榛子花芽转录组文库的Solexa测序及冷调节基因的表达谱分析
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摘要
温度是限制植物地理分布和生长发育的主要因素之一,低温会给植物的产量造成巨大的损失。榛子是重要的落叶木本植物,具有极大的经济价值和生态效益,而且与其他木本植物相比具有基因组小、结果早、易于研究的优势,可以作为研究桦木科乃至壳斗目植物代谢过程的模式物种;世界上广泛种植的是欧洲榛,具有果大皮薄等优点但不抗寒,在我国广泛分布的平榛能耐受-48℃的低温,但目前为止公共数据库中还没有榛子抗寒相关的分子生物学信息,本研究以寒冬时期的平榛花芽为试材,采用Solexa高通量测序技术进行转录组分析,从中筛选出大量的冷调节基因,采用RT-PCR技术研究了20个基因对不同低温条件的表达差异,并选择转录因子CBF和功能基因脱水素基因进行初步的功能分析,结果将为以后的榛子抗寒遗传改良提供基因资源,也为进一步了解榛子越冬分子机制奠定基础。主要研究结果如下:
1.利用平榛寒冬时期的花芽为材料建立cDNA文库,接着在Illumina上采用Solexa技术进行序列测定,总共进行了13,815,180次读数,共获得有效序列2,030,831,460 nt。序列经组装得到129,912个Contig,53,471个独立的Singleton,进一步拼接后,共获得43,285个Unigene。按照GO的分子功能、生物过程和细胞组分等3个不同分类角度对被注释的3,890个具有同源性匹配的基因序列进行分类。在具有功能注释的43,285个Unigene中,通过进一步Blastn比对,根据“基因结构相似,功能同源”的原理,在平榛花芽转录组文库中筛选到几十条冷调节相关的Unigene,分别涉及转录、信号转导、抗性、细胞壁代谢、碳代谢、氮代谢、脂肪酸代谢、多胺代谢、渗调物质等功能类别,对其中20个假定的冷调节基因(包括14-3-3、HSP22、GPAT、WCOR413-PM、ADF、bzip78、ICE、CIP、NAC、PHD、ENO、HD、DOF、SPDS、NIR、BADH、BAM、ERD7、KT、SATO)进行全长克隆并设计引物采用荧光定量PCR手段对越冬过程中的四个时间点进行了表达分析,2个基因在冷适应时期(CA)表达量最大,18个基因在寒冬时期(MW)出现最大表达丰度;采用半定量RT-PCR方法研究了20个基因对低温胁迫的响应,转录因子一般出现在低温胁迫后的2-4h之内,功能基因对低温的响应较慢;另外采用半定量RT-PCR方法还研究了20个基因在冷适应时期两种榛属植物平榛和欧洲榛中的表达差异,大部分基因在平榛中表达强度比欧洲榛高。
2.适当的内参基因选择是进行基因表达研究的前提,本研究采用半定量RT-PCR方法对ChACTIN、ChMDH、ChUBQ和ChEF1α四个内参基因在冷胁迫条件下和不同器官中的表达进行了研究,结果发现ChACTIN是最稳定的。
3.从平榛花芽转录组文库中筛选得到两个CBF基因:CBF1和CBF2,CBF1的最大表达量出现在寒冬时期,CBF2最大表达量出现在冷适应时期,推测两者分别主要参与平榛的抗冻和抗冷,共同完成平榛的越冬过程;CBF1和CBF2分别在4°C处理后8h和4h转录本累积量最大;空间表达结果表明CBF1主要在雄花序中表达,CBF2主要在花芽中表达。CBF2在两种生态型的榛属植物中存在序列多态性,这种氨基酸的突变可能导致转录结合活性的差异。同时pBI121-CBF2-GFP融合蛋白在洋葱表皮细胞中瞬时表达也验证了CBF2的核定位功能。
4.通过RACE技术获得两个DHN基因的全长序列,DHN1包含一个长度为339 bp的开放读码框,编码一个含112个氨基酸的蛋白质,5'非翻译区长60 bp,3'非翻译区长98 bp,属于K_2S类型DHN,在DHN1基因的3’非编码区域含有97bp的内含子,Genebank注册号:HM228388;DHN2包含一个长度为504bp的开放读码框,编码一个含167个氨基酸的蛋白质,5'非翻译区长41 bp,3'非翻译区长94 bp,该DHN2基因属于Y4SK2类型DHN,扩增基因组的对应区段发现在DHN2基因中间区段含有95bp的内含子序列,GenBank登录号为HM228389。冷激处理后ChDHN2基因表现逐渐地上调表达趋势,24 h达到最大表达量,在种子中高丰度表达。利用基因组步移法获取了两个DHN基因的5’侧翼序列,采用PLANTCARE软件分析顺式作用元件,除了含有胁迫响应元件MYB核心序列、ABRE或MYCR等元件外,DHN1还含有一个CRT低温响应元件。构建原核表达载体pET30(a+)-ChDHN2,在大肠杆菌BL21中表达出约30KDa的融合蛋白,过表达ChDHN2提高了大肠杆菌对低温胁迫的抗性。构建植物正义表达载体PBI121-ChDHN2,利用农杆菌介导转化烟草,低温处理后转基因烟草的冷害症状轻于野生型烟草,转基因烟草细胞电解质外渗较少和MDA累积量较低,这些实验结果说明DHN2的异源表达提高了植物的抗寒性。
Temperature is one of the most important environmental factors limiting the geographical distribution of plants and accounts for significant reductions in the yield of agriculturally important crops. Low temperature damages many plant species, especially those adapted to tropical climates. In contrast, some species from temperate regions are able to develop freezing tolerance in response to low non-freezing temperature, an adaptive process named cold acclimation. Numerous molecular, biochemical and physiological changes occur during cold acclimation, most of them being associated with significant changes in gene expression. hazelnut is one of ecologically and economically important woody plant species. Corylus avellana is a diploid with a small haploid genome, Hazelnut could serve as a model for genomics studies in the family Betulaceae and the order Fagales. The European hazelnut, C. avellana, is the species of commerce and is genetically diverse as its large size, thinner shells. However, some highly desirable and economically important traits such as cold hardiness, resistance to eastern filbert blight do not exist within C. avellana. Corylus heterophylla Fisch. is widely distributed in Northern China. Compared with C. avellana L, the nut