蓖麻钙依赖蛋白激酶29基因克隆与表达分析

doi:10.7668/hbnxb.20192958

摘要/Abstract

摘要：

为探讨蓖麻钙依赖蛋白激酶29基因(RcCDPK29)在蓖麻耐盐中的作用,以蓖麻叶片为材料,设计特异性引物,克隆蓖麻钙依赖蛋白激酶29基因(RcCDPK29),并对所得序列进行生物信息学分析。结果表明,蓖麻钙依赖蛋白激酶29基因(RcCDPK29)序列全长1 590 bp;编码528个氨基酸;蛋白分子量为59.74 ku;等电点(pI)值6.21;是典型的非跨膜蛋白;亲水性数值为负值,属于亲水性蛋白;RcCDPK29蛋白α-螺旋占比最高有228个;RcCDPK29与拟南芥CDPK(SMTL ID: 3q5i.1)相似度为40.41%,具有较高可信度(>30%)。将木薯、麻枫树、巴西橡胶树、柑橘、石榴、毛果杨与蓖麻RcCDPK29氨基酸序列进行同源性比对。其中,与麻枫树的同源性最高,为82.29%。RcCDPK29蛋白包含1个Ser/Thr蛋白激酶催化结构域和4个与Ca²⁺结合的EF-hand型结构域。通过qRT-PCR技术,分析RcCDPK29在不同水平盐胁迫下蓖麻不同组织中的表达,结果表明,RcCDPK29基因主要在茎中表达,盐处理12 h表达量最高。随着盐处理时间的延长,RcCDPK29基因根的表达量逐渐下降,分别在2,8,24 h达到最低,与0 h差异显著;叶的表达量在2 h的表达量最低且与0 h相比差异显著。根据RcCDPK29全长设计带有Sma Ⅰ和Xba Ⅰ酶切位点的引物扩增出序列全长,用Sma Ⅰ和Xba Ⅰ进行双酶切后与表达载体 pCG-3300连接。成功构建了CRcCDPK29的表达载体。因此,RcCDPK29在蓖麻受到盐胁迫时起重要作用。

关键词: 蓖麻, 钙依赖蛋白激酶, 基因克隆, 生物信息学分析, 表达载体

Abstract:

To investigate the role of RcCDPK29 in salt tolerance of castor, we designed specific primers to clone the calcium-dependent protein kinase 29 gene (RcCDPK29) from castor leaves and performed bioinformatics analysis of the resulting sequence. The results showed that the sequence of RcCDPK29 was 1 590 bp, encoding 528 amino acids, with a molecular weight of 59.74 ku and an isoelectric point (pI) value of 6.21. It is a typical non-transmembrane protein with a negative hydrophilic value and a hydrophilic protein with a maximum of 228 α-helices. The similarity of RcCDPK29 to Arabidopsis CDPK (SMTL ID: 3q5i.1) was 40.41% with high confidence(>30%). The amino acid sequences of Manihot esculenta, Jatropha curcas,Hevea Brasiliensis,Citrus sinensis,Punica granatum,Populus trichocarpa and Ricinus communis RcCDPK29 were compared for homology. Among them, the highest homology with Jatropha was 82.29%. The RcCDPK29 protein contained a Ser/Thr protein kinase catalytic domain and four Ca²⁺-binding EF-hand-type structural domains. The expression of RcCDPK29 in different tissues of castor under different levels of salt stress was analyzed by qRT-PCR. The results showed that the RcCDPK29 gene was mainly expressed in the stem, with the highest expression at 12 h of salt treatment. With the extension of salt treatment time, the expression of RcCDPK29 gene in roots gradually decreased and reached the lowest at 2, 8,24 h, respectively,with significant differences from 0 h;the expression of leaves was the lowest at 2 h and significantly different compared with 0 h. The full length of RcCDPK29 was amplified by designing primers with SmaⅠand XbaⅠdigestion sites, and then ligated with the expression vector pCG-3300 after double digestion with SmaⅠand XbaⅠ. The expression vector of RcCDPK29 was successfully constructed. Thus, RcCDPK29 plays an important role when castor is subjected to salt stress.

Key words: Castor, Calcium dependent protein kinase, Gene cloning, Bioinformatics analysis, Eexpression vector

王莹, 王晓宇, 孙德慧, 霍红雁, 刘海臣, 徐惠, 张继星. 蓖麻钙依赖蛋白激酶29基因克隆与表达分析[J]. 华北农学报, 2023, 38(2): 85-92. doi: 10.7668/hbnxb.20192958.

