丹参蛋白激酶SmSnRK2.4的克隆及表达分析(1)
[摘要]蔗糖非酵解型蛋白激酶家族2(SnRK2)在逆境植物信号转导途径中起着重要作用。该研究根据丹参转录组数据从丹参cDNA中克隆得到一个SnRK2家族基因，命名为SmSnRK24，属于Ⅰ类SnRK2。SmSnRK24基因包含8个内含子，9个外显子，其开放阅读框1 068 bp，编码355个氨基酸，推测其蛋白相对分子质量为4063 kDa，将其构建到原核表达载体pMALc2X上，原核诱导表达出与预测蛋白大小一致的目的蛋白。依据丹参基因组序列，PlantCARE分析SmSnRK24起始密码子ATG上游3 000 bp的启动子系列，结果显示该序列包含Ibox，GAmotif，HSE，LTR，TCrich repeats等逆境胁迫响应元件，以及GAREmotif，Pbox，ABRE，CGTCAmotif等激素响应元件。实时荧光定量PCR分析表明SmSnRK24在丹参根中的含量较高，在茎中和叶中含量相当，ABA和PEG胁迫响应分析表明，SmSnRK24对PEG渗透胁迫响应显著，对ABA胁迫响应微弱。该研究为进一步探讨SmSnRK24基因在丹参干旱胁迫下次生代谢产物积累机制中的作用奠定了基础。
, 百拇医药
[关键词]丹参； SmSnRK24； 原核表达； 启动子； 实时荧光定量PCR
Cloning and expression analysis of protein kinase
SmSnRK24 from Salvia miltiorrhiza
JIA Yanyan1， RU Mei2， JIN Weibo3， LIANG Zongsuo1，3*
(1 College of Life Science， Northwest Agriculture and Forestry University， Yangling 712100， China；
2 Institute of Soil and Water Conservation， Chinese Academy of Sciences and the Ministry of Water Resources， Yangling 712100， China；
, 百拇医药
3 College of Life Science， Zhejiang SciTech University， Hangzhou 310018， China)
[Abstract]Sucrose nonfermenting 1related protein kinase 2(SnRK2) plays a key role in abiotic stress signaling in plants In this study， we cloned a SmSnRK24 gene belonging to subclass I of SnRK2 from Salvia miltiorrhiza by screening its transcriptome database The SmSnRK24 gene contains 8 introns and 9 exons， with a 1 068 bp open reading frame encoding a polypeptide of 355 amino acids， the predicted molecular mass of which is 4063 kDa Prokaryotic expression of SmSnRK24 protein using pMALc2X as the expression vector displayed that the recombinant protein of SmSnRK24 gene in E coli was consistent with the predicted size A 3 000 bp promoter sequence of SmSnRK24 contained some stressresponsive elements and hormoneresponsive elements Quantitative realtime PCR analysis revealed that the expression of SmSnRK24 in root was much higher than that in stem and leaf， SmSnRK24 was strongly induced by PEG stress， weakly induced by ABA stress This research provided a basis for further study of the SmSnRK24 gene playing the role in accumulate mechanism of secondary metabolites in S miltiorrhiza under drought
, 百拇医药
[Key words]Salvia miltiorrhiza； SmSnRK24； prokaryotic expression； promoter； quantitative realtime PCR
丹參，为唇形科鼠尾草属多年生草本植物，是传统大宗药材之一，主要活性成分为水溶性酚酸类化合物和脂溶性丹参酮类。丹参基原植物具有生命力强、世代周期短、组织培养和转基因技术成熟、基因组小、染色体数目少等特点，并且随着丹参转录组信息、全基因组信息的逐步丰富，丹参已被认定为中药研究的理想模式植物[12]。
目前，干旱胁迫成为影响植物生长发育的最严重因子之一，植物在干旱胁迫的环境中会改变细胞内次生代谢产物的积累[3]。丹参是干旱敏感种，在干旱胁迫下通过酚酸类物质积累抵御干旱氧化胁迫[4]。中度干旱胁迫显著促进丹参活性成分的积累已得到研究者们的一致认同[59]，在此基础上，刘晓蕾从代谢途径着手，初步研究了中度胁迫下显著提高丹参叶片总酚酸含量积累的机制，即在干旱条件下通过启动水溶性代谢途径关键酶的活性从而调控酚酸类物质的积累[10]。, 百拇医药(贾彦彦 汝梅 金伟波 梁宗锁)
, 百拇医药
[关键词]丹参； SmSnRK24； 原核表达； 启动子； 实时荧光定量PCR
Cloning and expression analysis of protein kinase
SmSnRK24 from Salvia miltiorrhiza
JIA Yanyan1， RU Mei2， JIN Weibo3， LIANG Zongsuo1，3*
(1 College of Life Science， Northwest Agriculture and Forestry University， Yangling 712100， China；
2 Institute of Soil and Water Conservation， Chinese Academy of Sciences and the Ministry of Water Resources， Yangling 712100， China；
, 百拇医药
3 College of Life Science， Zhejiang SciTech University， Hangzhou 310018， China)
[Abstract]Sucrose nonfermenting 1related protein kinase 2(SnRK2) plays a key role in abiotic stress signaling in plants In this study， we cloned a SmSnRK24 gene belonging to subclass I of SnRK2 from Salvia miltiorrhiza by screening its transcriptome database The SmSnRK24 gene contains 8 introns and 9 exons， with a 1 068 bp open reading frame encoding a polypeptide of 355 amino acids， the predicted molecular mass of which is 4063 kDa Prokaryotic expression of SmSnRK24 protein using pMALc2X as the expression vector displayed that the recombinant protein of SmSnRK24 gene in E coli was consistent with the predicted size A 3 000 bp promoter sequence of SmSnRK24 contained some stressresponsive elements and hormoneresponsive elements Quantitative realtime PCR analysis revealed that the expression of SmSnRK24 in root was much higher than that in stem and leaf， SmSnRK24 was strongly induced by PEG stress， weakly induced by ABA stress This research provided a basis for further study of the SmSnRK24 gene playing the role in accumulate mechanism of secondary metabolites in S miltiorrhiza under drought
, 百拇医药
[Key words]Salvia miltiorrhiza； SmSnRK24； prokaryotic expression； promoter； quantitative realtime PCR
丹參，为唇形科鼠尾草属多年生草本植物，是传统大宗药材之一，主要活性成分为水溶性酚酸类化合物和脂溶性丹参酮类。丹参基原植物具有生命力强、世代周期短、组织培养和转基因技术成熟、基因组小、染色体数目少等特点，并且随着丹参转录组信息、全基因组信息的逐步丰富，丹参已被认定为中药研究的理想模式植物[12]。
目前，干旱胁迫成为影响植物生长发育的最严重因子之一，植物在干旱胁迫的环境中会改变细胞内次生代谢产物的积累[3]。丹参是干旱敏感种，在干旱胁迫下通过酚酸类物质积累抵御干旱氧化胁迫[4]。中度干旱胁迫显著促进丹参活性成分的积累已得到研究者们的一致认同[59]，在此基础上，刘晓蕾从代谢途径着手，初步研究了中度胁迫下显著提高丹参叶片总酚酸含量积累的机制，即在干旱条件下通过启动水溶性代谢途径关键酶的活性从而调控酚酸类物质的积累[10]。, 百拇医药(贾彦彦 汝梅 金伟波 梁宗锁)