|本期目录/Table of Contents|

 TIAN Lei,CHANG Feifei,QIN Shishang,et al.Cloning and expression analysis of a cold-inducible gene (DsKIN1) from Descurainia sophia[J].Chinese Journal of Applied & Environmental Biology,2017,23(03):467-473.[doi:2015.05019]





Cloning and expression analysis of a cold-inducible gene (DsKIN1) from Descurainia sophia
四川大学生命科学学院,微生物与代谢工程四川省重点实验室 成都 610065
TIAN Lei CHANG Feifei QIN Shishang TANG Ziyi CHEN Qiming MA Xiaorui XU Hui QIAO Dairong & CAO Yi
Microbiology and Metabolic Engineering Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu 610065, China
Descurainia sophia DsKIN1 gene cloning cold stress realtime-PCR
Q78 : Q945.78
播娘蒿是极端耐寒的冷诱导植物,研究其低温胁迫下的冷响应基因对于揭示其抗寒性具有重要意义. 根据拟南芥AtKIN1和AtKIN2同源比对,设计同源引物进行播娘蒿KIN基因克隆,通过RACE技术克隆得到DsKIN1基因,利用Vector NTI11.5软件对其进行序列分析,采用Realtime-PCR测定DsCBF1、DsCBF2、DsCOR和DsKIN1基因在不同处理方式[整株低温处理、同一植株局部低温处理和局部未受低温处理(两部分植株)]下各时间段(0.2 h、0.5 h、1 h、2 h、6 h、12 h、24 h)的表达,采用String数据库预测分析与DsKIN1相互作用的蛋白. 结果显示,DsKIN1基因序列全长为462 bp,开放阅读框ORF长度为198 bp,编码66个氨基酸,分子量(Mr)为6.61 × 103,理论等电点为9.10,亲水性好. 播娘蒿DsCBF1和DsCBF2在0.2 h开始表达,在2 h达到最大,随后下降. 在冷诱导0.2 h后,DsCBF调控的耐寒基因DsCOR和DsKIN1基因开始表达,均呈现上调的趋势. 整株、局部冷诱导植株与同一植株上未受低温胁迫部位的耐寒基因DsCOR和DsKIN1表达模式类似. 此外发现与DsKIN1相互作用的蛋白大部分为低温诱导蛋白、盐和渗透压胁迫响应的信号蛋白、与渗透调节有关的家族蛋白、RING-H2锌指蛋白2B以及未知蛋白等. 本研究说明播娘蒿局部冷诱导时,冷信号可进行传递,使未受冷诱导部位获得耐寒性能. (图7 表4 参27)
Descurainia sophia is a species of extremely hardy and cold-inducible plants. Research on cold-responsive genes of D. sophia under cold stress can help to elucidate the mechanisms underlying its cold resistance. Through homologous alignment of Arabidopsis thaliana AtKIN1 and AtKIN2, DsKIN1 was cloned based on homologous primers. The expression of DsKIN1 was examined by RT-PCR using different low-temperature processing methods (whole plant and partial plant treatment) and different periods (0.2 h, 0.5 h, 1 h, 2 h, 6 h, 12 h, and 24 h). Predicting and analyzing the proteins interacting with DsKIN1 involved the String database. Using RACE, a 462-bp DNA fragment was obtained as the full-length sequence of DsKIN1 containing a 198-bp open reading frame (ORF) encoding 66 amino acid residues. The sequence analysis in Vector NTI 11.5 showed that DsKIN1 has a molecular mass of 6.61 × 103, the theoretical isoelectric point of 9.10, and good hydrophilicity. The expression of DsCBF1 and DsCBF2 started at 0.2 h, reached the maximum at 2 h, and then dropped. The expression of cold-resistance genes DsCOR and DsKIN1 regulated by DsCBF started after cold induction for 0.2 h. This expression pattern showed an upregulation trend. In the whole-plant treatment group (various sites), partial-plant treatment group, and the group without treatment, expression patterns of DsCOR and DsKIN1 were similar. The proteins interacting with DsKIN1 were mostly low-temperature-inducible proteins, salt stress- and osmotic-stress-responsive signaling proteins, a family of proteins related to osmotic adjustment, RING-H2 type zinc-finger protein 2B, and some unknown proteins. This study indicates that a cold signal can be transmitted to give other sites (without cold induction) the capacity for cold resistance.


