枇杷幼果在低温胁迫下基因的差异表达
摘要
为了研究枇杷耐受低温的分子机理,以早熟易受冻害的早钟六号幼果为研究对象,利用mRNA差异显示(DDRT-PCR)技术,对枇杷在低温胁迫下的基因表达情况进行研究。
对SDS法进行改良,探索出快速、高效的提取枇杷幼果总RNA的方法,采用此方法提取的RNA适合mRNA差异显示分析。
通过优化试验,建立了适合枇杷幼果低温胁迫下DDRT-PCR分析的最佳体系,在20μl体系中,确定了各组分为:模板cDNA 1.0μl,锚定引物0.5μM,随机引物0.5μM,dNTPs 50μM,10×PCR buffer 2μl,Taq酶1U,退火温度40℃。在此基础上对枇杷幼果在0℃低温胁迫6h下mRNA的变化进行分析,共分离到19条差异表达的基因片段,经Reverse Northern杂交获得了4个阳性的枇杷低温诱导相关基因片段(编号为CSIGE 1~4—Cold Stress Induced Genes in Eriobotrya japonica 1~4)。对4个阳性差异片段进行测序,序列已提交GenBank,CSIGE 1~4登录号依次为GT617703、GT617704、GT617705和GT617706。
序列相似性分析表明:①CSIGE 1序列与Homo sapiens chromosome 8 clone Rp11-420F14 map p21-p11有高度同源性,是抗低温胁迫基因,这说明植物与动物基因组具有某些共同的编码序列,可能在基因表达方面具有一些共同的机制;②CSIGE 2与蓖麻乙醇脱氢酶(ADH)、拟南芥NADP依赖型氧化还原酶、大豆受水杨酸胁迫诱导相关基因等有不同程度的同源性,CSIGE 3与葡萄和黄瓜受逆境胁迫的EST有部分同源,该基因表达量的调节对枇杷响应低温逆境起到某种作用;③CSIGE 4序列未搜索到与任何基因有相似性,可能为新基因。
In order to analyze the molecular mechanism of cold tolerance in loquat, with early-maturing and susceptible-to-frost-damage Zao Zhong No.6 loquat fruit as the research object, DDRT-PCR technique was used to study the gene expression during the course of cold stress in loquat.
The SDS method was improved to find out a kind of fast and efficient extraction method of total RNA in loquat fruit, and the RNA extracted using this method was suitable for DDRT-PCR analysis.
By optimizing the test, the best DDRT-PCR analysis system for loquat fruit during the course of cold stress was established, In 20μl system, each component was identified as:Template cDNA 1.0μl,Primer 0.5μM,dNTPs 50μM, 10×PCR buffer 2μl, Taq enzyme 1U, annealing temperature 40℃. On the basis of this system, the mRNA changes of loquat fruit was analyzed under the condition of 0℃treated for 6 h. Nineteen differentially expressed gene segments induced by cold stress were obtained and four of them was confirmed as positive clones by Reverse Northern dot blotting(It was named CSIGE 1～4—Cold Stress Induced Genes in Eriobotrya japonica 1～4).
The four positive fragments were sequenced and have been submitted to GenBank. CSIGE 1~4 followed by the accession number of GT617703, GT617704, GT617705, GT617706.
The result of sequence similarity analysis showed that:①CSIGE 1 sequence has highly homologous to Homo sapiens chromosome 8 clone Rp11-420F14 map p21-p11, which was a cold-resistance gene. This shows that plant and animal genomes have certain common coding sequence, maybe they have some common gene expression mechanism;②CSIGE 2 has different degrees of homology with some genes, such as alcohol dehydrogenase(ADH) in ricinus communis,NADP-dependent oxidoreductases in arabidopsis thaliana, salicylic acid stress-related genes in soybean.,CSIGE 3 has some homologous to ESTs stressed by adversity in vitis vinifera and cucumber, the regulation of their expression play some roles in response to cold stress in loquat;③CSIGE 4 did not show homologous to any genes, which may be new genes.
对SDS法进行改良,探索出快速、高效的提取枇杷幼果总RNA的方法,采用此方法提取的RNA适合mRNA差异显示分析。
通过优化试验,建立了适合枇杷幼果低温胁迫下DDRT-PCR分析的最佳体系,在20μl体系中,确定了各组分为:模板cDNA 1.0μl,锚定引物0.5μM,随机引物0.5μM,dNTPs 50μM,10×PCR buffer 2μl,Taq酶1U,退火温度40℃。在此基础上对枇杷幼果在0℃低温胁迫6h下mRNA的变化进行分析,共分离到19条差异表达的基因片段,经Reverse Northern杂交获得了4个阳性的枇杷低温诱导相关基因片段(编号为CSIGE 1~4—Cold Stress Induced Genes in Eriobotrya japonica 1~4)。对4个阳性差异片段进行测序,序列已提交GenBank,CSIGE 1~4登录号依次为GT617703、GT617704、GT617705和GT617706。
序列相似性分析表明:①CSIGE 1序列与Homo sapiens chromosome 8 clone Rp11-420F14 map p21-p11有高度同源性,是抗低温胁迫基因,这说明植物与动物基因组具有某些共同的编码序列,可能在基因表达方面具有一些共同的机制;②CSIGE 2与蓖麻乙醇脱氢酶(ADH)、拟南芥NADP依赖型氧化还原酶、大豆受水杨酸胁迫诱导相关基因等有不同程度的同源性,CSIGE 3与葡萄和黄瓜受逆境胁迫的EST有部分同源,该基因表达量的调节对枇杷响应低温逆境起到某种作用;③CSIGE 4序列未搜索到与任何基因有相似性,可能为新基因。
In order to analyze the molecular mechanism of cold tolerance in loquat, with early-maturing and susceptible-to-frost-damage Zao Zhong No.6 loquat fruit as the research object, DDRT-PCR technique was used to study the gene expression during the course of cold stress in loquat.
The SDS method was improved to find out a kind of fast and efficient extraction method of total RNA in loquat fruit, and the RNA extracted using this method was suitable for DDRT-PCR analysis.
By optimizing the test, the best DDRT-PCR analysis system for loquat fruit during the course of cold stress was established, In 20μl system, each component was identified as:Template cDNA 1.0μl,Primer 0.5μM,dNTPs 50μM, 10×PCR buffer 2μl, Taq enzyme 1U, annealing temperature 40℃. On the basis of this system, the mRNA changes of loquat fruit was analyzed under the condition of 0℃treated for 6 h. Nineteen differentially expressed gene segments induced by cold stress were obtained and four of them was confirmed as positive clones by Reverse Northern dot blotting(It was named CSIGE 1～4—Cold Stress Induced Genes in Eriobotrya japonica 1～4).
The four positive fragments were sequenced and have been submitted to GenBank. CSIGE 1~4 followed by the accession number of GT617703, GT617704, GT617705, GT617706.
The result of sequence similarity analysis showed that:①CSIGE 1 sequence has highly homologous to Homo sapiens chromosome 8 clone Rp11-420F14 map p21-p11, which was a cold-resistance gene. This shows that plant and animal genomes have certain common coding sequence, maybe they have some common gene expression mechanism;②CSIGE 2 has different degrees of homology with some genes, such as alcohol dehydrogenase(ADH) in ricinus communis,NADP-dependent oxidoreductases in arabidopsis thaliana, salicylic acid stress-related genes in soybean.,CSIGE 3 has some homologous to ESTs stressed by adversity in vitis vinifera and cucumber, the regulation of their expression play some roles in response to cold stress in loquat;③CSIGE 4 did not show homologous to any genes, which may be new genes.
引文
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