WANG Ying, WANG Xiaoyu, SUN Dehui, HUO Hongyan, LIU Haichen, XU Hui, ZHANG Jixing. Cloning and Expression Analysis of Castor Calcium Dependent Protein Kinase 29 Gene[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(2): 85-92. doi: 10.7668/hbnxb.20192958.

http://www.hbnxb.net/CN/Y2023/V38/I2/85

图/表 14

表1 引物序列

Tab.1 Sequences of primers

引物名称 Primer	上游引物( 5' - 3') Forward primer( 5' - 3')	下游引物( 5'-3') Reverse primer( 5'-3')
RcCDPK29	ATGGGACTCTGTTTCACAAAATT	CTACAGCAAACCATTATTTTCATG
qRcCDPK29	TATCATAACCCACCACCG	CCTCTGCCCAGTTCTTTA
qRcSKIP	CATTGCTGAGGTCAGTGCAT	GACGGCCATGCTCTTCTAAC
qRcADP	TGCTGTCTTCTTCATTGCTT	CTCCTGCTCCATTTCCAT

表2 PCR反应体系及条件

Tab.2 PCR reaction system and conditions

PCR反应体系 PCR reaction system		PCR反应程序 PCR reaction procedure
试剂 Reagent	用量/μL Consumption	温度/℃ Temperature	时间 Time
cDNA 2×Es Taq Master Mix ddH₂O RcCML29-F RcCML29-R	2.0 12.5 8.5 1.0 1.0	94 94 55 72 72	10 min $30 s 30 s 1 m i n$ 35循环 5 min

表2 PCR反应体系及条件

Tab.2 PCR reaction system and conditions

PCR反应体系 PCR reaction system		PCR反应程序 PCR reaction procedure
试剂 Reagent	用量/μL Consumption	温度/℃ Temperature	时间 Time
cDNA 2×Es Taq Master Mix ddH₂O RcCML29-F RcCML29-R	2.0 12.5 8.5 1.0 1.0	94 94 55 72 72	10 min $30 s 30 s 1 m i n$ 35循环 5 min

表3 双酶切体系及连接体系

Tab.3 Double enzyme digestion system and ligation reaction system

双酶切体系 Double enzyme digestion system			连接体系 Ligation reaction system
试剂 Reagent	用量/μL Consumption	试剂 Reagent		用量/μL Consumption
重组质粒 Recombinant Plasmid 10×T Buffer 0.1% BSA SmaⅠ XbaⅠ ddH₂O	8 2 2 1 1 6	RcCDPK29 PCHF3300-eGFP 10×T₄ DNA Ligase Buffer T₄ DNA Ligase		7 5 1 1

图1 蓖麻RcCDPK29基因 PCR 扩增产物 M.DL2000 DNA Marker;1-2.PCR结果。图9-10同。

Fig.1 PCR products of RcCDPK29 gene in Ricinus communis M.DL2000 DNA Marker;1-2.PCR results.The same as Fig.9-10.

图2 RcCDPK29 基因CDS序列推导的氨基酸序列方框.EF-hand结构域。

Fig.2 RcCDPK29 CDS sequence and its deduced amino acid sequence The boxes.EF-hand domains.

图3 RcCDPK29蛋白保守区预测

Fig.3 Prediction of RcCDPK29 conserved domains

图4 蓖麻与其他植物CDPK29序列一致性对比

Fig.4 Identity comparison of CDPK29 sequences between Ricinus communis and other plants

图5 RcCDPK29蛋白结构预测 h.α-螺旋;e.延伸链;t.β-转角; c.无规则卷曲。

Fig.5 Secondary structure prediction of RcCDPK29 h. Alpha helix;e.Extended strand;t.Beta turn;c.Random coil.

图6 RcCDPK29蛋白的三级结构

Fig.6 RcCDPK29 protein tertiary structure prediction

图7 RcCDPK29蛋白的跨膜结构预测

Fig.7 Transmembrane structure prediction of RcCDPK29 protein

图8 RcCDPK29蛋白的疏水性分析

Fig.8 Hydrophobic analysis of RcCDPK29 protein

图9 表达载体重组质粒检测

Fig.9 Expression vector recombinant plasmid assay

图10 表达载体双酶切鉴定

Fig.10 Expression vector double digestion detection identification

图11 RcCDPK29 在蓖麻根(A)、茎(B)、叶(C)中不同处理时间的表达量测定不同小写字母表示差异显著(P<0.05)。

Fig.11 Determination of expression of RcCDPK29 at different processing times in Ricinus communis root(A),stem(B),leaf(C) Different lowercase letters indicate significant difference.

