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中间锦鸡儿(Caragana intermedia Kuang et H.C.Fu),俗称柠条,为豆科、锦鸡儿属旱生落叶灌木,分布在我国西部荒漠草原、干旱草原地区,是这些地区的建群植物种[1]。中间锦鸡儿耐寒、耐热、抗干旱、耐贫瘠,对沙地环境有很强的适应能力,可防风固沙、保持水土、改善局部小环境[2]。近年来,对中间锦鸡儿的研究日益增多,特别是对其干旱适应性方面。
目前,对中间锦鸡儿干旱适应性的研究大致可分为生理机制研究和分子机制研究两类。生理研究发现,柠条气孔开度较小,全天开启进行不间断的蒸腾,这与一般植物中气孔白天开、晚间闭有明显的差异[3];当土壤、叶片间的水分关系紧张系数低于1.0以后,柠条蒸腾速率会自动调低,以避免大量失水[4],另有文章进一步证实柠条的抗旱性与蒸腾速率、小叶水势和叶含水率呈负相关[5];在受到干旱胁迫时,柠条胞间二氧化碳体积分数先轻微下降,而后上升,这与净光合速率的变化趋势相反[6];在不同CO2浓度下,干旱胁迫均会造成柠条生物量的减少,且根、茎、叶中生物量下降的程度不同[7-8]。分子机制方面,柠条锦鸡儿水分胁迫下叶片均一化全长cDNA文库及叶片抑制性差减杂交文库已被构建[9-10],且已经筛选出适合干旱胁迫下进行基因表达分析的内参基因[11]。综上,目前对柠条抗旱性的研究大多集中在生理方面,而对分子方面的研究相对较少。为探究中间锦鸡儿适应干旱的分子机理及进行抗性育种,寻找中间锦鸡儿抗旱相关基因并对其功能进行解析显得十分必要。
大豆miR2118被证实与干旱胁迫显著相关,推测miR2118通过对其靶基因TG01的调控参与到大豆的干旱胁迫响应[12]。通过分析中间锦鸡儿小RNA测序数据,我们获得了中间锦鸡儿miR2118全长[13],并预测了其靶基因cDNA序列片段。为了明确中间锦鸡儿miR2118靶基因是否参与干旱胁迫响应,我们克隆了其靶基因CiDR1的cDNA全长序列,并分析了它在中间锦鸡儿遭受干旱胁迫时的表达模式。本研究为了解中间锦鸡儿CiDR1基因表达与耐旱性的关系提供了借鉴,并为进一步进行中间锦鸡儿抗性育种提供了候选基因。
中间锦鸡儿CiDR1的克隆及干旱胁迫下的表达分析
Cloning of CiDR1 from Caragana intermedia and Its Expression under Drought Stress
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摘要:
目的 研究并了解中间锦鸡儿CiDR1基因功能及其对干旱胁迫的响应,为抗性育种提供候选基因。 方法 通过RACE技术从中间锦鸡儿中克隆CiDR1基因的cDNA全长,利用生物信息学分析软件对其基因结构及功能进行分析和预测。再通过qRT-PCR技术对干旱胁迫后的幼苗中的CiDR1表达模式进行研究。 结果 从中间锦鸡儿中克隆到CiDR1基因的cDNA全长共计4 297 bp,GenBank登录号为KP277100。生物信息学分析表明,预测的CiDR1蛋白序列中含有1 243个氨基酸残基,具有抗病基因特征结构域TIR、NB-ARC、LRR等,其等电点为6.35,不稳定指数为42.91,不具备信号肽,为非分泌蛋白,定位于细胞质中。定量PCR检测发现,CiDR1基因在幼年期的茎中表达量较低,在成年期的叶片中表达量较高;在干旱胁迫处理后的幼苗中,CiDR1表达水平有明显下降,表明该基因的表达受干旱抑制,可能与中间锦鸡儿适应干旱相关。 结论 中间锦鸡儿在干旱胁迫后其根、茎和叶中CiDR1的表达均明显下降,表明CiDR1的表达受干旱抑制,可能与中间锦鸡儿适应干旱相关,进一步研究发现CiDR1在根、茎、叶中的表达水平可能受发育阶段调控。 -
关键词:
- 中间锦鸡儿
- / CiDR1
- / TIR-NBS-LRR
- / 干旱
Abstract:Objective To study the function of CiDR1 gene in Caragana intermedia and its response to drought stress. Method The full-length cDNA sequence of CiDR1 gene was cloned using RACE methods, and then the bioinformatics analysis were carried out to predict gene structure. Finally, CiDR1 expression patterns under drought stress was investigated by qRT-PCR. Result The full-length cDNA sequence of CiDR1 gene was 4 297 bp. The deduced amino-acid sequence for CiDR1 was 1 243 amino acids long and contained toll/interluekin receptor (TIR), nucleotide binding site (NBS) and leucine-rich repeats (LRR) motif, which are the characteristics of plant resistance genes. The theoretical isoelectric point and instability index of CiDR1 protein was 6.35 and 42.91, respectively. Further analysis showed that CiDR1 protein had no signal peptide, was a non-secretary protein and located in the cytoplasmic matrix. The qPCR analysis showed that the CiDR1 transcripts were expressed strongly in adult leaves and weakly in juvenile stems, and after drought treatments the levels of CiDR1 transcripts decreased. Conclusion The results indicate that CiDR1 might play an role in response of C. intermedia to drought and could be used for molecular breeding. -
Key words:
- Caragana intermedia
- / CiDR1
- / TIR-NBS-LRR
- / drought
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