weight of C. heterophylla Fisch. is smaller, and the yield is poor, but it has very good winter hardiness. There are not sufficient transcriptomic and genomic data in public databases for understanding of the molecular mechanism underlying overwintering of hazelnut. In this article, we report a transcriptome sequence of cold-hardy corylus species, C. heterophylla Fisch, derived by high-throughput Solexa sequencing. Transcript changes of 20 genes under the field and artificial-controlled cold stress condition were investigated by using real-time and semiRT-PCR method, respectively. The results provide a fairly comprehensive molecular biology background to the research on hazelnut development, particularly with overwintering of floral bud. In addition, we choosed CBF and DHN to do further functional identification. Overall, these sequences will provide valuable resources for the hazelnut community. Additionally, these results also suggested that transcriptome analysis based on Illumina paired-end sequencing is a powerful tool for gene discovery for non-model species. This work will improve our understanding on overwintering of hazelnut. And results will lay the foundation for carrying out genetic improvement of cold-tolerant hazelnut cultivar. Main results are as follows:
1. This study investigated the transcriptome profiles of hazelnut using the Solexa/Illumina RNA-seq technologies, high-throughput sequencing, assembly, and annotation of expressed sequences from hazelnut were obtained. The substantial amount of transcripts obtained provides a strong basis for future genomic research on hazelnut organisms. More than 13 million sequencing reads were generated using Illumina paired-end sequencing technology. De novo assembly yielded 43,285 Unigenes with an average length of 637 bp. These assembled unigenes were annotated with various methods, Out of these annotated Unigenes, 3,890 and 65,536 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Based on sequence similarity search with known proteins, a total of over 50 genes were identified to cold regulated. Among those genes some involved in osmo-regulation Substances biosynthesis, regulation of transcription, Nitrogen metabolism, antioxidant and stress responses or plant defence, were found, including 14-3-3, HSP22, GPAT, ERD7, WCOR413-PM, ADF, KT, bzip78, ICE, CIP, NAC, PHD, ENO, HD, DOF. SPDS, NIR, BADH, BAM, SATO. 20 genes of interest which belong to different categories were selected to clone and their expression was analyzed in floral buds during natural NA-CA-MW-DA and in leaves under controlled cold stress by quantitative real-time RT-PCR and semi RT-PCR, respectively. Furthermore, most genes accumulated to higher levels in the cold hardy genotype‘Corylus heterophylla Fisch’than in‘Corylus avellana L.’
2. Gene expression analysis by semi-quantitative RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in semi-quantitative RT-PCR for abiotic stress studies in hazelnut. Partial sequences of four Corylus heterophylla Fisch housekeeping genes such as ChACTIN, ChMDH, ChUBQ and ChEF1 were obtained by screening the transcriptome library. Primers for quantitative RT-PCR were designed based on these partial sequences. The housekeeping genes were evaluated for their expression stability in samples from plants subjected to cold stress and different organs. The analysis found that ChActin were the most stable.
