青天葵转录组特征研究
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摘要
本草基因组计划(Herb Genome Program,HGP)是针对有重要经济价值、资源濒危等药用植物开展的功能基因组学、蛋白组学、代谢组学及遗传代谢工程和分子遗传育种研究,旨在选育高品质、高产量、抗胁迫的药用植物,并建立现代中药有效成分生物工程体系,其中转录组是HGP最活跃的研究领域之一。在岭南地区有不少药用植物,如青天葵,都面临着资源濒危,已有的资源获得方法无法满足需求,而且功能基因等研究仍属空白的现状。
青天葵来源于兰科植物毛唇芋兰Nervilia fordii(Hance)Schltr.的叶片或全株。具有润肺止咳、清热解毒、散瘀止痛的功效,对小儿呼吸道疾病、小儿疳积疗效显著。青天葵不仅为岭南道地贵重药材,也是出口创汇中药,市场需求量大。由于人类过度开采和自身萌发条件苛刻,青天葵的野生资源已渐枯竭,其也己先后被列入《中国南部石灰岩稀有濒危植物名录》和《濒危动植物物种国际贸易公约》。组织培养和人工栽培是目前尝试获得青天葵资源的主要方法,但人工栽培和组织培养分别面临着种苗稀缺和“炼苗下田”、扩大种植等瓶颈问题,因此,通过这两种方法获得的青天葵远远不能满足市场和临床需求。在供需严重失衡的情况下,市场上出现了青天葵的众多混伪品、混淆种。此外,青天葵商品药材还根据叶片大小分为大、中、小叶3个等级。
本论文通过分析青天葵及其混伪品、同属植物的DNA条形码序列的差异信息,建立起青天葵的新型真伪鉴别体系。以种质明确的青天葵为材料,在RNA水平上进行青天葵转录组高通量测序,建立起青天葵的转录组特征及其数据平台,利用生物信息学挖掘青天葵次生代谢产物合成、生理功能等相关基因,并且通过对器官大小相关基因EBP1进行克隆和表达,验证青天葵转录组特征的可行性,为利用生物技术进行化学成分调控和品种选育提供基础数据。方法和结果概括如下：
1基于DNA条形码的青天葵真伪鉴别
利用MEGA软件分析3种叶型青天葵及其混伪品毛叶芋兰、车前草、红薯叶、积雪草的ITS2、rbcL、matK条形码序列信息,获得的ITS2序列的变异程度最高(89.3%),变异位点总数最多的则是matK基因(242个)。青天葵的种内遗传距离在0.000～0.004之间,不同序列的平均种间遗传距离变化范围为0.088-1.138,其中ITS2平均遗传距离最大,rbcL平均遗传距离最小。青天葵及其混伪品种间遗传距离远远大于青天葵的种内遗传距离,表明3条DNA条形码序列都有足够的差异来鉴别青天葵及其混伪品,基于3条序列构建的NJ聚类树也均能直观地区别青天葵及其混伪品。然而,3种叶型青天葵的7个样品rbcL和matK序列则完全一致,中、小叶型青天葵的核ITS2序列未获得合格序列,因此未能在以上3条形码序列中找到青天葵分型依据。
应用相应的通用引物分别进行PCR扩增并测序,进一步评估ITS2、rbcL、matK, LSU D1-D3等DNA片段对青天葵与芋兰属近缘物种的鉴别效果。各候选序列PCR扩增效率均达到100%。测序成功率以rbcL和LSU D1-D3最高,matK和ITS2稍低。在种内变异方面,matK, LSU D1-D3在各物种的居群内和不同居群间均没有变异,而ITS2和rbcL序列均存在微小的变异；ITS2种间变异则远远大于其他3个候选片段。matK序列种间和种内遗传变异没有重叠,存在明显的barcoding gap,能将供试的7个物种完全鉴别：ITS2种内和种间变异有部分重叠,但种间变异程度高,有利于区分青天葵及其同属植物。基于matK序列构建的系统发育树展示了7种芋兰属物种的亲缘关系。
综合PCR扩增和测序成功率,barcoding gap和鉴定效率评价,matK序列较其他条形码具有明显的优势,可作为青天葵真伪鉴别的DNA标志以及芋兰属潜在DNA条形码。
2青天葵转录组特征研究
