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33
2025年4月3日 星期四
农业生物技术学报  2024, Vol. 32 Issue (3): 546-558    DOI: 10.3969/j.issn.1674-7968.2024.03.005
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
铁皮石斛蔗糖合酶DoSUS2基因克隆与表达分析
刘羽佳1,2, 理雅2,3, 姜雅涵2, 何彬荣2, 陈嘉仪2, 于白音1,2,*, 李翔1,2, 刘博婷1,2,*
1 广东省粵北食药资源利用与保护重点实验室, 韶关 512005;
2 韶关学院 英东生物与农业学院,韶关 512005;
3 河南农业大学 农学院,郑州 450046
Cloning and Expression Analysis of Sucrose Synthase DoSUS2 from Dendrobium officinale
LIU Yu-Jia1,2, LI Ya2,3, JIANG Ya-Han2, HE Bin-Rong2, CHEN Jia-Yi2, YU Bai-Yin1,2,*, LI Xiang1,2, LIU Bo-Ting1,2,*
1 Guangdong Provincial Key Laboratory of Utilization and Conservation of Food and Medicinal Resources in Northerrn Region, Shaoguan 512005, China;
2 Henry Fok College of Biology and Agriculture, Shaoguan University, Shaoguan 512005, China;
3 College of Agriculture, Henan Agricultural University, Zhengzhou 450046, China
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摘要 蔗糖合酶是调控蔗糖代谢的关键酶,催化蔗糖进入各种代谢途径,在植物生长发育、信号转导和逆境抵御等过程中发挥重要作用。为探究铁皮石斛(Dendrobium officinale)蔗糖合酶2 (sucrose synthase 2, DoSUS2)在多糖合成代谢及非生物胁迫响应中的调控机制,本研究从铁皮石斛叶组织中克隆了DoSUS2基因,并对其进行了生物信息学分析和表达特性研究。结果显示,DoSUS2基因(GenBank No. OQ657188) ORF全长2 424 bp,编码807个氨基酸,与参考序列(基因ID: LOC110110417)有36个碱基差异,全部为同义替换。DoSUS2蛋白理论分子量92.05 kD,理论等电点为5.97,为疏水性稳定蛋白,无跨膜区域,含有1个信号肽和69个磷酸化位点,具有SUS蛋白保守的蔗糖合酶和糖基转移酶结构域,与拟南芥(Arabidopsis thaliana) AtSUS1蛋白的氨基酸序列同源性高达73.82%,属于单子叶植物SUSⅠ型亚组。DoSUS2基因在不同组织中的表达水平存在明显差异,在根和花器官中的表达量相对较高,在叶和茎中的表达量较低。DoSUS2基因启动子序列含有多个非生物胁迫响应相关元件,且低温、干旱和脱落酸(abscisic acid, ABA)能够诱导DoSUS2基因表达。在低温和ABA处理6 h后,DoSUS2基因表达量极显著上调(P<0.01),分别达到处理前的267倍和233倍;在干旱处理9 h后,DoSUS2基因表达量也达到最高值,为处理前的18.6倍(P<0.01),推测DoSUS2基因可能通过ABA信号通路参与低温和干旱胁迫响应过程。DoSUS2蛋白与多个糖代谢密切相关的酶存在互作关系,推测该基因可能通过调节相关酶基因的表达和糖的代谢途径,在非生物胁迫应答过程中发挥作用,从而提高铁皮石斛对逆境胁迫的耐受能力。本研究为后续进一步研究DoSUS2基因功能及其在多糖合成代谢和非生物胁迫响应中的作用机制提供理论依据。
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刘羽佳
理雅
姜雅涵
何彬荣
陈嘉仪
于白音
李翔
刘博婷
关键词 铁皮石斛DoSUS2克隆非生物胁迫表达分析    
Abstract:Sucrose synthase family is a crucial enzyme that regulates sucrose metabolism. It catalyzes the conversion of sucrose into various metabolic pathways and plays a significant role in plant growth and development, signal transduction, stress resistance and so on. In order to explore the regulatory mechanism of sucrose synthase gene 2 from Dendrobium officinale (DoSUS2), in polysaccharide anabolism and response to abiotic stress, the DoSUS2 gene (GenBank No. OQ657188) was cloned from leaves of D. officinale using homologous sequence method, the bioinformatics and expression characteristics were analyzed. The results showed that the ORF of DoSUS2 was 2 424 bp and encoded 807 amino acids, which had 36 bases differences compared with the reference sequence (Gene ID: LOC110110417), but all of them were synonymous substitutions. The theoretical molecular weight and isoelectric point of DoSUS2 protein were 92.05 kD and 5.97, respectively. It was a hydrophobic stable protein with only one signal peptide and 69 phosphorylation sites but no transmembrane domains, which contained conserved domain with SUS protein, such as sucrose synthase and glycosyl transferase domains. Tertiary structure modeling of DoSUS2 was highly homologous with AtSUS1 of Arabidopsis thaliana (73.82%), and belongs to the SUSⅠ subgroup of monocotyledonous. The expression of DoSUS2 had obvious differences among different tissues, which were relatively high in roots and floral organs while lower in leaves and stems. The promoter element analysis revealed that DoSUS2 contained several cis-acting elements in response to stress, and it was significantly induced by cold, drought stress and abscisic acid (ABA) treatment. The expression of DoSUS2 was up-regulated significantly (P<0.01) and peaked at 6 h after cold and ABA treatment, which reached 267 and 233 times compared with untreated. Similarly, the expression was also up-regulated accompanying with drought treatment time increased, and reached its peak at 9 h, its expression reached a extremely significant difference level significantly (P<0.01) which was 18.6 times then untreated group. The results showed that the DoSUS2 gene might participate in the response process to low temperature and drought stress through the ABA signaling pathway. At last, an DoSUS2 protein interaction network was generated with several enzymes closely related to glucose metabolism. These findings above suggested that DoSUS2 might respond to abiotic stress by regulating the expression of related enzyme genes and glucose metabolism pathways, thereby improving the stress resistance of D. officinale. The results would provide valuable theoretical basis for the further investigations of DoSUS2 function and its molecular mechanism in polysaccharide biosynthesis and abiotic stress response.
Key wordsDendrobium officinale    DoSUS2    Cloning    Abiotic stress    Expression analysis
收稿日期: 2023-01-28     
ZTFLH:  S184  
  Q78  
基金资助:国家自然科学基金(32000266); 广东省基础与应用基础研究基金(2020A1515011438); 广东省普通高校重点领域专项(2022ZDZX4044); 广东省科技创新战略专项资金(pdjh2023b0477); 韶关市科技计划项目(210726114532224); 韶关学院科研重点项目(SZ2019ZK04)
通讯作者: *lyjizbd@126.com;liuboting5566@163.com   
引用本文:   
刘羽佳, 理雅, 姜雅涵, 何彬荣, 陈嘉仪, 于白音, 李翔, 刘博婷. 铁皮石斛蔗糖合酶DoSUS2基因克隆与表达分析[J]. 农业生物技术学报, 2024, 32(3): 546-558.
LIU Yu-Jia, LI Ya, JIANG Ya-Han, HE Bin-Rong, CHEN Jia-Yi, YU Bai-Yin, LI Xiang, LIU Bo-Ting. Cloning and Expression Analysis of Sucrose Synthase DoSUS2 from Dendrobium officinale. 农业生物技术学报, 2024, 32(3): 546-558.
链接本文:  
https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2024.03.005     或     https://journal05.magtech.org.cn/Jwk_ny/CN/Y2024/V32/I3/546
 
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