油茶优良无性系组织培养、RAPD分子鉴别和cDNA文库构建的研究
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摘要
油茶是原产我国南方的重要木本油料树种，栽培历史悠久，现已被引种到国外。长期以来，由于品种良莠不齐，经营管理粗放，油茶单位面积产量普遍偏低。目前国内多应用油茶优良无性系造林，有利于大幅度提高产量。随着分子和细胞育种的深入开展，将来会产生更多更好的优良无性系，这些优良无性系的育成往往需要利用组织培养技术。随着无性系品种的增加，也往往需要从分子水平加以鉴别，便于新品种的登录、保护和分子标记辅助选择育种。另外油茶遗传改良和重要基因的分离克隆则有赖于分子生物学研究的深入，而开展这些工作的一个很重要的基础就是构建cDNA文库。因此，本文主要就油茶优良无性系的组织培养、RAPD鉴别和cDNA文库构建三方面的内容进行研究，为今后油茶品种改良、重要基因分离克隆及提高单位面积产量奠定基础。
以油茶优良无性系湘林4号腋芽和子叶为外植体，分别采用附加不同种类激素的MS培养基对其进行组织培养实验。研究结果表明：腋芽诱导再生丛芽的最适培养基是MS+6-BA3.0mg.L~(-1)+NAA1.0mg.L~(-1)；子叶形成胚性愈伤组织的最适合培养基为MS+2,4-D 2.0mg.L~(-1)+KT1.0mg.L~(-1)；经胚状体诱导产生不定芽分化的最适合培养基为MS+6-BA2.5mg.L~(-1)+IAA1.5mg.L~(-1)或6-BA3.0mg.L~(-1)+NAA0.05mg.L~(-1)”；油茶优良无性系的生根培养基以MS+NAA7.0mg.L~(-1)最适；通过对胚状体形成过程中细胞组织学显微观察，实验结果初步揭示了油茶胚状体的起源和大致的发生过程：即油茶胚状体起源于单细胞原胚或者多细胞团，胚状体多从表皮细胞的胚性愈伤组织诱导产生。另外，再生植株过程中各阶段的组培材料经RAPD鉴定分析，在DNA水平上均未发现变异，说明通过组织培养建立的油茶优良无性系再生植株同原无性系无差别，最终获得的组培苗木能够保持原无性系的优良特性，其遗传是稳定的。由腋芽培养产生的再生植株可用于优良无性系的快速扩繁，而以子叶诱导产生胚状体获得油茶优良无性系的再生植株，不仅能满足常规育种的需要，也将为今后开展油茶转基因育种工作打下良好的基础。
选择12个湘林系列的油茶优良无性系和1个普通油茶对照为实验材料，采用改良的CTAB法提取基因组DNA。对影响RAPD反应的主要因素(诸如Mg~(2+)等)进行了优化，获得了一个重复性高、稳定性好的RAPD反应体系。然后以该体系对上述材料进行RAPD检测和鉴别，实验结果表明：从180种10mer的随机寡核苷酸引物中筛选出22个稳定性好、多态性高、谱带清晰的引物；并以这些引物进行PCR扩增，最终通过反复实验共获得141个DNA谱带，其中多态性谱带91个，占扩增总数的64.54％。每个引物扩增的带数在3-10条之间，扩增片断长度介于305bp-2918bp之间。利用相似性系数进行分析，结果表明油茶
中南林学院博士学位论义
优良无性系与对照之间相似性水平低，说明这两者之间存在较大的差异，而各无
性系之间相似性水平也存在一定的差异。在此基础上通过LJ’PGMA力法进行
RAPD聚类分析，绘制了13个实验材料的聚类图。不同的D值其聚类结果不同。
当O值取0．4可将13种材料分为4组，组1：普通油茶对照：组2：湘林引小
组3：湘林刃、3、4、16号；组4：湘林厂、18。叩、53、％、69、82号。对
ILAPD结果做进一步分析，获得了26个特异性标记，可用于汕茶优良无怕；系的
鉴别。其中利川引物引106所产生的8个＊AP D标记可将所有汕茶优良无性系
和普通汕茶一次性区分开来。实验结果表明该方法是可行的。
以湘林4’G优良无性系组培的嫩芽和胚性愈伤组织为材料，利用改良CTAB
法提驭总 RNA后经 OligoNT）纤维素柱分离纯化得到 m洲A，再加入 NOll引
物接人，在 Superscript 11 RT反转录酶和其他酶的作用下合成一链 cDNA和二链
CDNA，经bbl接头修饰、限制性核酸内切酶Notl消化，再经柱层析纯化后同
质粒载体P＊＊VS卜ORT6连接，并转化感受态大肠杆菌＊H10B菌株，从而构建
了第一个仙茶 CDNA文库。经抽样检测，所构建的 CDNA文库包含 136X 10‘个
基囚兜隆，重组于的百分比为99．78％，克隆片段长度大于560hp，说明构建的文
咋质量较高，能够满足ESTS文库构建、从文库中分离筛选重要基因等研究工作
的需要，为今门进一步研究奠定了很好的物质和技术基础。
Oil-tea camellia or Camellia oleifera is one of the most important oil tree crops native to Southeast China and introductions have been made into several non-indigenous countries. The history of growing this edible oil plant is long in China; however, because of poor management and inadequacy in fine cultivars, the yield of oil per unit area is generally very low. Recently, many clones with good biological and genetic characters are widely used to improve the oil production. With the development of genetic studies and breeding technology at the molecular and cellular levels, a good number of improved varieties will be created. The extension of these varieties needs depending closely on the tissue culture technique and at the same time on the advancement of the molecular technology for logining and preserving these new variety entries. The molecular biotechnology is particularly instrumental when the molecule-assisted selection is applied. Besides, the construction of cDNA library in Camellia oleifera will be
