雨生红球藻钙依赖蛋白激酶（CDPK）家族鉴定与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0719

生物技术通报 ›› 2024, Vol. 40 ›› Issue (2): 300-312.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0719

雨生红球藻钙依赖蛋白激酶（CDPK）家族鉴定与表达分析

李亚男¹(), 张豪杰¹, 梁梦静¹, 罗涛¹, 李旺宁¹, 张春辉¹, 季春丽¹, 李润植¹, 薛金爱¹(), 崔红利¹^,²()

1.山西农业大学农学院分子农业与生物能源研究所，太谷 030801
2.中国科学院烟台海岸带研究所海岸带生物学与生物资源利用重点实验室，烟台 264003

收稿日期:2023-07-23 出版日期:2024-02-26 发布日期:2024-03-13
通讯作者: 薛金爱，女，博士，教授，研究方向：生物技术与组学工程；E-mail: 306214803@qq.com;
崔红利，男，博士，副教授，研究方向：微藻生物技术；E-mail: cuihongli@sxau.edu.cn
作者简介:李亚男，女，硕士研究生，研究方向：生物技术与组学工程；E-mail: Li_yanan_li@163.com
基金资助:
国家重点研发计划项目(2021YFD1901105);国家自然科学基金项目(31902394);国家自然科学基金项目(41876188);山西省高等学校科技创新计划(2021L119);山西农业大学科技创新基金(2018YJ16)

Identification and Expression Analysis of Calcium-dependent Protein Kinase（CDPK）Family in Haematococcus pluvialis

LI Ya-nan¹(), ZHANG Hao-jie¹, LIANG Meng-jing¹, LUO Tao¹, LI Wang-ning¹, ZHANG Chun-hui¹, JI Chun-li¹, LI Run-zhi¹, XUE Jin-ai¹(), CUI Hong-li¹^,²()

1. College of Agriculture, Shanxi Agricultural University, Institute of Molecular Agriculture and Bioenergy, Taigu 030801
2. Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai 264003

Received:2023-07-23 Published:2024-02-26 Online:2024-03-13

摘要/Abstract

摘要：

【目的】钙依赖蛋白激酶（CDPK或CPK）广泛参与植物生长发育和环境胁迫响应。为探究CDPK基因在雨生红球藻（Haematococcus pluvialis）生长发育及虾青素积累中的功能，对HpCDPK基因家族进行鉴定和表达分析。【方法】利用生物信息学方法鉴定雨生红球藻HpCDPK基因家族成员，分析其编码蛋白的理化特性、系统进化、保守基序和功能结构域，以及缺氮等胁迫条件下HpCDPK成员基因的表达谱。【结果】结果表明，共鉴定7个HpCDPK基因家族成员，所有HpCDPK蛋白都含有典型EF-hand基序和蛋白激酶结构域。系统发育分析表明，这些HpCDPK与来自莱茵衣藻的CrCDPK聚为一类，与高等植物拟南芥的AtCDPK分开，暗示在进化过程中发生了种属特异性的基因复制事件。转录组数据分析表明，HpCDPK基因表达受到多种胁迫诱导，其中HpCDPK7基因转录水平在缺氮条件下上调最为明显。此外，HpCDPK与类胡萝卜素合成相关基因的表达相关性分析结果显示，HpCDPK与多个类胡萝卜素合成相关基因表达密切关联，特别是HpCDPK2与虾青素合成关键基因（BKT和BCH）的表达显著正相关（P<0.05）。【结论】本研究鉴定了7个HpCDPK基因，HpCDPK7基因在缺氮条件下的表达上调最为明显，HpCDPK2可能在类胡萝卜素及虾青素合成积累中发挥重要调控作用。研究结果为后续深入解析HpCDPK介导雨生红球藻胁迫响应和类胡萝卜素合成积累的功能及分子机制提供参考依据。

关键词: 雨生红球藻, 钙依赖性蛋白激酶, 序列分析, 表达模式, 非生物逆境

Abstract:

【Objective】 Calcium-dependent protein kinases（CDPK or CPK）are widely involved in plant growth and development and environmental stress response. To explore their roles involved in Haematococcus pluvialis growth, development and astaxanthin accumulation, HpCDPK gene family was identified and analyzed. 【Method】 Bioinformatics tools were used to identify the HpCDPK gene family members, and to analyze the physical and chemical properties, phylogenetic evolution, conserved motifs and functional domains of the encoded proteins, as well as the gene expression profiles under nitrogen deficiency stress. 【Result】 The results showed that seven members of HpCDPK gene family were identified, and all HpCDPK proteins contained typical EF-hand motifs and protein kinase domains. Phylogenetic analysis showed that these HpCDPKs clustered with the CrCDPKs from Chlamydomonas reinhartii and separated from the AtCDPKs of Arabidopsis thaliana, suggesting that species-specific gene replication occurred in the process of evolution. Transcriptome data analysis showed that HpCDPK gene expression was induced by a variety of stresses, among which the transcription level of HpCDPK7 gene was most significantly up-regulated under nitrogen deficiency. In addition, the analysis of the correlation between HpCDPK and the expression of genes related to carotenoid synthesis showed that HpCDPK was closely related to the expressions of genes relevant to several carotenoid synthesis in H. pluvialis, especially HpCDPK2 was positively correlated with the expression of key genes of astaxanthin synthesis（BKT and BCH）. 【Conclusion】 Seven HpCDPK genes were identified, the expression of HpCDPK7 gene was most significantly up-regulated under nitrogen deficiency and HpCDPK2 may play an active role in the synthesis and accumulation of carotenoids and astaxanthin. The results provide a reference basis for further analysis of the function and molecular mechanism of stress response and carotenoids synthesis and accumulation mediated by HpCDPK in H. pluvialis.