通过Illumina Hiseq2000高通量测序从青天葵叶片和球茎中分别获得47,668，726和54,589,832个读序,总核苷酸数达9.20Gb,最终从青天葵叶片和球茎共组装出142,220条Unigene。从筛选所获得的5,223条Unigene序列中共鉴定出5,684个SSR模体,类型为二至六核苷酸模体,其中二核苷酸AG/CT出现频率最高。青天葵转录组共有38,640条Unigene (27.17%)被注释到COG数据库的25个功能类别；110,029条Unigene (77.37%)被归类到GO数据库生物学过程、在细胞中所处的位置、分子功能三大类别；28,970条Unigene可归入KEGG数据库的黄酮类生物合成等281个代谢途径。青天葵叶片和球茎转录组表达差异显著Unigene(错误发现率不大于0.001、Unigene在球茎表达量与在叶片表达量的比值的对数值不小于1),共为62,205条,包括了表达上调Unigene31,488条和表达下调Unigene31,117条。注释获得参与青天葵黄酮类生物合成途径10个关键酶、萜类生物合成途径的18个关键酶以及生理功能过程的27个功能基因,并明确了它们在叶片和球茎中的表达水平以及组织差异性。
3青天葵转录组的验证—器官生长调控基因NflFBP1的克隆与分析
根据从青天葵转录组Unigene数据库比对和注释,获得NfEBPl长度为1185bp的Unigene片段。应用RACE技术扩增cDNA序列全长,通过设计引物扩增并获得1188bp的NfEBP1编码区序列,该序列与转录组Unigene片段相似度达到99%。编码蛋白NfEBP1含有395个氨基酸,分子量为43.5KDa,不含信号肽、线粒体或叶绿体靶向转运肽,整个多肽链位于膜外。NfEBP1与来源于沙冬青和土豆的EBP1蛋白同源性分别为82%和86%,均具有PA2G4细胞增殖功能域,归属于APP MetAP超级家族。
以青天葵不同组织(叶片、叶柄和球茎)总RNA反转录产物为模板进行qPCR扩增,通过GeNorm和NormFinder软件评价5个常用内参基因18s rRNA.Actin、 Ubiquitin、EF-1α、β-tubulin的表达稳定性,确定β-tubulin作为内参基因用于校正qPCR分析NfEBP1基因在青天葵不同部位的相对表达量的差异。采用公式计算相对表达量F=2-ΔΔCt,NfEBP1基因在青天葵不同组织的表达量存在差异,其中在叶片的表达水平最高,其次为球茎,叶柄则最低。NfEBP1在青天葵不同组织中的表达趋势与其转录组特征一致,说明了青天葵转录组应用于后续研究中的可行性。
克隆获得的器官生长调控基因NfEBP1可为后续研究中利用植物基因工程技术促进青天葵叶片等器官生长、提高青天葵产量提供基础数据。
综上所述,本研究以通过DNA条形码鉴定的青天葵为材料,在RNA水平上建立起青天葵转录特征并验证其可行性,挖掘到参与青天葵次生代谢产物生物合成、生理过程的关键基因,为青天葵的基因调控研究和优育品种选育、产量提高奠定了坚实的基础,也为其他濒危南药的资源保护提供一种新的思路。
Herb Genome Program includes a serial of project on genomics, proteomics, metabolomics, genetic metabolic engineering and molecular breeding for greatly valuable and/or endangered medicinal plants, which aims to obtain good-quality, high-yield and anti-resistant herbal medicine and establish modern biological engineering system of pharmaceutical components. Among the field of HGP, transcriptome is one of the most remarkable and popular studies. In Lingnan area, many herbal medicines, such as Nervilia fordii, encounter resource endangerment, and the processes in existence are incapable to eaze the plight. In additional, transcriptome analysis is still blank in herbal medicines from Lingnan area.
