TMV侵染烟草基因差异表达的cDNA-AFLP分析
摘要
烟草普通花叶病是世界各烟草产区普遍发生的一种病害,发病田块可减产30-50%,导致重大的经济损失,选用抗病品种是最有效的防治手段。目前TMV的抗病基因主要来自野生种粘烟草(N.glutinosa)的单显性基因N基因,和普通烟草Ambalema品种中的隐性等位基因rm1和rm2,但以上抗病基因均与不良性状基因存在连锁或基因多效应性,在商业品种的利用上有较大困难,所以TMV抗病基因的标记与筛选,已经成为现今急待解决的课题。
本研究以TMV高抗品种龙江925为试验材料,利用cDNA-AFLP技术,对烟草叶片与TMV互作后的应答基因进行分析。通过240对引物组合的筛选,获得结果如下:
1、共得到约9500个转录基因片段,平均每对选择性扩增引物可获得20条转录基因片段,其中190个为组成型表达基因片段,42个为诱导表达基因片段,41个为抑制表达基因片段。经过克隆测序,最终获得了15个表达基因片段,其中包括12个TMV诱导表达基因片段的序列,两个组成型表达基因片段及1个抑制表达基因片段。
2、为验证表达基因的真实性,利用荧光定量PCR分析了一个诱导表达片段(TIF2)在0-72小时之间5个时间点的基因差异表达,并证实了所获得的基因序列为真实诱导的表达序列。对此序列进行RACE,最终获得全长cDNA序列。
3、利用NCBI上的GenBank等公共数据库对所获得的转录基因片段进行生物信息分析和功能预测。结果表明:TMV诱导烟草抗病机制涉及多方面生理生化反应,多个功能不同基因参与控制。本研究共获得15条表达基因片段,经过生物信息学分析和功能预测,得知12个诱导表达基因片段与核酸代谢、蛋白质合成与修饰、能量代谢、胁迫响应、细胞内运输、糖代谢等相关;证实2个组成型表达片段均为叶绿体DNA,1个抑制表达基因与细胞壁的代谢相关。
4、以TIF2序列为基础,应用RACE方法得到cDNA全序列857bp,预测的编码区为101bp到613bp之间。经Blastn、Blastp比对分析,在烟草中没有获得其同源序列,确定该基因是首次在烟草中发现的与抗TMV相关的新基因。
以上研究结果为烟草分子育种奠定物质和技术基础,也弥补了烟草与TMV互作后基因差异表达研究的不足。
Tobacco mosaic virus (TMV) is one of the main tobacco diseases in the world , and cause serious economic losses every year. Generally, the tobacco yield losses caused by TMV infection could reach up to 30%-50%. Identification of tobacco resistance gene is always an important object and one of crucial key topic in tobacco production.
The variety of Long Jiang 925 which was TMV high resistant was taken as tested plant material in this study. cDNA amplified fragment length polymorphism analysis (cDNA-AFLP) was used to display transcripts whose expression is rapidly altered during the treatment of TMV to tobacco leaf. Two hundred and forty primer combinations were used for selective amplification. The main results were as follows:
1. The results showed that there existed over 9500 expression bands appeared at different treatment times, and 20 expression bands were amplified by each primer pair on average. Among these, 190 fragments were constitutively expressed, 42 fragments were inducible expressed, 41 fragments were suppressive expressed. By clone and sequencing, the sequences of 15 expression bands were obtained, including two constitutively expressed fragments and one suppressive expressed fragment.
2. In order to validate the functions of these differentially expressed gene sequences obtained, the TIF2 fragments was investigated by Real-time PCR. The mRNA samples isolated from tobacco leaf infected by TMV, collected at 5 time points during 0-72 h time period after infection, were taken for Real-time PCR analysis. Supported by the result of Real-time PCR, the genes isolated in this study were resistance related. Then, both 5′-and 3′-rapid amplification of cDNA ends(RACE) were preformed, we got full length cDNA sequence of TIF2.
3. Then 15 of ESTs and one full length cDNA sequence were analyzed using methods of bioinformatics. Results of Blast analysis in GenBank showed that the TMV induced resistance involves many processes of tobacco biochemistry and physiology and are controlled by multi different genes in tobacco. According to the functions of homology genes, the twelve inducible expressed gene fragments functions involves in the Nucleic Acid Metabolism, protein synthesis and modulation, energy metabolism, stress responding, intracellular transport, Metabolism of Carbohydrates, etc. Two constitutively expression fragment were chloroplast DNA and one suppressive expression fragment was related to cell wall metabolism gene.