参考文献 31

[1]	李贵玲. 盐生植物耐盐机制概要及其在改良土壤中的作用[J]. 生物学通报, 2020, 55(9):7-10.doi:10.3969/j.issn.0006-3193.2020.09.003. doi: 10.3969/j.issn.0006-3193.2020.09.003
	Li G L. Summary of salt tolerance mechanism of halophytes and its role in improving soil[J]. Bulletin of Biology, 2020, 55(9):7-10.
[2]	范王涛. 土壤盐碱化危害及改良方法研究[J]. 农业与技术, 2020, 40(23):114-116.doi:10.19754/j.nyyjs.20201215035. doi: 10.19754/j.nyyjs.20201215035
	Fan W T. Study on the harm of soil salinization and its improvement methods[J]. Agriculture & Technology, 2020, 40(23):114-116.
[3]	付丽娜. 盐渍土的改良技术分析进展[J]. 农民致富之友, 2019(9): 119.doi:10.3969/j.issn.1003-1650.2019.09.116. doi: 10.3969/j.issn.1003-1650.2019.09.116
	Fu L N. Analysis and progress of improvement technology of saline soil[J]. Nongmin Zhifuzhiyou Yuekan, 2019(9):119.
[4]	张昆, 李明娜, 曹世豪, 孙彦. 植物盐胁迫下应激调控分子机制研究进展[J]. 草地学报, 2017, 25(2):226-235.doi:10.11733/j.issn.1007-0435.2017.02.002. doi: 10.11733/j.issn.1007-0435.2017.02.002
	Zhang K, Li M N, Cao S H, Sun Y. The research advances of molecular mechanisms of plant in responding to salt stress[J]. Acta Agrestia Sinica, 2017, 25(2):226-235.
[5]	Harmon A C, Gribskov M, Harper J F. CDPKs-a kinase for every Ca²+ signal?[J]. Trends in Plant Science, 2000, 5(4):154-159.doi:10.1016/s1360-1385(00)01577-6. doi: 10.1016/s1360-1385(00)01577-6 pmid: 10740296
[6]	Xu J, Tian Y S, Peng R H, Xiong A S, Zhu B, Jin X F, Gao F, Fu X Y, Hou X L, Yao Q H. AtCPK6,a functionally redundant and positive regulator involved in salt/drought stress tolerance in Arabidopsis[J]. Planta, 2010, 231(6):1251-1260.doi:10.1007/s00425-010-1122-0. doi: 10.1007/s00425-010-1122-0 URL
[7]	Asano T, Hayashi N, Kikuchi S, Ohsugi R. CDPK-mediated abiotic stress signaling[J]. Plant Signaling & Behavior, 2012, 7(7):817-821.doi:10.4161/psb.20351. doi: 10.4161/psb.20351
[8]	Mutlu H, Meier M A R. Castor oil as a renewable resource for the chemical industry[J]. European Journal of Lipid Science and Technology, 2010, 112(1):10-30.doi:10.1002/ejlt.200900138. doi: 10.1002/ejlt.200900138 URL
[9]	丛娇娇, 王晓宇, 李平, 李惠根, 张丽雪, 张继星. 蓖麻耐盐基因HKT的克隆及表达载体构建[J]. 分子植物育种, 2018, 16(14):4648-4657.doi:10.13271/j.mpb.016.004648. doi: 10.13271/j.mpb.016.004648
	Cong J J, Wang X Y, Li P, Li H G, Zhang L X, Zhang J X. Cloning and expression vector construction of salt tolerant gene HKT in Ricinus communis[J]. Molecular Plant Breeding, 2018, 16(14):4648-4657.
[10]	严兴初, 王力军. 蓖麻作为能源开发的现状与前景[J]. 安徽农业科学, 2007, 35(34):11165,11167.doi:10.3969/j.issn.0517-6611.2007.34.101. doi: 10.3969/j.issn.0517-6611.2007.34.101
	Yan X C, Wang L J. The status in quo and foreground of exploiting Castor-oil plant as energy sources[J]. Journal of Anhui Agricultural Sciences, 2007, 35(34):11165,11167.
[11]	王沛琦, 刘旭云, 胡学礼, 胡尊红, 杨谨, 郭丽芬, 李文昌. 蓖麻对重金属污染土壤治理的研究进展[J]. 分子植物育种, 2019, 17(6):2048-2054.doi:10.13271/j.mpb.017.002048. doi: 10.13271/j.mpb.017.002048
	Wang P Q, Liu X Y, Hu X L, Hu Z H, Yang J, Guo L F, Li W C. Advances and the effects of Castor for curing heavy metal contamination in soil[J]. Molecular Plant Breeding, 2019, 17(6):2048-2054.
