969/mpb.010.000311 Abs tract In this study, the AtPAL amino acids sequence was used as a query sequence to make tBLASTn comparison with cotton ( Gossypium hirsutum ) EST database in GenBank. The highly homologous ESTs were identified, aligned and assembled into a full-length cDNA sequence, which was used to design specific primers. Then a new full-length cDNA sequence of PAL gene was isolated from green cotton fibers with RT-PCR method. This new gene was named GhPAL1 (GenBank accession No.JN032297). Sequence analysis reveals that its full-length cDNA Sequence is 2 347 bp, and has a open reading frame (ORF) encoding 721 amino acids. QRT-PCR analysis shows that the expressed pattern of GhPAL1 gene in green cotton fibers is similar to that in white cotton fibers, but the amount of expression at the same time has a significant difference be