3. The CBF/DREB1 transcription factors control an important pathway for increased freezing tolerance in plants. We report here the isolation of CBF1 and CBF2, from freezing-tolerant hazelnut (Corylus heterophylla Fischer) and freezing-sensitive hazelnut(Corylus avellana L.). CBF2 expression pattern differ substantially between‘Corylus heterophylla Fischer’and‘Corylus avellana L.’Hydrophobic residues of C-terminal region may result in transcription activation. The tissue organ-specific expression pattern of the CBF1 and CBF2 gene showed that its transcripts were abundant in male flower and floral bud, respectively. Nuclear localization of CBF2 proteins was confirmed by transient introduction of green ?uorescent protein fusion constructs for a subset of protein genes into onion cells.
4. Two cDNA encoding dehydrin-like gene homologues was isolated from hazelnut(Corylus heterophylla Fisch.)by RACE-PCR and designated ChDHN1 (GenBank Accession No. HM228388) and ChDHN2 (GenBank Accession No. HM228389). Sequence analysis of ChDHN1 showed it contained a single open reading frame of 339 bp. The predicted ChDHN1 protein has 112 amino acids; Sequence analysis showed that cDNA of ChDHN2 was 639 bp long and contained a single open reading frame. The predicted ChDHN2 protein has 167 amino acids, qRT-PCR analysis showed that the expression of ChDHN2 was induced by low temperature and peaked at 24 h after exposed to low temperatures of 4℃. The transcripts of ChDHN2 appeared in many hazelnut tissues including male flower, bark, ?ower bud and seeds, but mostly accumulated in seeds. Genomic DNA sequence of DHN was obtained through TAIL-PCR, the results found that a 97-bp and 95-bp intron was present in the genomic sequence of DHN1 and DHN2, respectively..
Computer analysis using the PLANTCARE database predicted the presence of several putative cis-regulatory sequences, in addition to these MYC, ABRE and MYB recognition sites, CRT cis-elements were predicted in the DHN1 promoter. In order to ideniify the function of ChDHN2, in vitro functional analysis were performed using an E. coli heterologous expression system. Recombinant pET-ChDHN2 vector was constructed, SDS-PAGE analysis showed that the recombinant plasmid pET-30a(+)-DHN2 could be expressed a relative molecule mass of approximately 30 KDa, Western blot results showed that the recombinant fusion protein could be identified by anti-His antibody. Expression of pET-ChDHN2 in E. coli enhanced its tolerance to cold stress. Under low temperature, ChDHN2-overexpressing tobacco showed lower MDA content, electrolyte leakage and superoxide radical (O_2~(.-)). These results indicated heterologous expression of ChDHN2 in tobacco could enhance the ability to endure low temperature.
1.利用平榛寒冬时期的花芽为材料建立cDNA文库,接着在Illumina上采用Solexa技术进行序列测定,总共进行了13,815,180次读数,共获得有效序列2,030,831,460 nt。序列经组装得到129,912个Contig,53,471个独立的Singleton,进一步拼接后,共获得43,285个Unigene。按照GO的分子功能、生物过程和细胞组分等3个不同分类角度对被注释的3,890个具有同源性匹配的基因序列进行分类。在具有功能注释的43,285个Unigene中,通过进一步Blastn比对,根据“基因结构相似,功能同源”的原理,在平榛花芽转录组文库中筛选到几十条冷调节相关的Unigene,分别涉及转录、信号转导、抗性、细胞壁代谢、碳代谢、氮代谢、脂肪酸代谢、多胺代谢、渗调物质等功能类别,对其中20个假定的冷调节基因(包括14-3-3、HSP22、GPAT、WCOR413-PM、ADF、bzip78、ICE、CIP、NAC、PHD、ENO、HD、DOF、SPDS、NIR、BADH、BAM、ERD7、KT、SATO)进行全长克隆并设计引物采用荧光定量PCR手段对越冬过程中的四个时间点进行了表达分析,2个基因在冷适应时期(CA)表达量最大,18个基因在寒冬时期(MW)出现最大表达丰度;采用半定量RT-PCR方法研究了20个基因对低温胁迫的响应,转录因子一般出现在低温胁迫后的2-4h之内,功能基因对低温的响应较慢;另外采用半定量RT-PCR方法还研究了20个基因在冷适应时期两种榛属植物平榛和欧洲榛中的表达差异,大部分基因在平榛中表达强度比欧洲榛高。
2.适当的内参基因选择是进行基因表达研究的前提,本研究采用半定量RT-PCR方法对ChACTIN、ChMDH、ChUBQ和ChEF1α四个内参基因在冷胁迫条件下和不同器官中的表达进行了研究,结果发现ChACTIN是最稳定的。
3.从平榛花芽转录组文库中筛选得到两个CBF基因:CBF1和CBF2,CBF1的最大表达量出现在寒冬时期,CBF2最大表达量出现在冷适应时期,推测两者分别主要参与平榛的抗冻和抗冷,共同完成平榛的越冬过程;CBF1和CBF2分别在4°C处理后8h和4h转录本累积量最大;空间表达结果表明CBF1主要在雄花序中表达,CBF2主要在花芽中表达。CBF2在两种生态型的榛属植物中存在序列多态性,这种氨基酸的突变可能导致转录结合活性的差异。同时pBI121-CBF2-GFP融合蛋白在洋葱表皮细胞中瞬时表达也验证了CBF2的核定位功能。
4.通过RACE技术获得两个DHN基因的全长序列,DHN1包含一个长度为339 bp的开放读码框,编码一个含112个氨基酸的蛋白质,5'非翻译区长60 bp,3'非翻译区长98 bp,属于K_2S类型DHN,在DHN1基因的3’非编码区域含有97bp的内含子,Genebank注册号:HM228388;DHN2包含一个长度为504bp的开放读码框,编码一个含167个氨基酸的蛋白质,5'非翻译区长41 bp,3'非翻译区长94 bp,该DHN2基因属于Y4SK2类型DHN,扩增基因组的对应区段发现在DHN2基因中间区段含有95bp的内含子序列,GenBank登录号为HM228389。冷激处理后ChDHN2基因表现逐渐地上调表达趋势,24 h达到最大表达量,在种子中高丰度表达。利用基因组步移法获取了两个DHN基因的5’侧翼序列,采用PLANTCARE软件分析顺式作用元件,除了含有胁迫响应元件MYB核心序列、ABRE或MYCR等元件外,DHN1还含有一个CRT低温响应元件。构建原核表达载体pET30(a+)-ChDHN2,在大肠杆菌BL21中表达出约30KDa的融合蛋白,过表达ChDHN2提高了大肠杆菌对低温胁迫的抗性。构建植物正义表达载体PBI121-ChDHN2,利用农杆菌介导转化烟草,低温处理后转基因烟草的冷害症状轻于野生型烟草,转基因烟草细胞电解质外渗较少和MDA累积量较低,这些实验结果说明DHN2的异源表达提高了植物的抗寒性。