[12]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using Real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4):402-408.doi:10.1006/meth.2001.1262. doi: 10.1006/meth.2001.1262 pmid: 11846609
[13]	原增艳, 宋小锋, 朱畇昊. 地黄RgCDPK基因的克隆与表达分析[J]. 广西植物, 2020, 40(12):1816-1823.doi:10.11931/guihaia.gxzw201911042. doi: 10.11931/guihaia.gxzw201911042
	Yuan Z Y, Song X F, Zhu Y H. Cloning and expression analysis of calcium-dependent protein kinase genes in Rehmannia glutinosa[J]. Guihaia, 2020, 40(12):1816-1823.
[14]	雷蕾, 王璐, 姚丽娜, 郝心愿, 曾建明, 丁长庆, 王新超, 杨亚军. 茶树钙依赖性蛋白激酶基因CsCDPK17的鉴定及表达分析[J]. 茶叶科学, 2019, 39(3):267-279.doi:10.3969/j.issn.1000-369X.2019.03.005. doi: 10.3969/j.issn.1000-369X.2019.03.005
	Lei L, Wang L, Yao L N, Hao X Y, Zeng J M, Ding C Q, Wang X C, Yang Y J. Identification and expression analysis of calcium-dependent protein kinase CsCDPK17 in tea plant(Camellia sinensis)[J]. Journal of Tea Science, 2019, 39(3):267-279.
[15]	张丽雪, 王晓宇, 李平, 李惠根, 丛娇娇, 张继星. 蓖麻Na⁺/H⁺逆向转运蛋白基因NhaD克隆与表达载体构建[J]. 基因组学与应用生物学, 2019, 38(7):3132-3139.doi:10.13417/j.gab.038.003132. doi: 10.13417/j.gab.038.003132
	Zhang L X, Wang X Y, Li P, Li H G, Cong J J, Zhang J X. Cloning and expression vector construction of Na⁺/H⁺ antiporter in Castor(Ricinus communis)[J]. Genomics and Applied Biology, 2019, 38(7):3132-3139.
[16]	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.doi:10.13560/j.cnki.biotech.bull.1985.2018-1056. doi: 10.13560/j.cnki.biotech.bull.1985.2018-1056
	Liu Z S, Liu Y Y, Qin Y J, Bi Q, Wang S S, Zhu J B. Cloning and expression analysis of SikCML7 gene from Saussurea involucrata[J]. Biotechnology Bulletin, 2019, 35(6):48-54.
[17]	万丙良, 查中萍, 戚华雄. 钙依赖的蛋白激酶与植物抗逆性[J]. 生物技术通报, 2009(1):7-10.
	Wan B L, Zha Z P, Qi H X. Calcium-dependent protein kinases(CDPKs)and plant tolerance to environmental stresses[J]. Biotechnology Bulletin, 2009(1):7-10.
[18]	耿帅锋, 赵永亮, 李爱丽, 毛龙, 王卫国, 李磊. 植物钙依赖性蛋白激酶的进化和功能研究进展[J]. 河南工业大学学报(自然科学版), 2010, 31(5):86-92.doi:10.16433/j.cnki.issn1673-2383.2010.05.002. doi: 10.16433/j.cnki.issn1673-2383.2010.05.002
	Geng S F, Zhao Y L, Li A L, Mao L, Wang W G, Li L. Research progress of evolution and function of calcium-dependent protein kinase in plant[J]. Journal of Henan University of Technology (Natural Science Edition), 2010, 31(5):86-92.
[19]	余晓丛, 娜仁, 张少英. 钙信号在植物抗病性中的作用研究进展[J]. 中国农学通报, 2012, 28(3):12-16.doi:10.3969/j.issn.1000-6850.2012.03.003. doi: 10.3969/j.issn.1000-6850.2012.03.003
	Yu X C, Na R, Zhang S Y. Research progress about calcium signal involved in plant resistance to disease[J]. Chinese Agricultural Science Bulletin, 2012, 28(3):12-16. doi: 10.11924/j.issn.1000-6850.2011-2329
[20]	费小钰, 李红丽, 王俊皓. 植物钙依赖蛋白激酶CDPK基因功能综述[J]. 吉林农业, 2017(9):104-105.doi:10.14025/j.cnki.jlny.2017.09.064. doi: 10.14025/j.cnki.jlny.2017.09.064
	Fei X Y, Li H L, Wang J H. Review on the function of plant calcium-dependent protein kinase CDPK gene[J]. Jilin Nongye, 2017(9):104-105.
