对比根系转录组研究棉花盐胁迫应答及耐受机理
发布时间：2018-10-21 07:41
【摘要】：土壤盐渍化是限制农作物生产的全球性非生物胁迫因素之一,土壤饱和浸提液电导率等于4.0dS m-1的土壤即为盐渍化土壤(相当于40mM NaC1)。提高农作物的耐盐抗旱能力已成为现代农业生产中一个亟待解决的问题。陆地棉(Gossypium hirsutum L.)作为全球种植面积最大的纤维作物,在饱和浸提液电导率大于7.7dS m-1的盐渍化土壤中才开始表现出减产,具有较高的耐盐能力,被视为开发利用盐碱地的“先锋作物”以及研究植物耐盐机制的模式植物。因此,棉花耐盐分子机理的研究受到广泛的重视。 本研究选择一个相对敏盐陆地棉材料“中G5”,构建了其三叶一心期幼苗根系的7个全长cDNA文库,包括150mM NaCl盐胁迫后3h、12h和48h三个时间点的处理文库,以及3个对应时间点的对照文库,另加一个0h时间点对照文库。 从7个文库中随机挑选约3,300个克隆进行5’端焦磷酸测序,去掉载体接头及低质量序列后共获得20,358条高质量ESTs；利用CAP3拼接后得到8,516条niESTs,包括2,914个contigs和5,602个singletons。比对最新拼接棉花uniESTs结果发现1,795条uniESTs为本研究首次报道,另外6,721个τmiESTs (78.9%)有同源结果。 利用BLAST2GO对8,516条uniESTs进行了GO注释后,对比分析了三个时间点对照和胁迫文库中uniESTs的GO变化,发现盐胁迫后棉花根系激活了一系列应答调控进程,如生长调控、刺激应答、胁迫信号传导以及转录调控等以应答盐胁迫。基于对比对照和胁迫文库间转录因子ESTs发现,MYB、MYB-related、WRKY、bHLH、GRAS和ERF家族转录因子在盐胁迫后3h显著富集,而NAC家族转录因子则在盐胁迫后12和48h显著富集。 Fish er统计检验发现65个盐胁迫应答显著调控基因,主要功能涉及活性氧清除,赤霉素代谢,胁迫应答,水分及其他物质的跨膜转运,信号传导以及转录调控等。荧光定量PCR检测支持了Fis1ler检验的结果,并且在酵母中超表达部分差异应答基因显著提高了酵母的耐盐能力。GO和DEGs的分析结果均表明,ROS以及GAs信号通路积极参与了棉花的盐胁迫应答调控,结合模式植物中ROS与GAs间互作代谢的分析结果,我们提出一个ROS-GAs信号互作参与棉花盐胁迫应答的调控模型。 通过研究盐胁迫后多个时间点棉花根系的转录组应答调控,首次揭示了棉花盐胁迫动态的应答过程及多信号互作调控的耐盐应答机理,鉴定了一批具有耐盐功能的棉花基因。本实验构建的全长cDNA文库及随机测序获得的ESTs序列信息为克隆棉花基因提供了便利,并为进一步研究棉花耐盐机制、遗传改良棉花耐盐性提供了基因资源。
[Abstract]:Soil salinization is one of the global abiotic stress factors that limit crop production. Soil whose conductivity of saturated extract is equal to 4.0dS m-1 is salinized soil (equivalent to 40mM NaC1). Improving the salt tolerance and drought resistance of crops has become an urgent problem in modern agricultural production. Upland cotton (Gossypium hirsutum L.) As the largest fiber crop planted in the world, it is only in salinized soil where the conductivity of saturated extract is greater than 7.7dS m-1 that it begins to show reduced production and has higher salt tolerance. It is regarded as a pioneer crop and a model plant to study the mechanism of salt tolerance in saline-alkali land. Therefore, the molecular mechanism of salt tolerance in cotton has received extensive attention. In this study, 7 full-length cDNA libraries were constructed from the root system of three leaf seedlings of a relatively salt-sensitive upland cotton cultivar "ZhongG5", including three treatment libraries at 3 h, 12 h and 48 h after 150mM NaCl salt stress. And 3 reference libraries corresponding to time points, plus a 0 h time point library. About 3300 clones were randomly selected from 7 libraries for 5 'terminal pyrosequencing. 20358 high quality ESTs; were obtained after removing the vector connector and low quality sequence. 8516 niESTs, including 2914 contigs and 5602 singletons. were obtained by using CAP3 splicing. Compared with the latest cotton uniESTs results, 1795 uniESTs were reported for the first time, and 6721 蟿 miESTs (78.9%) had homologous results. After 8516 uniESTs were annotated with GO by BLAST2GO, the changes of uniESTs GO in three time points control and stress library were compared and analyzed. It was found that cotton root system activated a series of response regulation processes, such as growth regulation and stimulative response, after salt stress. Stress signal transduction and transcriptional regulation in response to salt stress. MYB,MYB-related,WRKY,bHLH,GRAS and ERF family transcription factors were significantly enriched at 3 h after salt stress based on contrastive control and stress interlibrary transcription factor (ESTs). On the other hand, NAC family transcription factors were significantly enriched in. Fish er at 12 and 48 hours after salt stress. 65 genes were found to regulate salt stress response, and their main functions were active oxygen scavenging, gibberellin metabolism and stress response. Transmembrane transport, signal transduction and transcriptional regulation of water and other substances. Fluorescence quantitative PCR test supports the results of Fis1ler test. The results of GO and DEGs analysis showed that ROS and GAs signaling pathway were actively involved in the regulation of salt stress response in cotton. Based on the analysis of the interaction metabolism between ROS and GAs in model plants, we proposed a model for regulating the interaction of ROS-GAs signals in cotton salt stress response. By studying the transcriptional response regulation of cotton roots at different time points after salt stress, the dynamic response process of cotton salt stress and the mechanism of salt tolerance under the regulation of multi-signal interaction were revealed for the first time, and a number of salt tolerant genes were identified. The full-length cDNA library constructed in this study and the ESTs sequence information obtained by random sequencing provided genetic resources for cloning cotton gene and further studying the mechanism of cotton salt tolerance and genetic improvement of cotton salt tolerance.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S562
