柴胡全长cDNA文库和遗传图谱的构建及蚕豆病毒属病毒的鉴定
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摘要
柴胡为伞形科(Umbelliferae)柴胡属(Bupleurum L.)植物,具有解表合里、疏肝解郁、升举阳气之功效,是我国重要的药用植物。本研究构建柴胡(B. chinense DC.)全长cDNA文库,并从cDNA文库中随机挑选了3902个单克隆进行5'端测序,挖掘SSR位点,并在对文库测定序列的分析研究过程中,发现了与蚕豆萎蔫病毒2(Broad bean will virus2, BBWV-2)同源性很高的克隆,推测构建柴胡文库的试料可能被BBWV-2侵染。因此,利用RT-PCR和指示植物接种方法,鉴定了侵染柴胡的病毒。最后,利用ISSR和开发的SSR分子标记构建柴胡分子遗传图谱。为进一步开展柴胡功能基因组学及柴胡分子标记辅助育种等研究奠定基础。试验主要结果如下：
(1)利用SMART技术构建了柴胡根全长cDNA文库。从文库中随机选择了3902个克隆进行5'端单反应测序,获得了3111个高质量cDNA序列,包括377个序列重叠群和1273个单一序列共1650个独立基因。文库插入片段平均长度约1.1 kb,全长比率约51.5%。
(2)利用BlastX分析结果表明949(57.5%)个独立基因和已鉴定基因同源,680(41.2%)个独立基因与GenBank中未知、未命名或推测的蛋白基因同源,21个独立基因没有找到同源基因。
(3)在1650个独立基因中82个含有SSR位点,其中4个独立基因有两个SSR位点,共挖掘了86个SSR位点,为柴胡遗传图谱的构建,遗传多样性的分析及种质鉴定提供有效的分子标记。
(4)在对文库测定序列的分析研究过程中,发现重叠群45与BBWV-2病毒的核酸序列有很高的同源性,推测构建柴胡文库的试料可能被BBWV-2侵染。
(5)首次在柴胡上发现了蚕豆萎蔫病毒2的侵染,利用检测蚕豆病毒属不同种的特异引物对染病柴胡进行了RT-PCR鉴定。并将染病柴胡上的病毒机械接种到苋色藜(Chenopodium Amaranticolor)指示植物上。结果证实了侵染柴胡的病毒为BBWV-2。
(6) ISSR和SSR分子标记的多态性：经父母本多态性筛选,从30条ISSR和44对SSR引物中筛选出28条ISSR和14对SSR多态性引物,扩增出168条多态性条带,其中有36个位点表现偏分离,频率为21.4%。
(7)遗传图谱的构建：通过对F1代进行基因型和连锁分析,初步构建了包含13个连锁群,80个(72个ISSR和8个SSR)位点的首张北柴胡遗传图谱。该图谱覆盖长度2633.9cM,平均图距33.4 cM,13个连锁群包含2-31个标记不等,连锁群遗传距离15.4 cM-1295.7 cM。
本研究旨在为进一步开展柴胡功能基因组学及柴胡性状基因定位、分子标记辅助选择育种等研究奠定了基础育种的研究奠定基础。
Radix Bupleuri (Chaihu), the dried root of Bupleurum L., is a Traditional Chinese Medicine with anti-inflammatory, anti-pyretic and anti-hepatotoxic efficacy.In this research, using the mRNA from the root of Bupleurum chinense DC., a full-length cDNAlibrary was constructed and 3902 clones were arbitrarily picked out for sequencing, and SSR loci were developed. Using RT-PCR with a pair of conserved primers and mechanical inoculation in indicator plant to Identify BBWV-2, when we discove overlap group 45 have the very high homology with the BBWV-2 virus's nucleic acid sequence. Finally, using ISSR and SSR markers constructed the genetic linkage map of Bupleurum chinense DC.