a crucial research emphasis in variety breeding and clone selection. Hence in this doctorate project, tissue culture, molecular identification using randomly amplified polymorphism DNA (RAPD) markers, and the construction of cDNA library in Camellia oleifera were studied, hoping to provide a sound basis for variety improvement, cloning of vital genes, and promotion of the oil production.
Auxiliary buds and cotyledons as explants from material 'xianglin-4' (XL-4) were cultured in MS medium with different hormones. Experiment results show that the optimal medium for shoot regeneration from auxiliary buds is MS with 6-BA 3.0 mg. L-1 and NAA 1.0 mg. L-1; the optimal medium for embryogenetic callus formation is MS with 2,4-D 2.0 mg. L-1 and KT 1.0 mg. L-1; the optimal medium for shoot development via embryos is MS with 6-BA 2.5 mg. L-1 and IAA 1.5 mg. L-1 or MS with 6-BA 3.0 mg. L-1 and NAA 0.05 mg. L-1; the optimal medium for root inducement is MS with NAA 7.0 mg. L-1. Somatic embryos were induced from cotyledons and its formation process was observed in the microscope. Results indicate that embryos were derived from single cell embryo or cell aggregate in embryogenetic callus and most of them were induced from embryogenetic callus of epidermis cells. In addition, some materials from different processes of plantlet regeneration were discriminated, but no mutations were discovered. Therefore, it
can be concluded that plantlets from tissue culture do not have any difference with quondam clone and can well maintain the hereditary consistency. Regenerated plantlets from auxiliary buds could be used in rapid propagation, and those from embryos induced from cotyledons could not only satisfy the need for general breeding, they could also serve as a good basis for gene transformation in the future.
After the genomic DNA was isolated with the modified CTAB process from 12 selected clones from Hunan province as target samples and one common cultivar of Camellia oleifera as control, their genetic diversity and molecular identity were ready for analysis by RAPD markers. Firstly, an optimized PCR program for RAPD analysis
with high stability and good repetition was developed by modifying such important factors as the Mg2+, which affected the RAPD-PCR reaction. Then, 22 random oligo primers screened from 180 arbitrary 10 mer primers were used for PCR amplification, which produced an array of polymorphic profiles. Finally, 22 pieces of DNA fingerprint profiles containing 141 bands with 91 distinct polymorphic bands (64.54%) were obtained. Many repetition experiments were conducted to verify the findings.