Nervilia fordii, belonging to Orchidaceae family, is a famous and valuable herbal medicine in Lingnan Area. The whole plant or leaf was used as a traditional Chinese medicine named Qingtiankui, for its benefits to human, such as moistening the lung to stop the cough, clearing away heat and relieving toxin, promoting circulation to relieve pains, and significant effect in curing pediatric respiratory diseases. Wild resource of N. fordii has endangered due to human excessive harvest and its rigorous requirements of germination and growth. The species has been listed in "Catalogue of Rare and Endangered Plants Grown on Limestone in South China" and "International Trade Convention for Endangered Plants and Animals". In order to ease the plight, existing studies have been attempting to obtain N. fordii resource by domestic cultivation and tissue culture proliferation. However, these two methods encounter their bottleneck with seedling scarcity, seedling hardening, and cultivation expanding, respectively; the resource of N. fordii acquired from these approaches is far from satisfactory of the market and clinic demand. Under the circumstance that imbalance of supply and demand and, driven by economic interests, many adulterants are mistaken as N. fordii in the market. In addition, dry N. fordii are defined to three types according to different leaf size.
In order to resolve species confusion of N. fordii and provide a scientific foundation for resource protection and biological research of N. fordii, DNA barcoding and transcriptome analysis were carried out in this thesis. N fordii was acquired that with accurately identification using DNA barcoding; and then, transcriptome features of N. fordii were explored, genes involved in synthesis of secondary metabolites and plant physiological processes were mined by high-throughput sequencing and bioinformatics'annotation, and the reliability of transcriptome features was validated using gene cloning and expression of EBP1gene, a key regulator functionally associated with plant organ size, as a sample. The methods and conclusions were summarized below.
1Molecular identification of Nervilia fordii using DNA barcodes
Three DNA sequences (ITS2, rbcL, matK) acquired from three types of N. fordii and their common adulterants. Nervilia plicata, Plantago asiatica, Centella asiatisa, Ipomoea batatas et al, were analyzed using MEGA software. The Results revealed that matK exhibited longest sequence while ITS2showed shortest, and ITS2contributed most average variation while rbcL contributed least. The interspecific divergence (0.088-1.138) between N. fordii and its adulterants was far higher than the intraspecific one in N. fordii (0.000-0.004). The NJ trees constructed based on three DNA barcoding sequence independently were capable to discriminate N. fordii and its adulterants intuitively.
To further evaluate the feasibility of ITS2, rbcL, matK and LSU D1-D3between N. fordii and its congeneric species, corresponding universal primers were used to amplify and sequence four candidate DNA barcodes respectively. All sequence exhibited well universality in Nervilia and were suitable selection of candidate barcodes in Nervilia. At intraspecific level, none variation were found in matK and LSUD1-D3regions, however,0.4%,7.2%of divergence of ITS2within N. aragoana, N. mackinnonii, and0.2%of variation of rbcL within N. fordii were detected. ITS2contributed most average interspecific variation (22.9%), followed by LSU D1-D3(4.6%), matK (3.7%) and rbcL (2.0%). Similar results were showed by wilcoxon test. However, matK gene exhibited significant barcoding gap between distribution of intra-and inter-specific variation and identified all Nervilia species used in this study. The phylogenetic tree constructed by matK gene illustrated the genetic relationship among Nervilia species.
Therefore, matK gene can served as standard DNA sequence for authentication of N. fordii and its adulterants and congeneric species, and as a potential DNA barcode for Nervilia.
2Transcriptome signature of Nervilia fordii
A total of142,220Unigenes were assembled from transcriptome sequencing data of N. fordii leaf and corm. A total of5,684SSR motifs were screened out from5,223Unigenes in the database, which were composed of di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide and hexa-nucleotide, and the di-nucleotide motif AG/CT repeat most. By means of bioinformatics annotation,38,640Unigenes were classified into25function categories of COG database, and110,029Unigenes were sorted into three main categories biological process, cellular component and molecular function of GO database, and28,970Unigenes distributed in281pathways in KEGG data library. Gene expression analysis of leaf and corm revealed that62,205Unigenes exhibited remarkable diversity (FDR(?)0.001,|Log2(Corm_RPKM/Leaf_RPKM)|(?)1), including31,488up-regulated genes and31,117down-regulated ones. In additional, expression trend and tissues specific of10genes encoding enzymes related to flavonoid biosynthesis,18genes encoding key enzymes involved in terpene backbone biosynthesis, and27functional genes participating plant physiological processes were excavated and illustrated in N. fordii from transcriptome data.