The result of this research canbe summarized as follows:
(1) A full-length cDNA library derived from the root of B. chinense was constructed for the first time by SMART technique to initiate the functional genomic research of this important medicinal plant. From the library, randomly selected 3902 clones were 5'single-pass sequenced, among which 3111 high quality cDNAs were generated and 1650 unigenes were identified with 377 contigs and 1273 singleton cDNAs. The estimated average cDNA insert size was 1.1kb and the fullness ratio was 51.5%.
(2) BlastX analysis of all unigenes resulted in 949 (57.5%) homology to previously identified genes,680 (41.2%) matched to unknown, unnamed or hypothetical protein genes and 21 clones had no hit.
(3) Through SSR searching within 1650 unigenes,86 potentially useful SSR loci were identified in 82 unigenes. The set of SSR loci would potentially be useful molecular markers for germplasm identification, genetic diversity analysis and gene mapping of Bupleurum.
(4) We supposed that the materials were infected with BBWV-2 using to construct cDNA library of B. chinense DC., when discove overlap group 45 have the very high homology with the BBWV-2 virus's nucleic acid sequence.
(5) Broad bean wilt virus 2 (BBWV-2) was first found in B. chinense. RT-PCR with a pair of conserved primers for specific detection of members of Fabavirus genus showed an expected-sized fragment was obtained. And mechanical inoculation in Chenopodium Amaranticolor showed local necrotic lesions on inoculated leaves. The result demonstrated that an isolate of BBWV-2 infecting B. chinense was firstly identified and reported.
(6) Polymorphism analysis of ISSR and SSR:with the theory of pseudo-testcross,96 F1 plants from an intraspecific cross of B. chinense were used as mapping populations.28 ISSR primers and 44 SSR primers were used to detect the polymorphisms between the parental plants, and of them,28 ISSRs and 14 SSRs were selected to analyze the F1 populations.168 polymorphic sites were produced in total.In the markers correspond to these168 polymorphic sites,36 markers'separate ratio didn't meet the Mendelian ratio,they were distorted separation markers.The frequency was 21.4%.
(7) Construction of genetic map:The map consisted of 13 linkage groups which included 80 (72 ISSRs and 8 SSRs) loci, and covered 2 633.9 cM with an average density of 33.4 cM. All 13 linkage groups consisted of 2-31 loci ranging in length from 15.4-1295.7 cM.
The study above metioned will provide the basis for studies on Functional Genomics, gene mapping, map-based cloning and maker-assisted selection of important traits in B. chinense.
(1)利用SMART技术构建了柴胡根全长cDNA文库。从文库中随机选择了3902个克隆进行5'端单反应测序,获得了3111个高质量cDNA序列,包括377个序列重叠群和1273个单一序列共1650个独立基因。文库插入片段平均长度约1.1 kb,全长比率约51.5%。
(2)利用BlastX分析结果表明949(57.5%)个独立基因和已鉴定基因同源,680(41.2%)个独立基因与GenBank中未知、未命名或推测的蛋白基因同源,21个独立基因没有找到同源基因。
(3)在1650个独立基因中82个含有SSR位点,其中4个独立基因有两个SSR位点,共挖掘了86个SSR位点,为柴胡遗传图谱的构建,遗传多样性的分析及种质鉴定提供有效的分子标记。
(4)在对文库测定序列的分析研究过程中,发现重叠群45与BBWV-2病毒的核酸序列有很高的同源性,推测构建柴胡文库的试料可能被BBWV-2侵染。
(5)首次在柴胡上发现了蚕豆萎蔫病毒2的侵染,利用检测蚕豆病毒属不同种的特异引物对染病柴胡进行了RT-PCR鉴定。并将染病柴胡上的病毒机械接种到苋色藜(Chenopodium Amaranticolor)指示植物上。结果证实了侵染柴胡的病毒为BBWV-2。
(6) ISSR和SSR分子标记的多态性：经父母本多态性筛选,从30条ISSR和44对SSR引物中筛选出28条ISSR和14对SSR多态性引物,扩增出168条多态性条带,其中有36个位点表现偏分离,频率为21.4%。
(7)遗传图谱的构建：通过对F1代进行基因型和连锁分析,初步构建了包含13个连锁群,80个(72个ISSR和8个SSR)位点的首张北柴胡遗传图谱。该图谱覆盖长度2633.9cM,平均图距33.4 cM,13个连锁群包含2-31个标记不等,连锁群遗传距离15.4 cM-1295.7 cM。
