王宏鹏, 成璐路, 滕彦娇, 陈成彬, 张力鹏*. 西藏大花红景天RcUDPGTs基因克隆与表达分析J. 药学学报, 2021,56(7): 2015-2024. doi: 10.16438/j.0513-4870.2021-0082
引用本文: 王宏鹏, 成璐路, 滕彦娇, 陈成彬, 张力鹏*. 西藏大花红景天RcUDPGTs基因克隆与表达分析J. 药学学报, 2021,56(7): 2015-2024. doi: 10.16438/j.0513-4870.2021-0082
WANG Hong-peng, CHENG Lu-lu, TENG Yan-jiao, CHEN Cheng-bin, ZHANG Li-peng*. Cloning and expression analysis of RcUDPGTs genes in Tibetan Rhodiola crenulataJ. Acta Pharmaceutica Sinica, 2021,56(7): 2015-2024. doi: 10.16438/j.0513-4870.2021-0082
Citation: WANG Hong-peng, CHENG Lu-lu, TENG Yan-jiao, CHEN Cheng-bin, ZHANG Li-peng*. Cloning and expression analysis of RcUDPGTs genes in Tibetan Rhodiola crenulataJ. Acta Pharmaceutica Sinica, 2021,56(7): 2015-2024. doi: 10.16438/j.0513-4870.2021-0082

西藏大花红景天RcUDPGTs基因克隆与表达分析

Cloning and expression analysis of RcUDPGTs genes in Tibetan Rhodiola crenulata

  • 摘要: 以西藏大花红景天为研究材料,利用生物信息学和实时荧光定量PCR技术对RcUDPGTs基因家族中18个成员的序列和表达模式进行分析,并构建RcUDPGT(JX228125.1)的诱饵载体以筛选拟南芥酵母文库中互作蛋白。结果表明,RcUDPGTs基因核苷酸序列长度为1 400 bp左右,编码452~498个氨基酸。一级结构中N端序列保守性较低,C端的保守性较高,均含有尿苷二磷酸葡萄糖结合结构域(PSPG盒)。蛋白质三级结构均呈现一定相似性,具有UDP糖供体识别中心,且含有多个可磷酸化位点。qRT-PCR结果显示RcUDPGTs基因在根、茎、叶三个器官中均有表达,且其表达量能够受到低温/紫外和多种植物激素(脱落酸、生长素、茉莉酸甲酯)的调控,但表达模式差异较大。与阴性对照相比,RcUDPGT基因没有自激活活性和细胞毒性,并筛选到两个互作明显的拟南芥基因AtKCR1(AT1G67730.1)与AtSNL4(AT1G70060)。以上结果丰富了RcUDPGTs基因的研究成果,为了解大花红景天与高原环境的适应性机制,同时为深入研究根部次生代谢产物的合成和积累提供一定理论基础。

     

    Abstract: UDP glucosyltransferase (UDPGT) catalyzes the synthesis of secondary metabolites and plant hormones to regulate plant growth and development, pathogen defense and environmental adaptability. In this study 18 members of the RcUDPGT gene family were cloned from Tibetan Rhodiola crenulata and analyzed using bioinformatics. The tissue-specific expression, abiotic stresses and plant hormones (abscisic acid, auxin, methyl jasmonate) induced expression patterns were identified by real-time quantitative PCR. The bait vector of RcUDPGT (JX228125.1) was constructed to select interacting proteins from an Arabidopsis yeast library. The results of the bioinformatics analysis revealed that RcUDPGT nucleotide sequences were about 1 400 bp and encoded 452-498 amino acids. In the primary protein sequences, C-terminal sequences were more conserved compared with N-terminal regions, which held a PSPG (plant secondary product glycosyltransferase) domain. In the tertiary structures, RcUDPGTs contained a UDP sugar donor recognition binding site. In addition, all genes had multiple phosphorylation sites. The results of qRT-PCR showed that RcUDPGTs genes were expressed in root, stem and leaf. The expression levels were regulated by low temperature/ultraviolet light and various plant hormones (ABA, IAA, MeJA), but the expression patterns were quite different among them. For example, RcUDPGT6, RcUDPGT11, and RcUDPGT17 had the highest expression in leaves and were induced by all three hormones, suggesting that the functions of these genes might be to respond to environmental changes. RcUDPGT9, RcUDPGT10, RcUDPGT14 were most abundantly expressed in roots and were significantly induced by ABA and MeJA hormones, indicating that these genes may be involved in the synthesis and accumulation of salidroside. Yeast two-hybrid results showed that RcUDPGT did not exhibit autoactivation and cell toxicity, and two significant interactional genes were identified, AtKCR1 (AT1G67730.1) and AtSNL4 (AT1G70060). The AtKCR1 gene encodes a β-ketoacyl reductase (KCR) involved in synthesis of very long chain fatty acids. The AtSNL4 gene encodes a homolog of the transcriptional repressor SIN3, which could participate in the ABA hormone signaling pathway and enhance the transcriptional repression of AP2/EREBP class factors in Arabidopsis. These results suggest that the accumulation of the secondary metabolite salidroside in Rhodiola crenulata might be affected by several regulatory mechanisms. The above results may lay the foundation for understanding the adaptive mechanism of Rhodiola crenulata in a high altitude environment and stimulate an in-depth study of the synthesis and accumulation of secondary metabolites in this species.

     

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