Each primer produced 3 to 10 bands with the band size ranging from 305 bp to 2,918 bp. Similarity coefficient analysis shows that there is a low similarity between the control and the target samples, and the difference among the target samples is small. On the basis of these findings, the cluster diagram with UPGMA was produced. When the D value is 0.4, the materials can be divided into four groups, with the c
以油茶优良无性系湘林4号腋芽和子叶为外植体，分别采用附加不同种类激素的MS培养基对其进行组织培养实验。研究结果表明：腋芽诱导再生丛芽的最适培养基是MS+6-BA3.0mg.L~(-1)+NAA1.0mg.L~(-1)；子叶形成胚性愈伤组织的最适合培养基为MS+2,4-D 2.0mg.L~(-1)+KT1.0mg.L~(-1)；经胚状体诱导产生不定芽分化的最适合培养基为MS+6-BA2.5mg.L~(-1)+IAA1.5mg.L~(-1)或6-BA3.0mg.L~(-1)+NAA0.05mg.L~(-1)”；油茶优良无性系的生根培养基以MS+NAA7.0mg.L~(-1)最适；通过对胚状体形成过程中细胞组织学显微观察，实验结果初步揭示了油茶胚状体的起源和大致的发生过程：即油茶胚状体起源于单细胞原胚或者多细胞团，胚状体多从表皮细胞的胚性愈伤组织诱导产生。另外，再生植株过程中各阶段的组培材料经RAPD鉴定分析，在DNA水平上均未发现变异，说明通过组织培养建立的油茶优良无性系再生植株同原无性系无差别，最终获得的组培苗木能够保持原无性系的优良特性，其遗传是稳定的。由腋芽培养产生的再生植株可用于优良无性系的快速扩繁，而以子叶诱导产生胚状体获得油茶优良无性系的再生植株，不仅能满足常规育种的需要，也将为今后开展油茶转基因育种工作打下良好的基础。
选择12个湘林系列的油茶优良无性系和1个普通油茶对照为实验材料，采用改良的CTAB法提取基因组DNA。对影响RAPD反应的主要因素(诸如Mg~(2+)等)进行了优化，获得了一个重复性高、稳定性好的RAPD反应体系。然后以该体系对上述材料进行RAPD检测和鉴别，实验结果表明：从180种10mer的随机寡核苷酸引物中筛选出22个稳定性好、多态性高、谱带清晰的引物；并以这些引物进行PCR扩增，最终通过反复实验共获得141个DNA谱带，其中多态性谱带91个，占扩增总数的64.54％。每个引物扩增的带数在3-10条之间，扩增片断长度介于305bp-2918bp之间。利用相似性系数进行分析，结果表明油茶
中南林学院博士学位论义
优良无性系与对照之间相似性水平低，说明这两者之间存在较大的差异，而各无
性系之间相似性水平也存在一定的差异。在此基础上通过LJ’PGMA力法进行
RAPD聚类分析，绘制了13个实验材料的聚类图。不同的D值其聚类结果不同。
当O值取0．4可将13种材料分为4组，组1：普通油茶对照：组2：湘林引小
组3：湘林刃、3、4、16号；组4：湘林厂、18。叩、53、％、69、82号。对
ILAPD结果做进一步分析，获得了26个特异性标记，可用于汕茶优良无怕；系的
鉴别。其中利川引物引106所产生的8个＊AP D标记可将所有汕茶优良无性系
和普通汕茶一次性区分开来。实验结果表明该方法是可行的。
以湘林4’G优良无性系组培的嫩芽和胚性愈伤组织为材料，利用改良CTAB
法提驭总 RNA后经 OligoNT）纤维素柱分离纯化得到 m洲A，再加入 NOll引
物接人，在 Superscript 11 RT反转录酶和其他酶的作用下合成一链 cDNA和二链
CDNA，经bbl接头修饰、限制性核酸内切酶Notl消化，再经柱层析纯化后同
质粒载体P＊＊VS卜ORT6连接，并转化感受态大肠杆菌＊H10B菌株，从而构建
了第一个仙茶 CDNA文库。经抽样检测，所构建的 CDNA文库包含 136X 10‘个
基囚兜隆，重组于的百分比为99．78％，克隆片段长度大于560hp，说明构建的文
咋质量较高，能够满足ESTS文库构建、从文库中分离筛选重要基因等研究工作
的需要，为今门进一步研究奠定了很好的物质和技术基础。
Oil-tea camellia or Camellia oleifera is one of the most important oil tree crops native to Southeast China and introductions have been made into several non-indigenous countries. The history of growing this edible oil plant is long in China; however, because of poor management and inadequacy in fine cultivars, the yield of oil per unit area is generally very low. Recently, many clones with good biological and genetic characters are widely used to improve the oil production. With the development of genetic studies and breeding technology at the molecular and cellular levels, a good number of improved varieties will be created. The extension of these varieties needs depending closely on the tissue culture technique and at the same time on the advancement of the molecular technology for logining and preserving these new variety entries. The molecular biotechnology is particularly instrumental when the molecule-assisted selection is applied. Besides, the construction of cDNA library in Camellia oleifera will be