本研究旨在为进一步开展柴胡功能基因组学及柴胡性状基因定位、分子标记辅助选择育种等研究奠定了基础育种的研究奠定基础。
Radix Bupleuri (Chaihu), the dried root of Bupleurum L., is a Traditional Chinese Medicine with anti-inflammatory, anti-pyretic and anti-hepatotoxic efficacy.In this research, using the mRNA from the root of Bupleurum chinense DC., a full-length cDNAlibrary was constructed and 3902 clones were arbitrarily picked out for sequencing, and SSR loci were developed. Using RT-PCR with a pair of conserved primers and mechanical inoculation in indicator plant to Identify BBWV-2, when we discove overlap group 45 have the very high homology with the BBWV-2 virus's nucleic acid sequence. Finally, using ISSR and SSR markers constructed the genetic linkage map of Bupleurum chinense DC.
The result of this research canbe summarized as follows:
(1) A full-length cDNA library derived from the root of B. chinense was constructed for the first time by SMART technique to initiate the functional genomic research of this important medicinal plant. From the library, randomly selected 3902 clones were 5'single-pass sequenced, among which 3111 high quality cDNAs were generated and 1650 unigenes were identified with 377 contigs and 1273 singleton cDNAs. The estimated average cDNA insert size was 1.1kb and the fullness ratio was 51.5%.
(2) BlastX analysis of all unigenes resulted in 949 (57.5%) homology to previously identified genes,680 (41.2%) matched to unknown, unnamed or hypothetical protein genes and 21 clones had no hit.
(3) Through SSR searching within 1650 unigenes,86 potentially useful SSR loci were identified in 82 unigenes. The set of SSR loci would potentially be useful molecular markers for germplasm identification, genetic diversity analysis and gene mapping of Bupleurum.
(4) We supposed that the materials were infected with BBWV-2 using to construct cDNA library of B. chinense DC., when discove overlap group 45 have the very high homology with the BBWV-2 virus's nucleic acid sequence.
(5) Broad bean wilt virus 2 (BBWV-2) was first found in B. chinense. RT-PCR with a pair of conserved primers for specific detection of members of Fabavirus genus showed an expected-sized fragment was obtained. And mechanical inoculation in Chenopodium Amaranticolor showed local necrotic lesions on inoculated leaves. The result demonstrated that an isolate of BBWV-2 infecting B. chinense was firstly identified and reported.
(6) Polymorphism analysis of ISSR and SSR:with the theory of pseudo-testcross,96 F1 plants from an intraspecific cross of B. chinense were used as mapping populations.28 ISSR primers and 44 SSR primers were used to detect the polymorphisms between the parental plants, and of them,28 ISSRs and 14 SSRs were selected to analyze the F1 populations.168 polymorphic sites were produced in total.In the markers correspond to these168 polymorphic sites,36 markers'separate ratio didn't meet the Mendelian ratio,they were distorted separation markers.The frequency was 21.4%.
(7) Construction of genetic map:The map consisted of 13 linkage groups which included 80 (72 ISSRs and 8 SSRs) loci, and covered 2 633.9 cM with an average density of 33.4 cM. All 13 linkage groups consisted of 2-31 loci ranging in length from 15.4-1295.7 cM.
The study above metioned will provide the basis for studies on Functional Genomics, gene mapping, map-based cloning and maker-assisted selection of important traits in B. chinense.
引文
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