棉花中一个钝叶醇14α-脱甲基酶基因同源基因(GhCYP51G1)的克隆、序列特征和表达分析

Molecular Identification and Expression Analysis of GhCYP51G1 Gene, a Homologue of Obtusifoliol-14α-demethylase Gene, from Upland Cotton (Gossypium hirsutum L.)

为研究植物固醇在棉花纤维细胞生长发育中的作用和信号传导机制,通过筛选棉花EST数据库并对目标EST序列进行整合和分析,从陆地棉栽培品种徐州142正在发育的纤维中克隆了植物固醇合成途径的重要酶基因——钝叶醇14α-脱甲基酶基因的同源基因,命名为GhCYP51G1(GenBank登录号为EU727154)。该基因编码486个氨基酸残基,其分子量和等电点分别为55.2kD和8.87。推导的氨基酸序列与烟草、马铃薯和葡萄等物种中CYP51家族成员有较高的同源性。而且具有钝叶醇14α-脱甲基酶序列中的典型保守结构域,如多个底物结合位点和血红素结合域。说明该克隆基因是钝叶醇14α-脱甲基酶基因的同源基因。实时定量RT-PCR的结果表明GhCYP51G1基因在快速伸长期的纤维中具有较高的表达水平,而在子叶、雌蕊和雄蕊以及开花后6d胚珠、开花后0d和2d的胚珠纤维中表达水平较低。在开花后8d的纤维细胞中GhCYP51G1的表达水平最高,这些结果说明该基因在纤维细胞的伸长生长中具有重要作用。同时在纤维生长过程中,由于生长素能够下调GhCYP51G1基因的表达,暗示植物固醇在植物激素,特别是油菜素类固醇物质和生长素的相互作用中具有一定作用。

Besides as a precursor of BL biosynthesis, more and more evidences support the hypothesis that phytosterols possess a BL-independent signaling pathway. Furthermore, the obtusifoliol is regarded as a signal molecule in sterol signaling. To understand the effects of phytosterols on the development of cotton （Gossypium hirsutum L.） fibers and the molecular basic of sterol signaling in cotton fiber growth, we cloned a gene encoding a homologue of obtusifoliol 14α-demethylase from developing fibers of upland cotton cv. Xuzhou 142 through screening cotton fiber EST （Express Sequence Tag） database and contigging the candi- date ESTs. The full length of GhCYP51G1 （GenBank accession No. EU727154） was 1 710 bp, including a 160 bp 5＇-untranslated region （UTR）, a 1 461 bp open reading frame （ORF）, and an 89 bp 3＇-UTR. The GhCYP51G1 encoded a polypeptide of 486 amino acid residues with a predicted molecular mass of 55.2 kD. The deduced amino acid sequences had high homology with the members of CYP51 family in plant kingdom. Moreover, many typical conserved regions were characterized as the obtusifoliol 14α-demethylase, such as substrate recognition sites （SRS） and heme-binding region presented in the deduced protein. Quantitative real-time RT-PCR analysis revealed that the higher expression levels of GhCYP51G1 gene were detected in 8-DPA, 12-DPA, 18-DPA fibers, and 12-DPA ovules. These results indicate that GhCYP15G1 gene plays an important role in fiber elongation. Furthermore, Auxin significantly down regulates the expression level of GhCYP51G1 in cotton fiber growth. This suggested that phytosterols play a role in the interaction of plant hormones, especially brassinosteroids and auxin.