山药块茎膨大期 DoCDPK1 基因生物信息学及低温胁迫下的表达分析

Bioinformatics and expression analysis of DoCDPK1 gene under low temperature stress during tuber expansion stage of yam

摘要

钙依赖性蛋白激酶(CDPK)在植物的生长发育及逆境胁迫方面发挥着重要作用。以大和长芋和毕克齐山药为试验材料，克隆钙依赖性蛋白激酶基因 DoCDPK1,并对其进行生物信息学分析，为 DoCDPK1 基因的功能研究奠定基础。采用生物信息学方法分析 DoCDPK1 基因结构，构建瞬时表达载体，对 DoCDPK1 蛋白进行亚细胞定位；采用 qRT-PCR 分析 DoCDPK1 基因在不同山药品种的不同发育阶段及低温胁迫处理下的表达模式。结果表明:(1) DoCDPK1 基因序列长度为 2023 bp, 编码 521 个氨基酸。(2) DoCDPK1 与几内亚薯蓣 CDPK 蛋白序列的一致性为 97%，且 DoCDPK1 存在 STKc_CAMK 结构域。(3)瞬时表达分析表明，DoCDPK1 蛋白定位于细胞核与细胞膜。(4)大和长芋山药中的 CDPK 活性更高， DoCDPK1 可能参与调控山药块茎膨大后期的生长发育。(5)经 4 ℃低温胁迫后，CDPK 活性降低， DoCDPK1 表达量上调，且表达水平在不同时间处理下存在差异。综上所述， DoCDPK1 可能参与调控山药块茎膨大后期的生长发育及低温胁迫响应过程。

Abstract

Calcium-dependent protein kinase plays an important role in plant growth and osmotic stress. Using Dahechangyu and Bikeqi yam as experimental materials, the calcium dependent protein kinase gene DoCDPK1 was cloned and bioinformatic analysis was performed to lay a foundation for the functional study of DoCDPK1. The structure of DoCDPK1 was analyzed by bioinformatics and subcellular localization of DoCDPK1 protein was performed by constructing transient expression vector. qRT-PCR was used to analyze the expression patterns of DoCDPK1 at different developmental stages and under low temperature stress in different yam varieties. The results indicated:(1) The length of DoCDPK1 gene sequence was 2023 bp, encoding 521 amino acids.(2) The consistency of the protein sequence between DoCDPK1 and CDPK protein sequence of Dioscorea cayenensis subsp. was 97%, and DoCDPK1 had STKc_CAMK domain.(3) Transient expression analysis showed that DoCDPK1 protein was localized in cell nucleus and membrane.(4) The CDPK activity in Dahechangyu was higher, and DoCDPK1 might be involved in regulating the growth and development of yam tuber at the later stage of enlargement.(5) After low temperature stress at 4 ℃, CDPK activity decreased, and the expression of DoCDPK1 was up-regulated, with differences in expression levels observed under different time treatments. In conclusion, DoCDPK1 gene may be involved in regulating the growth and development of yam tuber and its response to low temperature stress.

关键词

山药 / 钙依赖性蛋白激酶 / 亚细胞定位 / 低温胁迫 / 基因表达量

Key words

Yam / Calcium-dependent protein kinase / Subcellular localization / Low temperature stress / Gene expression

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基金资助

国家自然科学基金项目(32260759)

内蒙古自治区自然科学基金项目(2022MS03052)