1 Theocharis A, Clément C, Barka EA. Physiological and molecular changes in plants grown at low temperatures [J]. Planta, 2012, 235: 1091-1105
2 Chinnusamy V, Zhu J, Zhu JK. Cold stress regulation of gene expression in plants [J]. Trends Plant sci, 2007, 12: 444-451
3 Medina J, Catalá R, Salinas J. The CBFs: three arabidopsis transcription factors to cold acclimate [J]. Plant Sci, 2011, 180: 3-11
4 Thomashow MF. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms [J]. Annu Rev Plant Biol, 1999, 50: 571-599
5 陈超, 宋吉仁, 王晓楠, 付连双, 谢冬微, 李卓夫. 寒地冬小麦抗寒性评价方法的初探[J]. 中国农学通报, 2014, 30: 55-59 [Chen C, Song JR, Wang XX, Fu LS, Xie DW, Li ZF. Assessment method of cold-resistance on winter wheat [J]. Chin Agric Sci Bull, 2014, 30: 55-59]
6 贾明珍, 官春云. 油菜抗寒性生理生化机制及基因工程研究进展[J]. 中国油料作物学报, 2012, 34: 556-561 [Jia MZ, Guan CY. Review of physiology,biochemistry and gene engineering on cold resistance of rapeseed [J]. Chin J Oil Crop Sci, 2012, 34: 556-561]
7 邓雪柯, 乔代蓉, 李良, 于昕, 张乃胜, 雷高鹏, 曹毅. 低温胁迫对紫花苜蓿生理特性影响的研究[J]. 四川大学学报: 自然科学版, 2005, 42: 190-194 [Deng XK, Qiao DR, Li L, Yu X, Zhang NS, Leng GP, Cao Y. Research on the effects of low temperature stress on physiological characteristics of alfalfa [J]. J Sichuan Univ, 2005, 42: 190-194]
8 唐蓉. 我国主要农业气象灾害及灾害研究进展[J]. 安徽农业科学, 2007, 35: 9354-9362 [Tang R. Major agricultural meteorological disasters and research development of China [J]. J Anhui Agric Sci, 2007, 35: 9354-9362]
9 Khan M, Wang N. Descurainia sophia (l.): a weed with multiple medicinal uses [J]. Punjab Univ J Zool, 2012, 27: 45-51
10 Dai Xl, Huang M, Yi Xl, Huang Q, Wang Ll, Deng XK, Cao Y, Qiao DR. A study on physiological characters of Descurainia sophia in the condition of cold stress [J]. J Sichuan Univ (Nat Sci Ed), 2007, 1: 42
11 于延球. 野生资源植物播娘蒿的特征特性及其应用研究[J]. 现代农业科技, 2013, 18: 179-180 [Yu YQ. Characteristics and its application research of wild resource plant Descurainia sophia (L.) Webb. ex Prantl [J]. Mod Agric Sci Technol, 2013, 18: 179-180]
12 李红伟, 郑晓珂, 弓建红, 冯卫生. 独行菜和播娘蒿化学成分及药理作用研究进展[J]. 药物评价研究, 2013, 36 (3): 235-240 [Li HW, Zheng XK, Gong JG, Feng WS. Research progress in chemical constituents of Lepidium apetalum and Descurainia sophia and their pharmacological activities [J]. Drug Eval Res, 2013, 36 (3): 235-240 ]
13 Jiang F, Wang F, Wu Z, Li Y, Shi G, Hu J, Hou X. Components of the arabidopsis cbf cold-response pathway are conserved in non-heading chinese cabbage [J]. Plant Mol Biol Rep, 2011, 29: 525-532
14 Thomashow MF. Molecular basis of plant cold acclimation: insights gained from studying the cbf cold response pathway [J]. Plant Physiol, 2010, 154: 571-577
15 Shi Y, Ding Y, Yang S. Cold signal transduction and its interplay with phytohormones during cold acclimation [J]. Plant Cell Physiol, 2014: pcu115
16 吕胜男, 生吉萍, 赵丹莹, 申琳. CBF基因调控植物抗冷径途的研究进展 [J]. 西北植物学报, 2011, 31: 1275-1281 [Lü SN, Sheng JP, Zhao DY, Shen L. Advances of plant cold-resistance pathway regulated by CBF gene [J]. Acta Bot Bor-Occid Sin, 2011, 31: 1275-1281]
17 Gilmour SJ, Fowler SG, Thomashow MF. Arabidopsis transcriptional activators cbf1, cbf2, and cbf3 have matching functional activities [J]. Plant Mol Biol, 2004, 54: 767-781
18 Tao P, Peng L, Huang X, Wang J. Comparative analysis of the variable 3′ utr and gene expression of the kin and kin-homologous lea genes in Capsella bursa-pastoris [J]. Plant Cell Rep, 2012, 31: 1769-1777
19 Wang H, Datla R, Georges F, Loewen M, Cutler AJ. Promoters from kin1 and cor6.6, two homologous Arabidopsis thaliana genes: transcriptional regulation and gene expression induced by low temperature, aba, osmoticum and dehydration [J]. Plant Mol Biol, 1995, 28: 605-617
20 戴旭兰. 播娘蒿DREB/CBF基因的克隆和表达及其转基因拟南芥植株的筛选[D]. 成都: 四川大学, 2007: 3-18 [Dai XL. Cloning and expression of DREB1/CBF type transcription factors of Descurania sophia and expression in transgenic Arabidopsis thaliana [D]. Chengdu: Sichuan University, 2007: 3-18]
21 Li L, Cao Y, Deng XK, Zhang LY, Liao C, Zhang J, Qiao DR. Molecular cloning and sequence analysis of dscor gene from Descurania sophia (l.) [J]. J Sichuan Univ (Nat Sci Ed), 2004, 6: 44
22 Ruelland E, Djafi N, Zachowski A. The phosphoinositide dependent-phospholipase c pathway differentially controls the basal expression of dreb1 and dreb2 genes [J]. Plant Signal Behav, 2013, 8: 307
23 Oakenfull RJ, Baxter R, Knight MR. A c-repeat binding factor transcriptional activator (cbf/dreb1) from european bilberry (Vaccinium myrtillus) induces freezing tolerance when expressed in Arabidopsis thaliana [J]. PLoS ONE, 2013, 8: e54119
24 Akhtar M, Jaiswal A, Taj G, Jaiswal J, Qureshi M, Singh N. Dreb1/cbf transcription factors: their structure, function and role in abiotic stress tolerance in plants [J]. J Genet, 2012, 91: 385-395
25 唐艳梅. 拟南芥抗寒基因Kin1表达载体的构建及四季桔再生体系建立的研究[D]. 济南: 山东农业大学, 2006: 41-44 [Tang YM. The plant expression vector construction of a Arabidopsis antifreezing gene kin1 and the studies on establishment of the regeneration system of C. microcarpa [D]. Jinan: Shandong Agricultural University, 2006: 41-44]
26 Fraga D, Meulia T, Fenster S. Real-time PCR [J]. Curr Protocols Essential Lab Technol, 2014: 10.13. 11-10.13. 40
27 徐楠楠, 胡桂学. 实时荧光定量PCR技术的研究进展及应用[J]. 黑龙江畜牧兽医, 2012 (21): 24-27 [Xu NN, Hu GX. Research progress and application of real-time PCR [J]. Heilongjiang Anim Sci Vet Med, 2012 (21): 24-27]


