长叶红砂钙依赖蛋白激酶Rt...K16的非生物胁迫应答分析_张洁.pdf

第 32 卷第 2 期Vol.32，No.297-1092023 年 2 月草业学报ACTA PRATACULTURAE SINICA张洁，程凯，王迎春.长叶红砂钙依赖蛋白激酶 RtCDPK16的非生物胁迫应答分析.草业学报，2023，32（2）：97109.ZHANG Jie，CHENG Kai，WANG Ying-chun.Analysis of the calcium-dependent protein kinase RtCDPK16 response to abiotic stress in Reaumuriatrigyna.Acta Prataculturae Sinica，2023，32（2）：97109.长叶红砂钙依赖蛋白激酶 RtCDPK16的非生物胁迫应答分析张洁，程凯，王迎春*（省部共建草原家畜生殖调控与繁育国家重点实验室，内蒙古自治区牧草与特色作物生物技术重点实验室，内蒙古大学生命科学学院，内蒙古 呼和浩特 010040）摘要：植物受到逆境刺激后，钙离子结合蛋白能够感知钙信号并将其解码，激活下游靶蛋白，从而引发胁迫应答反应，钙调蛋白激酶在其中发挥了重要作用。长叶红砂为东阿拉善-西鄂尔多斯特有的珍稀泌盐植物，对干旱、盐碱等环境胁迫具有极强的适应性。本研究基于转录组数据，克隆了长叶红砂钙调蛋白激酶 RtCDPK16的开放阅读框（open reading frame，ORF），氨基酸序列比对和进化分析显示，其与拟南芥的同源性为 78.46%，与葡萄 VvCDPK16同源性最高（80.97%）。基因表达特性分析显示，RtCDPK16在根茎叶中均表达，且在根中表达量最高，同时其表达水平可被氯化钠（NaCl）/聚乙二醇（polyethylene glycol，PEG）/冷胁迫（cold stress，cold）/脱落酸（abscisic acid，ABA）等多种环境胁迫和氯化钙（calcium chloride，CaCl2）显著诱导。构建植物表达载体并转化拟南芥，对野生型拟南芥（WT）和过表达拟南芥（OE）株系进行干旱、盐和脱落酸胁迫，发现胁迫处理下 OE 株系的根长、鲜重和叶绿素含量等表型指标均显著高于 WT，表明 RtCDPK16的超表达使转基因株系获得了更强的胁迫耐受性；生理生化指标检测显示，胁迫处理下各株系中超氧化物歧化酶（superoxide dismutase，SOD）、过氧化物酶（peroxidase，POD）和过氧化氢酶（catalase，CAT）等抗氧化酶活性及脯氨酸（proline，Pro）、可溶性糖（soluble sugar，SS）等渗透调节物质的含量均被显著诱导，且超表达株系显著高于 WT，而过氧化氢（hydrogen peroxide，H2O2）/超氧阴离子（superoxideanion，O2-）的积累量显著低于 WT；实时荧光定量（quantitativereal-time，qRT-PCR）检测发现，胁迫条件下 OE 株系的相关抗氧化酶基因、ABA 合成和信号途径关键元件基因及脯氨酸合成基因均被显著诱导。以上结果表明，RtCDPK16在拟南芥中的超表达通过调控转基因植物中抗氧化和渗透调节系统相关基因的表达和酶活性，促进转基因植物的渗透平衡和活性氧（reactive oxygen species，ROS）稳态的维持，进而提高转基因植物的胁迫耐受性，这一过程可能是通过依赖于 ABA信号的途径实现的。关键词：长叶红砂；非生物胁迫；RtCDPK16；抗氧化；渗透调节Analysis of the calcium-dependent protein kinase RtCDPK16 response to abioticstress in Reaumuria trigynaZHANG Jie，CHENG Kai，WANG Ying-chun*State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock，Key Laboratory of Herbage&Endemic CropBiotechnology in Inner Mongolia，School of Life Science，Inner Mongolia University，Hohhot 010040，ChinaAbstract：Calmodulin kinase is a crucial component of calcium-binding proteins，which read and decode calciumDOI：10.11686/cyxb2022198http：/收稿日期：2022-05-05；改回日期：2022-07-28基金项目：内蒙古自治区自然科学基金重大项目（2020ZD07）和国家自然科学基金项目（32060404）资助。作者简介：张洁（1994-），女，内蒙古达拉特旗人，在读博士。E-mail： 通信作者 Corresponding author.E-mail：Vol.32，No.2ACTA PRATACULTURAE SINICA（2023）signals and activate downstream target proteins to initiate stress responses in plants after stress stimulation.Reaumuria trigyna，a rare and uncommon salt-producing plant found only in east Alxa and west Ordos，is veryresilient to environmental challenges including drought and salinity.Based on transcriptome information，the openreading frame（ORF）of RtCDPK16 was cloned in this study.The ORF shared 78.46%similarity with Arabidopsisthaliana and 80.97%identity with CDPK16 in Vitis vinifera，according to amino acid sequence alignment andevolutionary analyses.RtCDPK16 was found to be expressed in roots，stems，and leaves，with roots expressing it atthe highest level.Stress conditions such as sodium chloride（NaCl）/macrogol（polyethylene glycol，PEG）/coldstress（cold）/abscisic acid（ABA）and other environmental stresses and calcium chloride（CaCl2）were shown togreatly increase RtCDPK16 expression levels.Construction of plant expression vector and transformation of A.thaliana.Drought，salt and abscisic acid stress were applied to wild type Arabidopsis（WT）and overexpressionArabidopsis（OE）strains.The results showed that root length，fresh weight，chlorophyll content and otherphenotypic indicators of OE strain were significantly higher than WT under stress treatment，indicating that theoverexpression of RtCDPK16 made the transgenic strain obtain stronger stress tolerance.Physiological andbiochemical indexes showed that，in each strain under stress treatment，the activities of antioxidant enzymes such assuperoxide dismutase（SOD），Peroxidase（POD），catalase（CAT）and the content of osmotic regulators such asproline（Pro）and soluble sugar（SS）were significantly induced，and the OE strain was significantly higher thanWT，while the accumulation of hydrogen peroxide（H2O2）/superoxide anion（O2-）was significantly lower thanWT.Quantitative real-time（RT-PCR）detection found that the genes related to antioxidant enzymes，ABAsynthesis and key elements of signal pathway，and proline synthesis genes of OE strains were significantly inducedunder stress.The above results indicate that the overexpression of RtCDPK16 in Arabidopsis can promote theosmotic balance and ROS（reactive oxygen species）homeostasis by regulating the genes expression of antioxidantenzyme and osmotic regulation in transgenic plants，and thus improve the stress tolerance of transgenic plants.Thisprocess may be achieved through ABA signal dependent approaches.Key words：Reaumuria trigyna；abiotic stress；RtCDPK16；antioxidant；osmotic regulation植物在生长发育过程中往往会遭遇干旱、水涝、高温、冷害、高盐等非生物胁迫，使其正常生长受到限制，甚至可能造成其死亡1-2。在长期适应过程中，植物逐渐形成了各种调控机制来应对不利的生存环境，钙信号传导系统就是其中之一1，3。当植物受到逆境刺激后，钙离子结合蛋白能够感知钙信号并将其解码，进而调节下游靶蛋白，引发一系列的胞内反应1，4。在高等植物中，有 4类钙离子结合蛋白，包括钙调素（calmodulin，CaM）、钙调素类蛋白（calmodulin-like protein，CML）、钙依赖蛋白激酶（calmodulin-dependent protein kinases，CDPKs）及类钙调磷酸酶 B 亚基蛋白（calcineurin B-like prote