天山雪莲SikCML7的克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2018-1056

摘要/Abstract

摘要： 类钙调素蛋白（Calmodulin-like protein）是高等植物中最重要的Ca²⁺感受器之一,该家族蛋白参与植物对环境的适应过程。为了探究类钙调素蛋白是否参与到植物对逆境条件的响应过程,以天山雪莲低温转录组为依据,克隆获得SikCML7,生物信息学分析表明SikCML7的开放阅读框（ORF）为450 bp,编码149个氨基酸,含有4个钙结合区域（EF-hands）。系统进化树分析表明SikCML7与拟南芥AtCML9进化亲缘性较为接近。实时定量PCR结果显示,天山雪莲在经过不同低温（4℃/-2℃）、干旱和盐胁迫处理后,SikCML7的表达均呈上调趋势,但在冷害胁迫和冻害胁迫条件下的表达模式差异巨大,对干旱和盐胁迫也有一定的响应。SikCML7参与了天山雪莲对低温、干旱和盐胁迫响应的过程。

关键词: 天山雪莲, 类钙调素蛋白, 逆境胁迫, 表达分析

Abstract: Calmodulin-like protein is one of the most important Ca²⁺ receptors and participates in the adaptation process of plants to environment. In order to explore whether or not calmodulin-like proteins are involved in the plant response to stress,the SikCML7 gene was cloned based on the low temperature transcriptome of Saussurea involucrata. Bioinformatics analysis indicated that the open reading frame of SikCML7 was 450 bp and encoded 149 amino acids,and there were 4 calcium binding regions（EF-hands）in the encoded proteins. Phylogenetic tree analysis indicated that this gene was close to Arabidopsis AtCML9 in the evolutionary affinity. The results of real-time quantitative PCR showed that the expression of SikCML7gene was up-regulated after treatment with two different low temperatures（4℃/-2℃）,drought and salt stress respectively;however this gene demonstrated significantly different expression patterns under chilling stress and freezing stress as well as the certain responses to drought and salt stress. In sum,SikCML7 indeed participates in the responses process of S. involucrata to low temperature,draught,and salt stresses.

Key words: Saussurea involucrate, calmodulin-like protein, adverse stress, expression analysis

刘兆书, 刘元元, 秦玉杰, 毕权, 王赛赛, 祝建波. 天山雪莲SikCML7的克隆及表达分析[J]. 生物技术通报, 2019, 35(6): 48-54.

LIU Zhao-shu, LIU Yuan-yuan, QIN Yu-jie, BI Quan, WANG Sai-sai, ZHU Jian-bo. Cloning and Expression Analysis of SikCML7 Gene from Saussurea involucrata[J]. Biotechnology Bulletin, 2019, 35(6): 48-54.