3Cloning and analysis of plant organ size regulator EBP1gene from N. fordii
An1185-bp Unigene encoding EBP I gene was selected from transcriptome database, and then cloned from N. fordii using RACE-PCR. The acquired gene was named as NfEBP1, with an ORF contained1188base pairs and encoded395amino acids, which shared99%identity with the Unigene. Bioinformatics analysis revealed that molecular weight of NfEBPl protein was43.5kDa and the isoelectric point was6.12, and its overall structure exhibited hydrophilic; NfEBP1contains none of the signal peptide, chloroplast transit peptide, mitochondrial targeting peptide, and located entirely outside cell membrane. The predicted secondary structure of NfEBP1was composed of21α-helixes,21extend strands,18β-turners and28random coils. The putative model of three-dimensional structure exhibited high homology with the crystal structure of human EBP1. NfEBPl also showed high homology with EBP1protein from other plants, and possessed a functional domain PA2G4.
For expression analysis of NfEBP1, five common reference genes (18s rRNA, Actin, Ubiquitin, EF-1α,β-tubulin) were compared in different tissues (leaf, corm and petiole) of N. fordii. The stability of the candidate reference genes were evaluated by Ct value using GeNorm and NormFinder software. The analysis of GeNorm and NormFinder revealed that β-tubulin was more stable than others and could be served as reference gene for the normalization of gene expression in different tissues of N. fordii using real-time fluorescence PCR. The relative expression of NfEBP1in three tissues was calculated by formula F=2-ΔΔCt. NfEPB1was ubiquitously expressed in all tissues of N. fordii, and the relative expression level reached the highest in leaf, and the lowest in petiole, which was coincident with the trait of NfEBP1in transcriptome. The results validated the feasibility of transcriptome database in the subsequent study of N. fordii.
Manipulating organ size regulator NfEBPl acqured from this study would provided an approach for promoting organ size and yield of N. fordii.
In conclusion, the transcriptome profile of N. fordii with quick and accurate identification using DNA barcoding was demonstrated, and several key genes participated in synthesis of secondary metabolites and plant physiological processes were found and their expression trends were illustrated, which provide a firm foundation for variety breeding and resource protection of N. fordii by means of molecular biological approaches, and a novel and considerable research way for other endangered medicinal plants in Lingnan area.
青天葵来源于兰科植物毛唇芋兰Nervilia fordii(Hance)Schltr.的叶片或全株。具有润肺止咳、清热解毒、散瘀止痛的功效,对小儿呼吸道疾病、小儿疳积疗效显著。青天葵不仅为岭南道地贵重药材,也是出口创汇中药,市场需求量大。由于人类过度开采和自身萌发条件苛刻,青天葵的野生资源已渐枯竭,其也己先后被列入《中国南部石灰岩稀有濒危植物名录》和《濒危动植物物种国际贸易公约》。组织培养和人工栽培是目前尝试获得青天葵资源的主要方法,但人工栽培和组织培养分别面临着种苗稀缺和“炼苗下田”、扩大种植等瓶颈问题,因此,通过这两种方法获得的青天葵远远不能满足市场和临床需求。在供需严重失衡的情况下,市场上出现了青天葵的众多混伪品、混淆种。此外,青天葵商品药材还根据叶片大小分为大、中、小叶3个等级。
