铁皮石斛DcCDPK8基因启动子克隆及功能分析
|
摘要
铁皮石斛钙依赖性蛋白激酶基因DcCDPK8参与低温激素ABA和MeJA的信号转导过程,但转录调控尚不明确,为了研究铁皮石斛DcCDPK8基因的核心启动子区域,并对其转录调控机制进行探讨,采用PCR技术克隆铁皮石斛DcCDPK8基因启动子序列,并利用5'端缺失和GUS基因的融合表达研究启动子序列功能。结果表明DcCDPK8基因启动子序列含有1个低温响应元件LTR(-1 749～-614 bp),2个茉莉酸甲酯响应元件(-1 749～-230 bp)和1个ABA响应元件(-614～-230 bp),构建3个5'端不同缺失片段融合GUS报告基因的p BI121真核表达载体,瞬时转化烟草叶片,经低温胁迫后GUS活性强弱为DcCDPK8-p1>DcCDPK8-p2> DcCDPK8-p3,经外源ABA诱导后GUS活性DcCDPK8-p1> DcCDPK8-p2> DcCDPK8-p3,而经外源MeJA诱导后GUS活性DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3,推测DcCDPK8基因启动子序列中ABA响应元件(ARE)对外源ABA的响应起正调控作用,而MeJA顺式作用元件对外源茉莉酸甲酯响应起抑制作用。
DcCDPK8 involved in abiotic stress such as low temperature and signal transduction of hormones ABA and MeJA,but the transcriptional regulation is still unclear. In order to study the core promoter region of DcCDPK8 gene in Dendrobium catenatum and explore its transcriptional regulation mechanism,the DcCDPK8 gene promoter sequence was cloned by PCR from D. catenatum. Promoter sequence function was studied by fusion of 5 'terminal deletion and GUS gene. The results showed that the promoter sequence of DcCDPK8 gene has a low-temperature responsive element( LTR) between~(-1) 749 bp and-614 bp,two MeJA responsive elements between~(-1) 749 bp and-230 bp,and one ABA responsive elements between-614 bp and-230 bp. Three 5'-end different deletion fragments were constructed to fuse the eukaryotic expression vectors p BI121 with GUS,which were transformed into tobacco leaves. The GUS activity under cold stress treatment was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. GUS activity under exogenous ABA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3,and GUS activity under exogenous MeJA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. It is speculated that the ABA response element( ARE) in the promoter sequences of DcCDPK8 is positive regulatory role in response to exogenous ABA,the MeJA cis-acting element plays a negative role in response to exogenous MeJA.
DcCDPK8 involved in abiotic stress such as low temperature and signal transduction of hormones ABA and MeJA,but the transcriptional regulation is still unclear. In order to study the core promoter region of DcCDPK8 gene in Dendrobium catenatum and explore its transcriptional regulation mechanism,the DcCDPK8 gene promoter sequence was cloned by PCR from D. catenatum. Promoter sequence function was studied by fusion of 5 'terminal deletion and GUS gene. The results showed that the promoter sequence of DcCDPK8 gene has a low-temperature responsive element( LTR) between~(-1) 749 bp and-614 bp,two MeJA responsive elements between~(-1) 749 bp and-230 bp,and one ABA responsive elements between-614 bp and-230 bp. Three 5'-end different deletion fragments were constructed to fuse the eukaryotic expression vectors p BI121 with GUS,which were transformed into tobacco leaves. The GUS activity under cold stress treatment was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. GUS activity under exogenous ABA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3,and GUS activity under exogenous MeJA induction was DcCDPK8-p1>DcCDPK8-p2>DcCDPK8-p3. It is speculated that the ABA response element( ARE) in the promoter sequences of DcCDPK8 is positive regulatory role in response to exogenous ABA,the MeJA cis-acting element plays a negative role in response to exogenous MeJA.
引文
[1]斯金平,张媛,罗毅波,等.石斛与铁皮石斛关系的本草考证[J].中国中药杂志,2017,42(10):2001.
[2]斯金平,俞巧仙,宋仙水,等.铁皮石斛人工栽培模式[J].中国中药杂志,2013,38(4):481.
[3] Harmon A C,Gribskov M,Gubrium E,et al. The CDPK superfamily of protein kinases[J]. New Phytol,2010,151(1):175.
[4]刘贯山,陈珈.钙依赖蛋白激酶(CDPKs)在植物钙信号转导中的作用[J].植物学报,2003,20(2):160.