4. By RACE, we got full length cDNA sequence of TIF2, 857bp,and putative reading frame from 101bp to 613bp. Analysis of Blastn and Blastp showed that the gene was most probably the anti-TMV related novel gene.
Under the study, the substance and technique basis for advanced tobacco molecular breeding resistant to TMV was established. And part of the shortage in the area of gene differentially expressed research in tobacco-TMV interaction has been filled up.
本研究以TMV高抗品种龙江925为试验材料,利用cDNA-AFLP技术,对烟草叶片与TMV互作后的应答基因进行分析。通过240对引物组合的筛选,获得结果如下:
1、共得到约9500个转录基因片段,平均每对选择性扩增引物可获得20条转录基因片段,其中190个为组成型表达基因片段,42个为诱导表达基因片段,41个为抑制表达基因片段。经过克隆测序,最终获得了15个表达基因片段,其中包括12个TMV诱导表达基因片段的序列,两个组成型表达基因片段及1个抑制表达基因片段。
2、为验证表达基因的真实性,利用荧光定量PCR分析了一个诱导表达片段(TIF2)在0-72小时之间5个时间点的基因差异表达,并证实了所获得的基因序列为真实诱导的表达序列。对此序列进行RACE,最终获得全长cDNA序列。
3、利用NCBI上的GenBank等公共数据库对所获得的转录基因片段进行生物信息分析和功能预测。结果表明:TMV诱导烟草抗病机制涉及多方面生理生化反应,多个功能不同基因参与控制。本研究共获得15条表达基因片段,经过生物信息学分析和功能预测,得知12个诱导表达基因片段与核酸代谢、蛋白质合成与修饰、能量代谢、胁迫响应、细胞内运输、糖代谢等相关;证实2个组成型表达片段均为叶绿体DNA,1个抑制表达基因与细胞壁的代谢相关。
4、以TIF2序列为基础,应用RACE方法得到cDNA全序列857bp,预测的编码区为101bp到613bp之间。经Blastn、Blastp比对分析,在烟草中没有获得其同源序列,确定该基因是首次在烟草中发现的与抗TMV相关的新基因。
以上研究结果为烟草分子育种奠定物质和技术基础,也弥补了烟草与TMV互作后基因差异表达研究的不足。
Tobacco mosaic virus (TMV) is one of the main tobacco diseases in the world , and cause serious economic losses every year. Generally, the tobacco yield losses caused by TMV infection could reach up to 30%-50%. Identification of tobacco resistance gene is always an important object and one of crucial key topic in tobacco production.
The variety of Long Jiang 925 which was TMV high resistant was taken as tested plant material in this study. cDNA amplified fragment length polymorphism analysis (cDNA-AFLP) was used to display transcripts whose expression is rapidly altered during the treatment of TMV to tobacco leaf. Two hundred and forty primer combinations were used for selective amplification. The main results were as follows:
1. The results showed that there existed over 9500 expression bands appeared at different treatment times, and 20 expression bands were amplified by each primer pair on average. Among these, 190 fragments were constitutively expressed, 42 fragments were inducible expressed, 41 fragments were suppressive expressed. By clone and sequencing, the sequences of 15 expression bands were obtained, including two constitutively expressed fragments and one suppressive expressed fragment.
2. In order to validate the functions of these differentially expressed gene sequences obtained, the TIF2 fragments was investigated by Real-time PCR. The mRNA samples isolated from tobacco leaf infected by TMV, collected at 5 time points during 0-72 h time period after infection, were taken for Real-time PCR analysis. Supported by the result of Real-time PCR, the genes isolated in this study were resistance related. Then, both 5′-and 3′-rapid amplification of cDNA ends(RACE) were preformed, we got full length cDNA sequence of TIF2.
3. Then 15 of ESTs and one full length cDNA sequence were analyzed using methods of bioinformatics. Results of Blast analysis in GenBank showed that the TMV induced resistance involves many processes of tobacco biochemistry and physiology and are controlled by multi different genes in tobacco. According to the functions of homology genes, the twelve inducible expressed gene fragments functions involves in the Nucleic Acid Metabolism, protein synthesis and modulation, energy metabolism, stress responding, intracellular transport, Metabolism of Carbohydrates, etc. Two constitutively expression fragment were chloroplast DNA and one suppressive expression fragment was related to cell wall metabolism gene.
4. By RACE, we got full length cDNA sequence of TIF2, 857bp,and putative reading frame from 101bp to 613bp. Analysis of Blastn and Blastp showed that the gene was most probably the anti-TMV related novel gene.
Under the study, the substance and technique basis for advanced tobacco molecular breeding resistant to TMV was established. And part of the shortage in the area of gene differentially expressed research in tobacco-TMV interaction has been filled up.
引文
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