柑橘中冷胁迫相关miRNAs的克隆与鉴定
摘要
我国拥有丰富的柑橘品种和悠久的栽培历史,是柑橘的原产地和生产大国。柑橘业是我国区域经济发展,新农村建设的支柱产业。冷胁迫是柑橘生长发育和地理分布的重要制约因素,严重影响柑橘的产量和品质。
MiRNA是一类长约21-22nt的单链非编码RNA,通过切割靶基因或抑制其翻译,能协调许多基因的表达,调节植物的激素分泌和信号转导,增强植物对恶劣环境的适应能力。关于miRNA参与植物适应非生物胁迫的研究已有报道,但是miRNA与冷胁迫的关系研究较少。研究柑橘中冷胁迫相关的miRNA,为发现柑橘中新的miRNA及其基因调控模式,改良柑橘品种,提高柑橘对冷胁迫的耐受性,促进柑橘产业的发展,具有十分重要的意义。主要研究成果如下：1以前期研究发现的柑橘中27个miRNAs为研究对象,通过整合基因芯片数据、对miRNAs前体结构及靶基因功能的预测和启动子区顺式作用元件的分析,筛选出柑橘中5个冷胁迫相关的miRNAs。
2提取金橘幼嫩叶片中总RNA,用通用引物Oligd(T)进行逆转录合成cDNA,再PCR扩增,克隆到miRNAs 167a、miRNAs 167d和miRNAs171的前体。
3通过设计特殊的茎环引物进行逆转录,采用Stem-loop RT-PCR的方法和从总RNA中分离出小分子miRNAs,给小分子miRNAs加poly(A)尾,再采用RT-PCR这两种方法均检测到miRNA 156和(?)niRNA166这两种miRNAs成熟体。
4将金橘在4℃条件下分别冷处理0、0.5h、4h、8h和14天后,提取其叶片中的RNA,用半定量RT-PCR法检测这5个miRNAs的表达变化,发现在冷胁迫下miRNA 167a和miRNA167d的表达模式相似,miRNA156、miRNA 166和miRNA171的表达模式各不相同。
China is a big country of citrus production where citrus originated, possessing abundant kinds of citrus and long history of planting. Citrus industry is key to regional economy development as well as our new country construction. Cold stress is an important environmental factors which limiting citrus plant growth, development, geographical distribution and gravely affect citrus production and quality.
MicroRNA is an class of single-strand noncoding RNAs with about 21-22 nt length.By cleavaging target mRNA or inhibiting its translation, microRNA can coordinate expression of many genes invoving regulating plant hormone and signal conduction to adapte to adversity. There are some reports about plant adaptions to abiotic stress,but rare is about relationship between miRNA and cold stress. Exploring cold related miRNAs in citrus to find new miRNAs and their patterns of gene regulation is of great significance to development of citrus industry, while it can optimize citrus species, improving their adaption to cold. The main results are as follows:
1 Focusing on 27 citrus miRNAs in previous study, we screen 5 cold stress related miRNAs by integrating microarray dates, predicting pre-miRNAs structure and analyzing cis-acting element in miRNAs promoter region.
2 After total RNA extracted from young leaves of kumquat, reverse transcription reactions were performed using universal primer Olig(T), then we clone precursors of miRNA167a、miRNA167d and miRNAs171 by PCR amplification.
3 Mature sequence of miRNA 156 and miRNA 166 are detected by two means. One is stem loop RT-PCR by designing special stem loop primer, the other is that adding poly(A) to small molecule miRNAs isolated from total RNA
4 RNA are isolated from different samples kumquat; untreated, treated with cold stress 4°C for 0.5h、4h、8h and 14 days prior to extraction. We study expression difference of 5 miRNAs by semi-quantitative PCR and find that the expression patterns of miRNA167a and miRNA167d are similar, while miRNA156、miRNA166 and miRNA171 are different from each other.
MiRNA是一类长约21-22nt的单链非编码RNA,通过切割靶基因或抑制其翻译,能协调许多基因的表达,调节植物的激素分泌和信号转导,增强植物对恶劣环境的适应能力。关于miRNA参与植物适应非生物胁迫的研究已有报道,但是miRNA与冷胁迫的关系研究较少。研究柑橘中冷胁迫相关的miRNA,为发现柑橘中新的miRNA及其基因调控模式,改良柑橘品种,提高柑橘对冷胁迫的耐受性,促进柑橘产业的发展,具有十分重要的意义。主要研究成果如下：1以前期研究发现的柑橘中27个miRNAs为研究对象,通过整合基因芯片数据、对miRNAs前体结构及靶基因功能的预测和启动子区顺式作用元件的分析,筛选出柑橘中5个冷胁迫相关的miRNAs。
2提取金橘幼嫩叶片中总RNA,用通用引物Oligd(T)进行逆转录合成cDNA,再PCR扩增,克隆到miRNAs 167a、miRNAs 167d和miRNAs171的前体。
3通过设计特殊的茎环引物进行逆转录,采用Stem-loop RT-PCR的方法和从总RNA中分离出小分子miRNAs,给小分子miRNAs加poly(A)尾,再采用RT-PCR这两种方法均检测到miRNA 156和(?)niRNA166这两种miRNAs成熟体。
4将金橘在4℃条件下分别冷处理0、0.5h、4h、8h和14天后,提取其叶片中的RNA,用半定量RT-PCR法检测这5个miRNAs的表达变化,发现在冷胁迫下miRNA 167a和miRNA167d的表达模式相似,miRNA156、miRNA 166和miRNA171的表达模式各不相同。
China is a big country of citrus production where citrus originated, possessing abundant kinds of citrus and long history of planting. Citrus industry is key to regional economy development as well as our new country construction. Cold stress is an important environmental factors which limiting citrus plant growth, development, geographical distribution and gravely affect citrus production and quality.
MicroRNA is an class of single-strand noncoding RNAs with about 21-22 nt length.By cleavaging target mRNA or inhibiting its translation, microRNA can coordinate expression of many genes invoving regulating plant hormone and signal conduction to adapte to adversity. There are some reports about plant adaptions to abiotic stress,but rare is about relationship between miRNA and cold stress. Exploring cold related miRNAs in citrus to find new miRNAs and their patterns of gene regulation is of great significance to development of citrus industry, while it can optimize citrus species, improving their adaption to cold. The main results are as follows:
1 Focusing on 27 citrus miRNAs in previous study, we screen 5 cold stress related miRNAs by integrating microarray dates, predicting pre-miRNAs structure and analyzing cis-acting element in miRNAs promoter region.
2 After total RNA extracted from young leaves of kumquat, reverse transcription reactions were performed using universal primer Olig(T), then we clone precursors of miRNA167a、miRNA167d and miRNAs171 by PCR amplification.
3 Mature sequence of miRNA 156 and miRNA 166 are detected by two means. One is stem loop RT-PCR by designing special stem loop primer, the other is that adding poly(A) to small molecule miRNAs isolated from total RNA
4 RNA are isolated from different samples kumquat; untreated, treated with cold stress 4°C for 0.5h、4h、8h and 14 days prior to extraction. We study expression difference of 5 miRNAs by semi-quantitative PCR and find that the expression patterns of miRNA167a and miRNA167d are similar, while miRNA156、miRNA166 and miRNA171 are different from each other.
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