[21]	Zhang H C, Yin W L, Xia X L. Calcineurin B-Like family in populus:comparative genome analysis and expression pattern under cold,drought and salt stress treatment[J]. Plant Growth Regulation, 2008, 56(2):129-140.doi:10.1007/s10725-008-9293-4. doi: 10.1007/s10725-008-9293-4 URL
[22]	Kudla J, Batistic O, Hashimoto K. Calcium signals:The lead currency of plant information processing[J]. The Plant Cell, 2010, 22(3):541-563.doi:10.1105/tpc.109.072686. doi: 10.1105/tpc.109.072686 pmid: 20354197
[23]	Liese A, Romeis T. Biochemical regulation of in vivo function of plant calcium-dependent protein kinases(CDPK)[J]. Biochimica et Biophysica Acta, 2013, 1833(7):1582-1589.doi:10.1016/j.bbamcr.2012.10.024. doi: 10.1016/j.bbamcr.2012.10.024 pmid: 23123193
[24]	Li J X, Lee Y R J, Assmann S M. Guard cells possess a calcium-dependent protein kinase that phosphorylates the KAT1 potassium channel[J]. Plant Physiology, 1998, 116(2):785-795.doi:10.1104/pp.116.2.785. doi: 10.1104/pp.116.2.785 pmid: 9489023
[25]	Yahalom A, Lando R, Katz A, Epel B L. A calcium-dependent protein kinase is associated with maize mesocotyl plasmodesmata[J]. Journal of Plant Physiology, 1998, 153(3/4):354-362.doi:10.1016/S0176-1617(98)80162-4. doi: 10.1016/S0176-1617(98)80162-4 URL
[26]	Nishiyama R, Mizuno H, Okada S, Yamaguchi T, Takenaka M, Fukuzawa H, Ohyama K. Two mRNA species encoding calcium-dependent protein kinases are differentially expressed in sexual organs of Marchantia polymorpha through alternative splicing[J]. Plant and Cell Physiology, 1999, 40(2):205-212.doi:10.1093/oxfordjournals.pcp.a029529. doi: 10.1093/oxfordjournals.pcp.a029529 pmid: 10202816
[27]	Li A L, Zhu Y F, Tan X M, Wang X, Wei B, Guo H Z, Zhang Z L, Chen X B, Zhao G Y, Kong X Y, Jia J Z, Mao L. Evolutionary and functional study of the CDPK gene family in wheat(Triticum aestivum L.)[J]. Plant Molecular Biology, 2008, 66(4):429-443.doi:10.1007/s11103-007-9281-5. doi: 10.1007/s11103-007-9281-5 URL
[28]	Myers C, Romanowsky S M, Barron Y D, Garg S, Azuse C L, Curran A, Davis R M, Hatton J, Harmon A C, Harper J F. Calcium-dependent protein kinases regulate polarized tip growth in pollen tubes[J]. Plant J, 2009, 59(4):528-539.doi:10.1111/j.1365-313x.2009.03894.x. doi: 10.1111/j.1365-313x.2009.03894.x URL
[29]	Dodd A N, Kudla J, Sanders D. The language of calcium signaling[J]. Annual Review of Plant Biology, 2010, 61:593-620.doi:10.1146/annurev-arplant-070109-104628. doi: 10.1146/annurev-arplant-070109-104628 pmid: 20192754
[30]	张秋平, 文李, 王峰, 廖志强, 李慧, 刘睿洋, 官春云. 油菜钙依赖蛋白激酶BnCDPK1的克隆和表达分析[J]. 植物遗传资源学报, 2014, 15(6):1320-1326.doi:10.13430/j.cnki.jpgr.2014.06.021 doi: 10.13430/j.cnki.jpgr.2014.06.021
	Zhang Q P, Wen L, Wang F, Liao Z Q, Li H, Liu R Y, Guan C Y. Molecular cloning and expression analysis of calcium-dependent protein kinase BnCDPK1 in Brassica napus[J]. Journal of Plant Genetic Resources, 2014, 15:(6):1320-1326.
[31]	张海斐. 甜瓜CDPK和CRK基因家族的鉴定及表达分析[D]. 杨凌: 西北农林科技大学, 2017.
	Zhang H F. Identification and expression analysis of CDPK and CRK gene families in melon[D]. Yangling: Northwest A&F University, 2017.

选择文件类型/文献管理软件名称

选择包含的内容