Temperature is one of the most important environmental factors limiting the geographical distribution of plants and accounts for significant reductions in the yield of agriculturally important crops. Low temperature damages many plant species, especially those adapted to tropical climates. In contrast, some species from temperate regions are able to develop freezing tolerance in response to low non-freezing temperature, an adaptive process named cold acclimation. Numerous molecular, biochemical and physiological changes occur during cold acclimation, most of them being associated with significant changes in gene expression. hazelnut is one of ecologically and economically important woody plant species. Corylus avellana is a diploid with a small haploid genome, Hazelnut could serve as a model for genomics studies in the family Betulaceae and the order Fagales. The European hazelnut, C. avellana, is the species of commerce and is genetically diverse as its large size, thinner shells. However, some highly desirable and economically important traits such as cold hardiness, resistance to eastern filbert blight do not exist within C. avellana. Corylus heterophylla Fisch. is widely distributed in Northern China. Compared with C. avellana L, the nut weight of C. heterophylla Fisch. is smaller, and the yield is poor, but it has very good winter hardiness. There are not sufficient transcriptomic and genomic data in public databases for understanding of the molecular mechanism underlying overwintering of hazelnut. In this article, we report a transcriptome sequence of cold-hardy corylus species, C. heterophylla Fisch, derived by high-throughput Solexa sequencing. Transcript changes of 20 genes under the field and artificial-controlled cold stress condition were investigated by using real-time and semiRT-PCR method, respectively. The results provide a fairly comprehensive molecular biology background to the research on hazelnut development, particularly with overwintering of floral bud. In addition, we choosed CBF and DHN to do further functional identification. Overall, these sequences will provide valuable resources for the hazelnut community. Additionally, these results also suggested that transcriptome analysis based on Illumina paired-end sequencing is a powerful tool for gene discovery for non-model species. This work will improve our understanding on overwintering of hazelnut. And results will lay the foundation for carrying out genetic improvement of cold-tolerant hazelnut cultivar. Main results are as follows:
1. This study investigated the transcriptome profiles of hazelnut using the Solexa/Illumina RNA-seq technologies, high-throughput sequencing, assembly, and annotation of expressed sequences from hazelnut were obtained. The substantial amount of transcripts obtained provides a strong basis for future genomic research on hazelnut organisms. More than 13 million sequencing reads were generated using Illumina paired-end sequencing technology. De novo assembly yielded 43,285 Unigenes with an average length of 637 bp. These assembled unigenes were annotated with various methods, Out of these annotated Unigenes, 3,890 and 65,536 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Based on sequence similarity search with known proteins, a total of over 50 genes were identified to cold regulated. Among those genes some involved in osmo-regulation Substances biosynthesis, regulation of transcription, Nitrogen metabolism, antioxidant and stress responses or plant defence, were found, including 14-3-3, HSP22, GPAT, ERD7, WCOR413-PM, ADF, KT, bzip78, ICE, CIP, NAC, PHD, ENO, HD, DOF. SPDS, NIR, BADH, BAM, SATO. 20 genes of interest which belong to different categories were selected to clone and their expression was analyzed in floral buds during natural NA-CA-MW-DA and in leaves under controlled cold stress by quantitative real-time RT-PCR and semi RT-PCR, respectively. Furthermore, most genes accumulated to higher levels in the cold hardy genotype‘Corylus heterophylla Fisch’than in‘Corylus avellana L.’