本论文通过分析青天葵及其混伪品、同属植物的DNA条形码序列的差异信息,建立起青天葵的新型真伪鉴别体系。以种质明确的青天葵为材料,在RNA水平上进行青天葵转录组高通量测序,建立起青天葵的转录组特征及其数据平台,利用生物信息学挖掘青天葵次生代谢产物合成、生理功能等相关基因,并且通过对器官大小相关基因EBP1进行克隆和表达,验证青天葵转录组特征的可行性,为利用生物技术进行化学成分调控和品种选育提供基础数据。方法和结果概括如下：
1基于DNA条形码的青天葵真伪鉴别
利用MEGA软件分析3种叶型青天葵及其混伪品毛叶芋兰、车前草、红薯叶、积雪草的ITS2、rbcL、matK条形码序列信息,获得的ITS2序列的变异程度最高(89.3%),变异位点总数最多的则是matK基因(242个)。青天葵的种内遗传距离在0.000～0.004之间,不同序列的平均种间遗传距离变化范围为0.088-1.138,其中ITS2平均遗传距离最大,rbcL平均遗传距离最小。青天葵及其混伪品种间遗传距离远远大于青天葵的种内遗传距离,表明3条DNA条形码序列都有足够的差异来鉴别青天葵及其混伪品,基于3条序列构建的NJ聚类树也均能直观地区别青天葵及其混伪品。然而,3种叶型青天葵的7个样品rbcL和matK序列则完全一致,中、小叶型青天葵的核ITS2序列未获得合格序列,因此未能在以上3条形码序列中找到青天葵分型依据。
应用相应的通用引物分别进行PCR扩增并测序,进一步评估ITS2、rbcL、matK, LSU D1-D3等DNA片段对青天葵与芋兰属近缘物种的鉴别效果。各候选序列PCR扩增效率均达到100%。测序成功率以rbcL和LSU D1-D3最高,matK和ITS2稍低。在种内变异方面,matK, LSU D1-D3在各物种的居群内和不同居群间均没有变异,而ITS2和rbcL序列均存在微小的变异；ITS2种间变异则远远大于其他3个候选片段。matK序列种间和种内遗传变异没有重叠,存在明显的barcoding gap,能将供试的7个物种完全鉴别：ITS2种内和种间变异有部分重叠,但种间变异程度高,有利于区分青天葵及其同属植物。基于matK序列构建的系统发育树展示了7种芋兰属物种的亲缘关系。
综合PCR扩增和测序成功率,barcoding gap和鉴定效率评价,matK序列较其他条形码具有明显的优势,可作为青天葵真伪鉴别的DNA标志以及芋兰属潜在DNA条形码。
2青天葵转录组特征研究
通过Illumina Hiseq2000高通量测序从青天葵叶片和球茎中分别获得47,668，726和54,589,832个读序,总核苷酸数达9.20Gb,最终从青天葵叶片和球茎共组装出142,220条Unigene。从筛选所获得的5,223条Unigene序列中共鉴定出5,684个SSR模体,类型为二至六核苷酸模体,其中二核苷酸AG/CT出现频率最高。青天葵转录组共有38,640条Unigene (27.17%)被注释到COG数据库的25个功能类别；110,029条Unigene (77.37%)被归类到GO数据库生物学过程、在细胞中所处的位置、分子功能三大类别；28,970条Unigene可归入KEGG数据库的黄酮类生物合成等281个代谢途径。青天葵叶片和球茎转录组表达差异显著Unigene(错误发现率不大于0.001、Unigene在球茎表达量与在叶片表达量的比值的对数值不小于1),共为62,205条,包括了表达上调Unigene31,488条和表达下调Unigene31,117条。注释获得参与青天葵黄酮类生物合成途径10个关键酶、萜类生物合成途径的18个关键酶以及生理功能过程的27个功能基因,并明确了它们在叶片和球茎中的表达水平以及组织差异性。
3青天葵转录组的验证—器官生长调控基因NflFBP1的克隆与分析
根据从青天葵转录组Unigene数据库比对和注释,获得NfEBPl长度为1185bp的Unigene片段。应用RACE技术扩增cDNA序列全长,通过设计引物扩增并获得1188bp的NfEBP1编码区序列,该序列与转录组Unigene片段相似度达到99%。编码蛋白NfEBP1含有395个氨基酸,分子量为43.5KDa,不含信号肽、线粒体或叶绿体靶向转运肽,整个多肽链位于膜外。NfEBP1与来源于沙冬青和土豆的EBP1蛋白同源性分别为82%和86%,均具有PA2G4细胞增殖功能域,归属于APP MetAP超级家族。
以青天葵不同组织(叶片、叶柄和球茎)总RNA反转录产物为模板进行qPCR扩增,通过GeNorm和NormFinder软件评价5个常用内参基因18s rRNA.Actin、 Ubiquitin、EF-1α、β-tubulin的表达稳定性,确定β-tubulin作为内参基因用于校正qPCR分析NfEBP1基因在青天葵不同部位的相对表达量的差异。采用公式计算相对表达量F=2-ΔΔCt,NfEBP1基因在青天葵不同组织的表达量存在差异,其中在叶片的表达水平最高,其次为球茎,叶柄则最低。NfEBP1在青天葵不同组织中的表达趋势与其转录组特征一致,说明了青天葵转录组应用于后续研究中的可行性。
克隆获得的器官生长调控基因NfEBP1可为后续研究中利用植物基因工程技术促进青天葵叶片等器官生长、提高青天葵产量提供基础数据。
综上所述,本研究以通过DNA条形码鉴定的青天葵为材料,在RNA水平上建立起青天葵转录特征并验证其可行性,挖掘到参与青天葵次生代谢产物生物合成、生理过程的关键基因,为青天葵的基因调控研究和优育品种选育、产量提高奠定了坚实的基础,也为其他濒危南药的资源保护提供一种新的思路。
Herb Genome Program includes a serial of project on genomics, proteomics, metabolomics, genetic metabolic engineering and molecular breeding for greatly valuable and/or endangered medicinal plants, which aims to obtain good-quality, high-yield and anti-resistant herbal medicine and establish modern biological engineering system of pharmaceutical components. Among the field of HGP, transcriptome is one of the most remarkable and popular studies. In Lingnan area, many herbal medicines, such as Nervilia fordii, encounter resource endangerment, and the processes in existence are incapable to eaze the plight. In additional, transcriptome analysis is still blank in herbal medicines from Lingnan area.
