Molecular Identification and Expression Analysis of GhHYDRA1 Gene, a Homologue of HYDRA1 Gene From Upland Cotton (Gossypium hirsutum L.)

Beside as precursors of BRs biosynthesis, more and more evidences supported that phytosterols play an important role in plant growth and development. To investigate the effects of phytosterols on the fiber development of upland cotton （Gossypium hirsutum L.） and the molecular base of sterol regulating cotton fiber growth, a homologue of HYDRA1 was cloned from upland cotton （cv. Xuzhou 142） by screening cotton fiber EST database and contigging the candidate ESTs. The GhHYDRA1 encoded a polypeptide of 218 amino acid residues and the deduced amino acid sequences had high homology with the members of HYDRA1 in Populus trichocarpa, Solanum tuberosum, and Arabidopsis thaliana. Moreover, GhHYDRA1 had comparable transmembrane regions to AtHYDRA1 in sequence, length, order, and spacing, except for a C-terminal polylysine cluster. Quantitative real-time RT-PCR analysis revealed that the higher expression levels of GhHYDRA1 gene were detected in 6 to 12 DPA （days post anthesis） fibers, while the lower levels were observed in 0 DPA ovule （with fibers） and 16 to 18 DPA fibers. These results indicated that GhHYDRA1 is the homologue of HYDRA1 gene and plays a crucial role in fiber elongation. Furthermore, auxin and BL up-regulated the expression level of GhHYDRA1 while ABA and KT down-regulated the expression level of GhHYDRA1 in cotton ovule and fiber growth. The result suggested that phytosterols play a role in the interaction of plant hormones.

Phytosterol content and the campesterol:sitosterol ratio influence cotton fiber development: role of phytosterols in cell elongation

Phytosterols play an important role in plant growth and development, including cell division, cell elongation, embryogenesis, cellulose biosynthesis, and cell wall formation. Cotton fiber, which undergoes synchronous cell elongation and a large amount of cellulose synthesis, is an ideal model for the study of plant cell elongation and cell wall biogenesis. The role of phytosterols in fiber growth was investigated by treating the fibers with tfidemorph, a sterol biosynthetic inhibitor. The inhibition of phy- tosterol biosynthesis resulted in an apparent suppression of fiber elongation in vitro or in planta. The determination of phy- tosterol quantity indicated that sitosterol and campesterol were the major phytosterols in cotton fibers; moreover, higher con- centrations of these phytosterols were observed during the period of rapid elongation of fibers. Furthermore, the decrease and increase in campesterol：sitosterol ratio was associated with the increase and decease in speed of elongation, respectively, dur- ing the elongation stage. The increase in the ratio was associated with the transition from cell elongation to secondary cell wall synthesis. In addition, a number of phytosterol biosynthetic genes were down-regulated in the short fibers of ligon lintless-1 mutant, compared to its near-isogenic wild-type TM-1. These results demonstrated that phytosterols play a crucial role in cot- ton fiber development, and particularly in fiber elongation.

Cloning and Expression Analysis of a Brassinosteroid Biosynthetic Enzyme Gene,GhDWF1,from Cotton (Gossypium hirsuturm L.)

Brassinosteroids （BRs） are an important class of plant steroidal hormones that are essential in a wide variety of physiological processes. To determine the effects of BRs on the development of cotton fibers, through screening cotton fiber EST database and contigging the candidate ESTs, a key gene （GhDWF1） involved in the upstream biosynthetic pathway of BRs was cloned from developing fibers of upland cotton （Gossypium hirsutum L.） cv. Xuzhou 142. The full length of the cloned cDNA is 1 849 bp, including a 37 bp 5＇-untranslated region, an ORF of 1 692 bp, and a 120 bp 3＇-untranslated region. The cDNA encodes a polypeptide of 563 amino acid residues with a predicted molecular mass of 65 kD. The deduced amino acid sequence has high homology with the BR biosynthetic enzyme, DWARF1/DIMINUTO, from rice, maize, pea, tomato, and Arabidopsis. Furthermore, the typical conserved structures, such as the transmembrane domain, the FAD- dependent oxidase domain, and the FAD-binding site, are present in the GhDWF1 protein. The Southern blot indicated that the GhDWF1 gene is a single copy in upland cotton genome. RT-PCR analysis revealed that the highest level of GhDWF1 expression was detected in 0 DPA （day post anthesis） ovule （with fibers） while the lowest level was observed in cotyledon. The GhDWF1 gene presents high expression levels in root, young stem, and fiber, especially, at the fiber developmental stage of secondary cell wall accumulation. Moreover, the expression level was higher in ovules （with fibers） of wildtype （Xuzhou 142） than in ovules of fuzzless-lintless mutant at the same developmental stages （0 and 4 DPA）. The results suggest that the GhDWF1 gene plays a crucial role in fiber development.

棉花油菜素类固醇合成酶基因GhDWF1的克隆和表达分析

【目的】研究油菜素类固醇物质(BRs)在棉花纤维生长发育过程中的作用。【方法】通过棉花EST序列的筛选和整合,从陆地棉徐州142纤维中克隆了一个BRs合成酶基因GhDWF1。【结果】其cDNA序列全长1849bp,包含一个1692bp的开放阅读框,推导编码563个氨基酸,与水稻、玉米、豌豆、番茄和拟南芥的BRs合成酶DWARF1/DIMINUTO有较高的同源性,具有该类蛋白的典型结构。GhDWF1基因在开花当天的胚珠和纤维中表达最强,在根、幼茎和纤维发育的各个时期表达较高。与徐州142野生型相比,无绒无絮突变体胚珠中表达量较低。【结论】暗示GhDWF1基因的表达与棉花纤维的生长发育有密切关系。

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

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。