Key words: Haematococcus pluvialis, calcium-dependent protein kinase, sequence analysis, expression pattern, abiotic stress

李亚男, 张豪杰, 梁梦静, 罗涛, 李旺宁, 张春辉, 季春丽, 李润植, 薛金爱, 崔红利. 雨生红球藻钙依赖蛋白激酶（CDPK）家族鉴定与表达分析[J]. 生物技术通报, 2024, 40(2): 300-312.

LI Ya-nan, ZHANG Hao-jie, LIANG Meng-jing, LUO Tao, LI Wang-ning, ZHANG Chun-hui, JI Chun-li, LI Run-zhi, XUE Jin-ai, CUI Hong-li. Identification and Expression Analysis of Calcium-dependent Protein Kinase（CDPK）Family in Haematococcus pluvialis[J]. Biotechnology Bulletin, 2024, 40(2): 300-312.

图/表 11

表1 雨生红球藻CDPK基因家族成员信息

Table 1 Members of CDPK gene family in H. pluvialis

基因名称 Gene name	基因编号 Gene No.	蛋白长度 Protein length/aa	分子量 MW/Da	等电点 pI	EF手型数量 EF-hand number	N-肉豆蔻酰化 N- myristoylator	N-棕榈酰化 N-palmitoylation	亚细胞定位 Subcellular localization
HpCDPK1	comp60148_c2	605	67 397.89	5.92	4	No	Yes	Cytoplasmic
HpCDPK2	comp60654_c0	545	59 849.50	6.08	4	No	Yes	Chloroplast
HpCDPK3	comp57813_c0	478	53 280.35	5.71	4	No	No	Cytoplasmic
HpCDPK4	comp60035_c0	676	73 764.11	6.03	2	No	Yes	Cytoplasmic
HpCDPK5	comp58900_c0	716	76 076.86	6.39	4	No	Yes	Nuclear
HpCDPK6	comp61638_c1	1 479	153 784.74	7.29	4	No	Yes	Nuclear
HpCDPK7	comp60433_c0	633	70 805.48	5.79	4	Yes	Yes	Mitochondrial

图1 雨生红球藻CDPK蛋白三级结构预测

Fig. 1 Prediction of tertiary structures of CDPK proteins in H. pluvialis

图2 雨生红球藻、莱茵衣藻和拟南芥CDPK基因系统发育树

Fig. 2 Phylogenetic tree of CDPK genes in H. pluvialis, C. reinhardtii and A. thaliana

图3 雨生红球藻HpCDPK家族成员的保守基序和功能结构域不同的图案用特定的颜色表示；紫色框表示丝氨酸/苏氨酸蛋白激酶结构域（SM000220），绿色框表示EF-Hand（SM000054）

Fig. 3 Conserved motif and functional domain of HpCDPK family members of H. pluvialis Different motif is represent by specific color. Purple hollow box indictes the Serine/ Threonine protein kinases domain（SM000220）, and green hollow box indicted the EF-hand（SM000054）

图4 MEME预测的雨生红球藻HpCDPK保守基序横坐标是氨基酸的位置，纵坐标是氨基酸在一个部位的比例

Fig. 4 Conserved motifs of HpCDPKs in H. pluvialis predicted by MEME The abscissa indicates amino acid position and the ordinate indicates the proportion of amino acid at a site

图5 雨生红球藻HpCDPK蛋白保守PDK结构域的多序列比对

Fig. 5 Multiple sequence alignments of conserved PDK domains of HpCDPK proteins in H. pluvialis

图6 雨生红球藻HpCDPK蛋白保守EF基序的多序列比对

Fig. 6 Multiple sequence alignments of conserved EF motifs of HpCDPK proteins in H. pluvialis

图7 雨生红球藻HpCDPK的保守结构域和重要位点分析

Fig. 7 Conserved domain and important site analysis of HpCDPKs in H. pluvialis

图8 雨生红球藻HpCDPK基因在非生物胁迫下的表达模式 A:正常光（N），蓝光（B），高白光（W）；B:正常光（L），正常光和紫外灯200 W（LU200）, 正常光和紫外灯400 W（LU400）, 黑暗（D），黑暗和紫外灯200 W（DU200），黑暗和紫外灯400 W（DU400）；C:高光（CK），高光和α-酮戊二酸（OG），高光和缺氮（ND），高光、缺氮和α-酮戊二酸（ND-OG）

Fig. 8 Expression patterns of HpCDPK genes in H. pluvialis in response to abiotic stresses A: Normal light（N）, blue light（B）, and high white light（W）. B: Normal light（L）, normal light and UV-B 200 W（LU200）, normal light and UV-B 400 W（LU400）, dark（D）, dark and UV-B 200 W（DU200）, dark and UV-B 400 W（DU400）. C: High light（CK）, high light and α-ketoglutaric acid（OG）, high light and nitrogen deficiency（ND）, high light, nitrogen deficiency and α-ketoglutaric acid（ND-OG）

图9 HpCDPK基因与虾青素合成及积累基因的表达量相关性分析

Fig. 9 Correlation between HpCDPKs and gene expressions for astaxanthin synthesis and accumulation

图10 HpCDPK2、HpCDPK3和HpCDPK5基因与类胡萝卜素及虾青素合成及积累基因的表达量相关性

Fig. 10 Correlation between HpCDPK2, HpCDPK3, HpCDPK5 genes and gene expressions for the biosynthesis and accumulation of carotenoid and astaxanthin

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雨生红球藻钙依赖蛋白激酶（CDPK）家族鉴定与表达分析

Identification and Expression Analysis of Calcium-dependent Protein Kinase（CDPK）Family in Haematococcus pluvialis

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