Nervilia fordii, belonging to Orchidaceae family, is a famous and valuable herbal medicine in Lingnan Area. The whole plant or leaf was used as a traditional Chinese medicine named Qingtiankui, for its benefits to human, such as moistening the lung to stop the cough, clearing away heat and relieving toxin, promoting circulation to relieve pains, and significant effect in curing pediatric respiratory diseases. Wild resource of N. fordii has endangered due to human excessive harvest and its rigorous requirements of germination and growth. The species has been listed in "Catalogue of Rare and Endangered Plants Grown on Limestone in South China" and "International Trade Convention for Endangered Plants and Animals". In order to ease the plight, existing studies have been attempting to obtain N. fordii resource by domestic cultivation and tissue culture proliferation. However, these two methods encounter their bottleneck with seedling scarcity, seedling hardening, and cultivation expanding, respectively; the resource of N. fordii acquired from these approaches is far from satisfactory of the market and clinic demand. Under the circumstance that imbalance of supply and demand and, driven by economic interests, many adulterants are mistaken as N. fordii in the market. In addition, dry N. fordii are defined to three types according to different leaf size.
In order to resolve species confusion of N. fordii and provide a scientific foundation for resource protection and biological research of N. fordii, DNA barcoding and transcriptome analysis were carried out in this thesis. N fordii was acquired that with accurately identification using DNA barcoding; and then, transcriptome features of N. fordii were explored, genes involved in synthesis of secondary metabolites and plant physiological processes were mined by high-throughput sequencing and bioinformatics'annotation, and the reliability of transcriptome features was validated using gene cloning and expression of EBP1gene, a key regulator functionally associated with plant organ size, as a sample. The methods and conclusions were summarized below.
1Molecular identification of Nervilia fordii using DNA barcodes
Three DNA sequences (ITS2, rbcL, matK) acquired from three types of N. fordii and their common adulterants. Nervilia plicata, Plantago asiatica, Centella asiatisa, Ipomoea batatas et al, were analyzed using MEGA software. The Results revealed that matK exhibited longest sequence while ITS2showed shortest, and ITS2contributed most average variation while rbcL contributed least. The interspecific divergence (0.088-1.138) between N. fordii and its adulterants was far higher than the intraspecific one in N. fordii (0.000-0.004). The NJ trees constructed based on three DNA barcoding sequence independently were capable to discriminate N. fordii and its adulterants intuitively.