本文编号：2284415
[Abstract]:Soil salinization is one of the global abiotic stress factors that limit crop production. Soil whose conductivity of saturated extract is equal to 4.0dS m-1 is salinized soil (equivalent to 40mM NaC1). Improving the salt tolerance and drought resistance of crops has become an urgent problem in modern agricultural production. Upland cotton (Gossypium hirsutum L.) As the largest fiber crop planted in the world, it is only in salinized soil where the conductivity of saturated extract is greater than 7.7dS m-1 that it begins to show reduced production and has higher salt tolerance. It is regarded as a pioneer crop and a model plant to study the mechanism of salt tolerance in saline-alkali land. Therefore, the molecular mechanism of salt tolerance in cotton has received extensive attention. In this study, 7 full-length cDNA libraries were constructed from the root system of three leaf seedlings of a relatively salt-sensitive upland cotton cultivar "ZhongG5", including three treatment libraries at 3 h, 12 h and 48 h after 150mM NaCl salt stress. And 3 reference libraries corresponding to time points, plus a 0 h time point library. About 3300 clones were randomly selected from 7 libraries for 5 'terminal pyrosequencing. 20358 high quality ESTs; were obtained after removing the vector connector and low quality sequence. 8516 niESTs, including 2914 contigs and 5602 singletons. were obtained by using CAP3 splicing. Compared with the latest cotton uniESTs results, 1795 uniESTs were reported for the first time, and 6721 蟿 miESTs (78.9%) had homologous results. After 8516 uniESTs were annotated with GO by BLAST2GO, the changes of uniESTs GO in three time points control and stress library were compared and analyzed. It was found that cotton root system activated a series of response regulation processes, such as growth regulation and stimulative response, after salt stress. Stress signal transduction and transcriptional regulation in response to salt stress. MYB,MYB-related,WRKY,bHLH,GRAS and ERF family transcription factors were significantly enriched at 3 h after salt stress based on contrastive control and stress interlibrary transcription factor (ESTs). On the other hand, NAC family transcription factors were significantly enriched in. Fish er at 12 and 48 hours after salt stress. 65 genes were found to regulate salt stress response, and their main functions were active oxygen scavenging, gibberellin metabolism and stress response. Transmembrane transport, signal transduction and transcriptional regulation of water and other substances. Fluorescence quantitative PCR test supports the results of Fis1ler test. The results of GO and DEGs analysis showed that ROS and GAs signaling pathway were actively involved in the regulation of salt stress response in cotton. Based on the analysis of the interaction metabolism between ROS and GAs in model plants, we proposed a model for regulating the interaction of ROS-GAs signals in cotton salt stress response. By studying the transcriptional response regulation of cotton roots at different time points after salt stress, the dynamic response process of cotton salt stress and the mechanism of salt tolerance under the regulation of multi-signal interaction were revealed for the first time, and a number of salt tolerant genes were identified. The full-length cDNA library constructed in this study and the ESTs sequence information obtained by random sequencing provided genetic resources for cloning cotton gene and further studying the mechanism of cotton salt tolerance and genetic improvement of cotton salt tolerance.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S562
【参考文献】
相关期刊论文 前2条
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2 张国伟;路海玲;张雷;陈兵林;周治国;;棉花萌发期和苗期耐盐性评价及耐盐指标筛选[J];应用生态学报;2011年08期
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