[5] Schenk P W,Snaarjagalska B E. Signal perception and transduction:the role of protein kinases[J]. Biochim Biophys Acta,1999,1449(1):1.
[6] Nishiyama R,Mizuno H,Okada S,et al. Two mRNA species encoding calcium-dependent protein kinases are differentially expressed in sexual organs of Marchantia polymorpha through alternative splicing[J]. Plant Cell Physiol,1999,40(2):205.
[7] Cordingley M G,Riegel A T,Hager G L. Steroid-dependent interaction of transcriptionfactors with the inducible promoter of mouse mammary tumor virus in vivo[J]. Cell,1987,48(2):261.
[8] Valdivia R H,Falkow S. Bacterial genetics by flow cytometry:rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction[J]. Mol Microbio,1996,22(2):367.
[9]盛况,高燕会,斯金平,等.铁皮石斛Do CDPK6基因及启动子的克隆与表达分析[J].农业生物技术学报,2017,25(4):588.
[10]盛况,高燕会,斯金平,等.铁皮石斛Do CDPK基因的克隆与表达分析[J].园艺学报,2016,43(12):2412.
[11] Bendahmane A,Querci M,Kanyuka K,et al. Agrobacterium transient expression system as a tool fo the isolation of disease resistance genes:application to the Rx2 locus in potato[J]. Plant J,2010,21(1):73.
[12] Jefferson R A,Kavanagh T A,Bevan M W. GUS fusions:betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants.[J]. Embo J,1987,6(13):3901.
[13]盛况.铁皮石斛Dc CDPKs基因的克隆及表达分析[D].临安:浙江农林大学,2017.
[14]刘亚茹,吕丽莎,程瑾,等. CDPKs在植物适应非生物胁迫过程中的调节作用[J].植物生理学报,2015(9):1387.
[15]武志刚,武舒佳,王迎春,等.植物中钙依赖蛋白激酶(CDPK)的研究进展[J].草业学报,2018(1):204.
[2]斯金平,俞巧仙,宋仙水,等.铁皮石斛人工栽培模式[J].中国中药杂志,2013,38(4):481.
[3] Harmon A C,Gribskov M,Gubrium E,et al. The CDPK superfamily of protein kinases[J]. New Phytol,2010,151(1):175.
[4]刘贯山,陈珈.钙依赖蛋白激酶(CDPKs)在植物钙信号转导中的作用[J].植物学报,2003,20(2):160.
[5] Schenk P W,Snaarjagalska B E. Signal perception and transduction:the role of protein kinases[J]. Biochim Biophys Acta,1999,1449(1):1.
[6] Nishiyama R,Mizuno H,Okada S,et al. Two mRNA species encoding calcium-dependent protein kinases are differentially expressed in sexual organs of Marchantia polymorpha through alternative splicing[J]. Plant Cell Physiol,1999,40(2):205.
[7] Cordingley M G,Riegel A T,Hager G L. Steroid-dependent interaction of transcriptionfactors with the inducible promoter of mouse mammary tumor virus in vivo[J]. Cell,1987,48(2):261.
[8] Valdivia R H,Falkow S. Bacterial genetics by flow cytometry:rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction[J]. Mol Microbio,1996,22(2):367.
[9]盛况,高燕会,斯金平,等.铁皮石斛Do CDPK6基因及启动子的克隆与表达分析[J].农业生物技术学报,2017,25(4):588.
[10]盛况,高燕会,斯金平,等.铁皮石斛Do CDPK基因的克隆与表达分析[J].园艺学报,2016,43(12):2412.
[11] Bendahmane A,Querci M,Kanyuka K,et al. Agrobacterium transient expression system as a tool fo the isolation of disease resistance genes:application to the Rx2 locus in potato[J]. Plant J,2010,21(1):73.
[12] Jefferson R A,Kavanagh T A,Bevan M W. GUS fusions:betaglucuronidase as a sensitive and versatile gene fusion marker in higher plants.[J]. Embo J,1987,6(13):3901.
[13]盛况.铁皮石斛Dc CDPKs基因的克隆及表达分析[D].临安:浙江农林大学,2017.
[14]刘亚茹,吕丽莎,程瑾,等. CDPKs在植物适应非生物胁迫过程中的调节作用[J].植物生理学报,2015(9):1387.
[15]武志刚,武舒佳,王迎春,等.植物中钙依赖蛋白激酶(CDPK)的研究进展[J].草业学报,2018(1):204.