陆地棉PIP亚家族基因的克隆及表达特征分析

该研究以陆地棉苯基香豆满苄基醚还原酶(phenylcoumaran benzylic ether reductase,PCBER)氨基酸序列为探针,利用Blastp从陆地棉基因组数据库中发现了6个同源性较高的基因。根据6个基因序列设计引物,利用RT-PCR技术从陆地棉纤维细胞中克隆出了这6个基因的全长cDNA序列,分别命名为GhPCBER1、GhPCBER2、GhPCBER3、GhIFR、GhPLR1和GhPLR2。多重序列比对和进化树分析发现,6个蛋白均含有PIP类型蛋白的所有保守性基序和活性残基,属于PIP亚家族。实时荧光定量PCR结果显示,除GhPLR1之外其他5个PIP亚家族基因均在纤维细胞中优势或特异表达;在纤维发育过程中,GhPCBER1、GhPCBER2、GhPCBER3和GhIFR的表达均表现为先上升后下降,GhPCBER1和GhPCBER2在花后21d表达量达到最高,GhPCBER3和GhIFR在花后18d达到最高,GhPLR1和GhPLR2在纤维中的表达量呈持续上升趋势。根据基因的表达特征,推测PIP亚家族可能在棉纤维的发育过程中发挥着重要作用。

Cloning and Expression Analysis of PIP Subfamily Genes in Gossypium hirsutum

Using the protein sequence of GhPCBER as a probe, six of genes showing high sequence homology were obtained from a genome database of Gossypium hirsutum L.tetraploid cotton with Blastp search. These sequences were used to design primers, and then six genes were isolated from cotton fiber of G. hirsutum L. by using RT PCR technique and designated as GhPCBER1, GhPCBER2, GhPCBER3, GhIFR, GhPLR1 and GhPLR2, respectively. Multiple sequence alignment and phylogenetic tree analysis revealed that all of the 6 proteins belong to PIP subfamily since the conserved motifs and active residues in their sequences. Real time fluorescent quantitative PCR results showed that in addition to GhPLR1, all the other 5 PIP subfamily genes were specifically or preferentially expressed in fiber cells. Of 6 genes, the expression pattern of GhPCBER1, GhPCBER2, GhPCBER3 and GhIFR showed increasing firstly and then decreasing trend during cotton fiber developing stage. The expression level of GhPCBER1 and GhPCBER2 reached a maximum at 21 day post anthesis, and GhIFR and GhPCBER3 reached a maximum at 18 day post anthesis. The expression pattern of GhPLR1 and GhPLR2 showed an ascendant trend with cotton fiber developing. According to the characteristics of gene expression, it is speculated that the PIP subfamily may play an important role in cotton fiber development.