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

Cloning and Expression Analysis of a Phenylalanine Ammonia-lyase Gene GhPAL1 from Natural Green Cotton Fiber

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2347bp,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6d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。

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 eDNA sequence, which was used to design specific primers. Then a new full-length eDNA sequence of PAL gene was isolated from green cotton fibers with RT-PCR method. This new gene was named GhPA L1 （GenBank accession No.JN032297）. Sequence analysis reveals that its full-length eDNA 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 between them. At 6 days post-anthesis, the expression level of GhPAL1 gene in green cotton fibers is six times more than in white cotton fibers at the same period. It shows that GhPAL1 gene might play an important role in green pigmentation of natural green cotton fibers.