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以3年生水仙(Narcissus tazetta var.chinensis)鳞茎主芽为材料,采用蒽酮-硫酸法测定可溶性糖含量;采用PCR法从主芽中分离克隆NtTIM基因并对其序列进行生物信息学分析;采用qRT-PCR方法分析主芽不同生长发育时期和温度诱导下的表达变化。可溶性糖含量测定结果表明,水仙鳞茎膨大期,主芽可溶性糖含量逐渐降低;休眠期先升高后降低;花芽分化期其含量在均衡的水平(4%左右)。生物信息学分析表明:分离克隆到了1个NtTIM基因,该基因含有765 bp开放阅读框,编码254个氨基酸,属于TIM-phosphatebinding基因家族,推导的氨基酸序列与油菜、芜菁、拟南芥、甘蓝等植物具有85%以上的同源性;编码的蛋白为稳定的呈酸性的疏水蛋白,无信号肽位点但含有1个跨膜结构域、2个N-糖基化位点。qRT-PCR分析表明,水仙生长发育过程中鳞茎膨大期主芽NtTIM基因表达量略升高;休眠期表达量呈阶段性降低;花芽分化期表达量略升高;20℃、25℃、30℃、35℃温度处理时,主芽NtTIM基因表达量随着温度升高而降低,且处理时间越长,其表达量越低。推测NtTIM基因参与调控水仙鳞茎主芽休眠。为进一步研究NtTIM基因的分子功能,以及深入研究水仙夏休眠的分子机制提供帮助。
The soluble sugar content was determined by the anthrone-sulfuric acid method using the main bulb of 3-year-old Narcissus tazetta var. Chinensis as material. The NtTIM gene was isolated and cloned by PCR from the main buds and the sequence was analyzed by bioinformatics. The qRT-PCR method was used to analyze the expression changes induced by different growth stages and temperatures. The results of soluble sugar content showed that the content of soluble sugar in main bud decreased gradually when daffodil bulbs, the dormancy firstly increased and then decreased, and the flower bud differentiation stage was at equilibrium level (about 4%). Bioinformatics analysis showed that an NtTIM gene was isolated and cloned, which contains a 765 bp open reading frame and encodes a protein of 254 amino acids. It belongs to the TIM-phosphatebinding gene family. The deduced amino acid sequence is homologous to rape, turnip, Arabidopsis thaliana, And other plants have more than 85% homology. The encoded protein is a stable acidic hydrophobin with no signal peptide site but one transmembrane domain and two N-glycosylation sites. qRT-PCR analysis showed that the expression of NtTIM gene in bulblets increased slightly during dormancy stage of daffodil and decreased gradually in dormancy stage, and slightly increased in flower bud differentiation stage. , 35 ℃ temperature treatment, the main bud NtTIM gene expression decreased with increasing temperature, and the longer the treatment, the lower the expression level. It is speculated that NtTIM gene is involved in the regulation of daffodil bulb dormancy. To further study the molecular function of NtTIM gene, as well as in-depth study of the molecular mechanism of Narcissus summer to help.