2. Gene expression analysis by semi-quantitative RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in semi-quantitative RT-PCR for abiotic stress studies in hazelnut. Partial sequences of four Corylus heterophylla Fisch housekeeping genes such as ChACTIN, ChMDH, ChUBQ and ChEF1 were obtained by screening the transcriptome library. Primers for quantitative RT-PCR were designed based on these partial sequences. The housekeeping genes were evaluated for their expression stability in samples from plants subjected to cold stress and different organs. The analysis found that ChActin were the most stable.
3. The CBF/DREB1 transcription factors control an important pathway for increased freezing tolerance in plants. We report here the isolation of CBF1 and CBF2, from freezing-tolerant hazelnut (Corylus heterophylla Fischer) and freezing-sensitive hazelnut(Corylus avellana L.). CBF2 expression pattern differ substantially between‘Corylus heterophylla Fischer’and‘Corylus avellana L.’Hydrophobic residues of C-terminal region may result in transcription activation. The tissue organ-specific expression pattern of the CBF1 and CBF2 gene showed that its transcripts were abundant in male flower and floral bud, respectively. Nuclear localization of CBF2 proteins was confirmed by transient introduction of green ?uorescent protein fusion constructs for a subset of protein genes into onion cells.
4. Two cDNA encoding dehydrin-like gene homologues was isolated from hazelnut(Corylus heterophylla Fisch.)by RACE-PCR and designated ChDHN1 (GenBank Accession No. HM228388) and ChDHN2 (GenBank Accession No. HM228389). Sequence analysis of ChDHN1 showed it contained a single open reading frame of 339 bp. The predicted ChDHN1 protein has 112 amino acids; Sequence analysis showed that cDNA of ChDHN2 was 639 bp long and contained a single open reading frame. The predicted ChDHN2 protein has 167 amino acids, qRT-PCR analysis showed that the expression of ChDHN2 was induced by low temperature and peaked at 24 h after exposed to low temperatures of 4℃. The transcripts of ChDHN2 appeared in many hazelnut tissues including male flower, bark, ?ower bud and seeds, but mostly accumulated in seeds. Genomic DNA sequence of DHN was obtained through TAIL-PCR, the results found that a 97-bp and 95-bp intron was present in the genomic sequence of DHN1 and DHN2, respectively..
Computer analysis using the PLANTCARE database predicted the presence of several putative cis-regulatory sequences, in addition to these MYC, ABRE and MYB recognition sites, CRT cis-elements were predicted in the DHN1 promoter. In order to ideniify the function of ChDHN2, in vitro functional analysis were performed using an E. coli heterologous expression system. Recombinant pET-ChDHN2 vector was constructed, SDS-PAGE analysis showed that the recombinant plasmid pET-30a(+)-DHN2 could be expressed a relative molecule mass of approximately 30 KDa, Western blot results showed that the recombinant fusion protein could be identified by anti-His antibody. Expression of pET-ChDHN2 in E. coli enhanced its tolerance to cold stress. Under low temperature, ChDHN2-overexpressing tobacco showed lower MDA content, electrolyte leakage and superoxide radical (O_2~(.-)). These results indicated heterologous expression of ChDHN2 in tobacco could enhance the ability to endure low temperature.
引文
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