To further evaluate the feasibility of ITS2, rbcL, matK and LSU D1-D3between N. fordii and its congeneric species, corresponding universal primers were used to amplify and sequence four candidate DNA barcodes respectively. All sequence exhibited well universality in Nervilia and were suitable selection of candidate barcodes in Nervilia. At intraspecific level, none variation were found in matK and LSUD1-D3regions, however,0.4%,7.2%of divergence of ITS2within N. aragoana, N. mackinnonii, and0.2%of variation of rbcL within N. fordii were detected. ITS2contributed most average interspecific variation (22.9%), followed by LSU D1-D3(4.6%), matK (3.7%) and rbcL (2.0%). Similar results were showed by wilcoxon test. However, matK gene exhibited significant barcoding gap between distribution of intra-and inter-specific variation and identified all Nervilia species used in this study. The phylogenetic tree constructed by matK gene illustrated the genetic relationship among Nervilia species.
Therefore, matK gene can served as standard DNA sequence for authentication of N. fordii and its adulterants and congeneric species, and as a potential DNA barcode for Nervilia.
2Transcriptome signature of Nervilia fordii
A total of142,220Unigenes were assembled from transcriptome sequencing data of N. fordii leaf and corm. A total of5,684SSR motifs were screened out from5,223Unigenes in the database, which were composed of di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide and hexa-nucleotide, and the di-nucleotide motif AG/CT repeat most. By means of bioinformatics annotation,38,640Unigenes were classified into25function categories of COG database, and110,029Unigenes were sorted into three main categories biological process, cellular component and molecular function of GO database, and28,970Unigenes distributed in281pathways in KEGG data library. Gene expression analysis of leaf and corm revealed that62,205Unigenes exhibited remarkable diversity (FDR(?)0.001,|Log2(Corm_RPKM/Leaf_RPKM)|(?)1), including31,488up-regulated genes and31,117down-regulated ones. In additional, expression trend and tissues specific of10genes encoding enzymes related to flavonoid biosynthesis,18genes encoding key enzymes involved in terpene backbone biosynthesis, and27functional genes participating plant physiological processes were excavated and illustrated in N. fordii from transcriptome data.
3Cloning and analysis of plant organ size regulator EBP1gene from N. fordii
An1185-bp Unigene encoding EBP I gene was selected from transcriptome database, and then cloned from N. fordii using RACE-PCR. The acquired gene was named as NfEBP1, with an ORF contained1188base pairs and encoded395amino acids, which shared99%identity with the Unigene. Bioinformatics analysis revealed that molecular weight of NfEBPl protein was43.5kDa and the isoelectric point was6.12, and its overall structure exhibited hydrophilic; NfEBP1contains none of the signal peptide, chloroplast transit peptide, mitochondrial targeting peptide, and located entirely outside cell membrane. The predicted secondary structure of NfEBP1was composed of21α-helixes,21extend strands,18β-turners and28random coils. The putative model of three-dimensional structure exhibited high homology with the crystal structure of human EBP1. NfEBPl also showed high homology with EBP1protein from other plants, and possessed a functional domain PA2G4.
For expression analysis of NfEBP1, five common reference genes (18s rRNA, Actin, Ubiquitin, EF-1α,β-tubulin) were compared in different tissues (leaf, corm and petiole) of N. fordii. The stability of the candidate reference genes were evaluated by Ct value using GeNorm and NormFinder software. The analysis of GeNorm and NormFinder revealed that β-tubulin was more stable than others and could be served as reference gene for the normalization of gene expression in different tissues of N. fordii using real-time fluorescence PCR. The relative expression of NfEBP1in three tissues was calculated by formula F=2-ΔΔCt. NfEPB1was ubiquitously expressed in all tissues of N. fordii, and the relative expression level reached the highest in leaf, and the lowest in petiole, which was coincident with the trait of NfEBP1in transcriptome. The results validated the feasibility of transcriptome database in the subsequent study of N. fordii.
Manipulating organ size regulator NfEBPl acqured from this study would provided an approach for promoting organ size and yield of N. fordii.
In conclusion, the transcriptome profile of N. fordii with quick and accurate identification using DNA barcoding was demonstrated, and several key genes participated in synthesis of secondary metabolites and plant physiological processes were found and their expression trends were illustrated, which provide a firm foundation for variety breeding and resource protection of N. fordii by means of molecular biological approaches, and a novel and considerable research way for other endangered medicinal plants in Lingnan area.
引文
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