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为了揭示BpGT14基因的分子结构特征,明确糖基转移酶BpGT14基因的表达模式及其对非生物胁迫的响应机制,初步探索其在白桦生长发育中的功能,在克隆白桦GT14基因全长序列的基础上,利用生物信息学对其理化性质进行了分析,应用实时荧光定量PCR技术分析BpGT14基因在野生白桦植株雄花序、木质部、韧皮部及叶片4个不同部位不同月份的表达差异;同时,选用白桦茎段悬浮细胞进行了水杨酸(SA)、盐胁迫(NaCl)、重金属镉(CdCl_2)及4℃低温非生物胁迫处理,检测目的基因对逆境的响应情况。研究结果表明,BpGT14基因开放阅读框为1 302 bp,编码433个氨基酸。分析其氨基酸序列发现,该蛋白含有乙酰氨基葡糖转移酶结构域,属于14家族的重要结构域。该蛋白与其他物种GT14蛋白比对分析表明,这些蛋白在100—350氨基酸区域内保守性较高,而且该基因与毛果杨的GT14家族基因同源性高达79%。不同部位不同月份的表达模式分析结果表明,BpGT14基因的表达具有部位及时间特异性,其各部位表达量均与月份相关,同时发现其在木质部、韧皮部及叶片中的表达量较高,而在雄花序中表达量较低。非生物胁迫处理结果显示,BpGT14基因对不同处理均产生响应,但其响应模式不尽相同:SA、CdCl_2及低温处理结果显示,处理初期(6 h)目的基因上调表达,6 h处理时分别达到了对照组的51.2、48.9及3.3倍,24 h后相对表达量与对照组相比均下调,只有低温处理在96 h恢复上调表达;NaCl处理使白桦BpGT14基因的表达量全部呈现下调趋势,24 h处理后,BpGT14基因表达量下调明显,24 h后比对照组降低了93.7%。
In order to reveal the molecular structural characteristics of BpGT14 gene and clarify the expression pattern of glycosyltransferase BpGT14 gene and its mechanism of response to abiotic stress, we explored the function of BpGT14 gene in growth and development of birch, On the basis of bioinformatics, the physical and chemical properties of BpGT14 gene were analyzed by real-time fluorescence quantitative PCR. The expression differences of BpGT14 gene in four different parts of male birch plants from different periods of male inflorescence, xylem, phloem and leaf were analyzed by real-time fluorescence quantitative PCR. Segment cells were treated with salicylic acid (SA), salt stress (NaCl), cadmium (CdCl 2) and abiotic stress at 4 ℃ to detect the response of the target gene to stress. The results showed that the BpGT14 gene open reading frame was 1 302 bp encoding 433 amino acids. Analysis of its amino acid sequence found that the protein contains acetylglucosaminyltransferase domain, belonging to the 14 family of important domains. The alignment of this protein with the GT14 protein of other species showed that these proteins were highly conserved in the 100-350 amino acid region and had 79% higher homology with the GT14 family of Populus trichocarpa. The analysis of expression patterns of different parts in different months showed that the expression of BpGT14 gene was site-specific and time-specific. The expression of BpGT14 gene was correlated with the month, and the expression of BpGT14 gene was higher in xylem, phloem and leaf. Lower expression in male inflorescences. Abiotic stress treatment results showed that BpGT14 gene responded to different treatments, but the response patterns were different: SA, CdCl 2 and low temperature treatment showed that the target gene was up-regulated at 6 h and reached the level at 6 h The control group at 51.2,48.9 and 3.3 times, 24 h after the relative expression was down compared with the control group, only low temperature treatment at 96 h up-regulated expression; NaCl treatment so that the expression level of BpGT14 gene all showed a downward trend, 24 h after treatment, the expression of BpGT14 gene was significantly down-regulated and decreased by 93.7% after 24 h.