秋石斛钙依赖蛋白激酶基因的克隆及表达分析  

Cloning and Expression Analysis of Calcium-dependent Protein Kinase Gene in Dendrobium spp.

作　　者：林榕燕 钟淮钦[1] 陈艺荃 方能炎 孔兰 叶秀仙[1] LIN Rongyan;ZHONG Huaiqin;CHEN Yiquan;FANG Nengyan;KONG Lan;YE Xiuxian(Crops Research Institute,Fujian Academy of Agricultural Sciences/Fujian Engineering Research Center for Characteristic Floriculture,Fuzhou 350013,China;Agricultural Engineering and Technology Research Institute,Fujian Academy of Agricultural Sciences,Fuzhou 350003,China)

机构地区：[1]福建省农业科学院作物研究所/福建省特色花卉工程技术研究中心,福州350013 [2]福建省农业科学院农业工程技术研究所,福州350003

出　　处：《西北植物学报》2022年第11期1818-1826,共9页Acta Botanica Boreali-Occidentalia Sinica

基　　金：福建省省属公益类科研院所项目(2022R1031009);福建省种业工程项目(ZYCX-LY-202102);福建省农业科学院科技创新平台专项(CXPT202102);福建省农业科学院科技创新团队建设项目(CXTD2021010-2);福建省农业高质量发展超越“5511”协同创新工程项目(XTCXGC2021016)。

摘　　要：钙依赖蛋白激酶(calcium-dependent protein kinase,CDPK)在植物的生长发育及逆境胁迫方面发挥着重要作用。该研究以秋石斛品种‘水芙蓉’为试验材料,采用RT-PCR方法克隆钙依赖蛋白激酶基因,并对其进行生物信息学分析;采用qRT-PCR方法对CDPK s基因在‘水芙蓉’不同组织及不同低温胁迫下的表达情况进行分析,为秋石斛CDPK基因的功能验证以及抗寒分子机制研究奠定基础。结果表明:(1)成功克隆获得了DenCDPK 1(GenBank登录号为MZ322902)、DenCDPK 2(GenBank登录号为MZ322903)、DenCDPK 3(GenBank登录号为MZ322904)基因,序列长度依次为1934、1971和2302 bp,分别编码534、541和536个氨基酸。(2)序列一致性结果显示,DenCDPKs蛋白质与铁皮石斛相应CDPK蛋白质序列的一致性均高于97%,且DenCDPKs均存在STKc_CAMK结构域和4个EF-hand结构域。(3)qRT-PCR分析显示,DenCDPK 1、DenCDPK 2和DenCDPK 3基因均在叶中高表达,分别在茎、花、茎中低表达;且‘水芙蓉’叶片中DenCDPK 1和DenCDPK 2的表达量高于其他3个供试品种;经8℃胁迫12 h后,叶片中DenCDPK s的表达量均显著高于对照组(25℃条件下),且DenCDPK 3的表达量在0℃胁迫12 h时达到最高值。(4)常温(25℃)下‘水芙蓉’叶片中的相对电导率显著低于其他3个供试品种;经低温(8℃、0℃)胁迫12 h后,叶片中的相对电导率均显著高于对照组,且随温度的降低呈上升趋势。研究认为,DenCDPK s基因可能参与秋石斛低温胁迫的响应过程,特别是DenCDPK 2可能在秋石斛抗寒过程中发挥着重要作用。CDPK(calcium-dependent protein kinase)gene was proved to play an important role in the biological and abiotic stress of plants.In this study,Dendrobium‘Rainbow-compactum’was used as the experimental material,calcium-dependent protein kinase genes were cloned by RT-PCR and their bioinformatics analysis were carried out.The expression of CDPKs gene in different tissues and under different low temperature stress was analyzed by qRT-PCR,which laid the foundation for the functional verification of the CDPK gene and the research on the molecular mechanism of cold resistance of Dendrobium spp..The results show that:(1)DenCDPK1(GenBankaccession number:MZ322902),DenCDPK2(GenBank accession number:MZ322903),DenCDPK3(GenBank accession numbe:rMZ322904)were cloned,with 1934,1971 and 2302 bp in length,encoding 534,541 and 536 amino acids,respectively.(2)Sequence identity analyses showed that the identity of DenCDPKs protein and corresponding CDPK protein of Dendrobium officinale were both higher than 97%,and DenCDPKs contained a STKc_CAMK domain and 4 EF-hand domains.(3)qRT-PCR analysis showed that DenCDPK 1,DenCDPK 2 and DenCDPK 3 were all highly expressed in leaves,and lowly expressed in stems,flowers and stems,respectively.The expression levels of DenCDPK 1 and DenCDPK 2 in leaves of Den.‘Rainbow-compactum’were higher than those of other three tested varieties.After being stressed at 8℃for 12 h,the expression of DenCDPK s in leaves was significantly higher than that in the control group,and the expression of DenCDPK 3 reached the highest value at 0℃for 12 h.(4)The relative electrical conductivity in the leaves of Den.‘Rainbow-compactum’was lower than those of other three tested varieties under normal temperature(25℃).After low temperature(8℃,0℃)stress for 12 h,the relative electrical conductivity in the leaves was significantly higher than that of the control group,and showed an upward trend with the decrease of temperature.It is speculated that DenCDPK s genes may be involved in the response to low tem

关 键 词：秋石斛 CDPK基因 克隆 QRT-PCR 

分 类 号：Q785[生物学—分子生物学] Q786[农业科学—观赏园艺] Q789[农业科学—园艺学] S682.31