 YANG Ge,LI Jing,LÜ,et al.Purification of DsCOR in Descurainia sophia, Preparation of Antibody and Its Response to Cold Inducement[J].Chinese Journal of Applied & Environmental Biology,2010,16(03):337.[doi:10.3724/SP.J.1145.2010.00337]
 WANG Le,LI Jing,CAO Yu,et al.Interaction of DsCBF of Descurainia sophia with CRT/DRE Element[J].Chinese Journal of Applied & Environmental Biology,2010,16(03):609.[doi:10.3724/SP.J.1145.2010.00609]
[3]陈麒名 常菲菲 李 茜 刘 博 田 磊 陈天盈 徐 辉 乔代蓉.播娘蒿转录因子DsCBF1突变体的构建及其蛋白质的EMSA试验分析[J].应用与环境生物学报,2016,22(02):179.[doi:10.3724/SP.J.1145.2015.05015]
 CHEN Qiming,CHANG Feifei,LI Qian,et al.Construction of transcription factor DsCBF1 mutant and EMSA analysis of itsprotein*[J].Chinese Journal of Applied & Environmental Biology,2016,22(03):179.[doi:10.3724/SP.J.1145.2015.05015]
[4]林 强 陈麒民 田 磊 乔代蓉.表达外源DsCOR转基因拟南芥的筛选及其抗寒性[J].应用与环境生物学报,2016,22(02):184.[doi:10.3724/SP.J.1145.2015.07039]
 LIN Qiang,CHEN Qimin,TIAN Lei & QIAO Dairong**.Heterologous expression of DsCOR enhances cold resistance in Arabidopsis*[J].Chinese Journal of Applied & Environmental Biology,2016,22(03):184.[doi:10.3724/SP.J.1145.2015.07039]

更新日期/Last Update: 2017-06-25