a crucial research emphasis in variety breeding and clone selection. Hence in this doctorate project, tissue culture, molecular identification using randomly amplified polymorphism DNA (RAPD) markers, and the construction of cDNA library in Camellia oleifera were studied, hoping to provide a sound basis for variety improvement, cloning of vital genes, and promotion of the oil production.
Auxiliary buds and cotyledons as explants from material 'xianglin-4' (XL-4) were cultured in MS medium with different hormones. Experiment results show that the optimal medium for shoot regeneration from auxiliary buds is MS with 6-BA 3.0 mg. L-1 and NAA 1.0 mg. L-1; the optimal medium for embryogenetic callus formation is MS with 2,4-D 2.0 mg. L-1 and KT 1.0 mg. L-1; the optimal medium for shoot development via embryos is MS with 6-BA 2.5 mg. L-1 and IAA 1.5 mg. L-1 or MS with 6-BA 3.0 mg. L-1 and NAA 0.05 mg. L-1; the optimal medium for root inducement is MS with NAA 7.0 mg. L-1. Somatic embryos were induced from cotyledons and its formation process was observed in the microscope. Results indicate that embryos were derived from single cell embryo or cell aggregate in embryogenetic callus and most of them were induced from embryogenetic callus of epidermis cells. In addition, some materials from different processes of plantlet regeneration were discriminated, but no mutations were discovered. Therefore, it
can be concluded that plantlets from tissue culture do not have any difference with quondam clone and can well maintain the hereditary consistency. Regenerated plantlets from auxiliary buds could be used in rapid propagation, and those from embryos induced from cotyledons could not only satisfy the need for general breeding, they could also serve as a good basis for gene transformation in the future.
After the genomic DNA was isolated with the modified CTAB process from 12 selected clones from Hunan province as target samples and one common cultivar of Camellia oleifera as control, their genetic diversity and molecular identity were ready for analysis by RAPD markers. Firstly, an optimized PCR program for RAPD analysis
with high stability and good repetition was developed by modifying such important factors as the Mg2+, which affected the RAPD-PCR reaction. Then, 22 random oligo primers screened from 180 arbitrary 10 mer primers were used for PCR amplification, which produced an array of polymorphic profiles. Finally, 22 pieces of DNA fingerprint profiles containing 141 bands with 91 distinct polymorphic bands (64.54%) were obtained. Many repetition experiments were conducted to verify the findings.
Each primer produced 3 to 10 bands with the band size ranging from 305 bp to 2,918 bp. Similarity coefficient analysis shows that there is a low similarity between the control and the target samples, and the difference among the target samples is small. On the basis of these findings, the cluster diagram with UPGMA was produced. When the D value is 0.4, the materials can be divided into four groups, with the c
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