参考文献

[1] Dodd AN, Kudla J, Sanders D.The language of calcium signaling[J]. Annual Review of Plant Biology, 2010, 61(1):593-620.
[2] 郭新勇, 程晨, 王爱英, 等. 天山雪莲冷调节蛋白基因siCOR转化烟草植株的抗旱性分析[J]. 植物学报, 2012, 47(2):111-119.
[3] Ishida H, Vogel HJ.Protein-peptide interaction studies demonstrate the versatility of calmodulin target protein binding[J]. Protein & Peptide Letters, 2006, 13(5):455-465.
[4] Ishida H, Huang H, Yamniuk AP, et al.The solution structures of two soybean calmodulin isoforms provide a structural basis for their selective target activation properties[J]. Journal of Biological Chemistry, 2008, 283(21):14619-14628.
[5] Perochon A, Dieterle SC, Aldon D, et al.Interaction of a plant pseudo-response regulator with a calmodulin-like protein[J]. Biochemical & Biophysical Research Communications, 2010, 398(4):747-751.
[6] Magnan F, Benoît R, Charpenteau M, et al.Mutations in AtCML9, a calmodulin-like protein from Arabidopsis thaliana, alter plant responses to abiotic stress and abscisic acid[J]. The Plant Journal, 2008, 56(4):575-589.
[7] 赵桂红, 张妮妮, 高帅帅, 等. 菘蓝CYP83A1基因的克隆、表达特性及原核表达分析[J]. 基因组学与应用生物学, 2018, 37(1):_302-312
[8] Li DF, Li J, Ma L, et al.Calmodulin isoform-specific activation of a rice calmodulin-binding kinase conferred by only three amino-acids of OsCaM61[J]. FEBS Letters, 2006, 580(18):4325-4331.
[9] Ogunrinde A, Munro K, Davidson A, et al.Arabidopsis calmodulin-like proteins, CML15 and CML16 possess biochemical properties distinct from calmodulin and show non-overlapping tissue expression patterns[J]. Front Plant Sci, 2017, 8:2175.
[10] Astegno A, Bonza MC, Vallone R, et al.Arabidopsis calmodulin-like protein CML36 is a calcium(Ca²+)sensor that interacts with the plasma membrane Ca²+-ATPase Isoform ACA8 and stimulates its activity[J]. Journal of Biological Chemistry, 2017, 292:15049-15061.
[11] Lokdarshi A, Conner WC, Mcclintock C, et al.Arabidopsis CML38, a calcium sensor that localizes to ribonucleoprotein complexes under hypoxia stress[J]. Plant Physiology, 2016, 170(2):1046-1059.
[12] Vadassery J, Reichelt M, Hause B, et al.CML42-mediated calcium signaling coordinates responses to spodoptera herbivory and abiotic stresses in Arabidopsis[J]. Plant Physiology, 2012, 159(3):1159-1175.
[13] Scholz SS, Reichelt M, Vadassery J, et al.Calmodulin-like protein CML37 is a positive regulator of ABA during drought stress in Arabidopsis[J]. Plant Signaling & Behavior, 2015, 10(6):e1011951
[14] Tsai YC, Delk NA, Chowdhury NI, et al.Arabidopsis potential calcium sensors regulate nitric oxide levels and the transition to flowering[J]. Plant Signaling & Behavior, 2007, 2(6):446-454.
[15] Köhler C, Neuhaus G.Characterisation of calmodulin binding to cyclic nucleotide-gated ion channels from Arabidopsis thaliana[J]. FEBS Letters, 2000, 471(2):133-136.
[16] Hua W, Liang S, Lu YT.A tobacco(Nicotiana tabaccum)calmodulin-binding protein kinase, NtCBK2, is regulated differentially by calmodulin isoforms[J]. Biochemical Journal, 2003, 376(1):291.
[17] Mccormack E, Braam J.Calmodulins and related potential calcium sensors of Arabidopsis[J]. New Phytologist, 2003, 159(3):585-598.
[18] Boonburapong B, Buaboocha T.Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins[J]. BMC Plant Biology, 2007, 7(1):4.
[19] Mccormack E, Tsai YC, Braam J.Handling calcium signaling:Arabidopsis CaMs and CMLs[J]. Trends in Plant Science, 2005, 10(8):383-389.
[20] Mantyla E, Lang V, Palva ET.Role of abscisic acid in drought-induced freezing tolerance, cold acclimation, and accumulation of LT178 and RAB18 proteins in Arabidopsis thaliana[J]. Plant Physiology, 1995, 107(1):141-148.
[21] Magnan F, Ranty BM, Sotta B, et al.Mutations in AtCML9, a calmodulin-like protein from Arabidopsis thaliana, alter plant responses to abiotic stress and abscisic acid[J]. The Plant Journal, 2010, 56(4):575-589.
[22] Delk NA, Johnson KA, Chowdhury NI, et al.CML24, regulated in expression by diverse stimuli, encodes a potential Ca²+ sensor that functions in responses to abscisic acid, daylength, and ion stress[J]. Plant Physiology, 2005, 139(1):240-253.
[23] Ji CY, Jin R, Xu Z, et al.Overexpression of Arabidopsis P3B increases heat and low temperature stress tolerance in transgenic sweetpotato[J]. BMC Plant Biology, 2017, 17(1):139.
[24] Storey KB, Storey JM.Molecular biology of freezing tolerance[M]//Comprehensive Physiology. John Wiley & Sons, Inc. 2013:1283-1308.
[25] Yamaguchi T, Aharon GS, Sottosanto JB, et al.Vacuolar Na+/H+ antiporter cation selectivity is regulated by calmodulin from within the vacuole in a Ca²+- and pH-dependent manner[J]. Proc Natl Acad Sci USA, 2005, 102(44):16107-16112.
[26] Deswal R, Sopory SK.Glyoxalase I from Brassica juncea is a calmodulin stimulated protein[J]. Biochimica Et Biophysica Acta, 1999, 1450(3):460.
[27] Veena, Reddy VS, Sopory SK. Glyoxalase I from Brassica juncea:molecular cloning, regulation and its over-expression confer tolerance in transgenic tobacco under stress[J]. The Plant Journal, 2010, 17(4):385-395.
[28] Vanderbeld B, Snedden WA.Developmental and stimulus-induced expression patterns of Arabidopsis calmodulin-like genes CML37, CML38 and CML39[J]. Plant Molecular Biology, 2007, 64(6):683-697.