吉林人参BIBAC基因组文库的构建及其在人参功能基因组研究上的应用
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摘要
人参(Panax ginseng C. A. Mey.)是我国古老而名贵的中药材,因其大补元气,提高机体免疫力,抗衰老,抗肿瘤,宁神益智,调解二型糖尿病和改善性功能等多种功效现已广泛用于制药、保健、美容、食品、饮料等诸多行业,成为我国国民经济中最具特色和优势的产业之一。然而,人参生物学研究,特别是现代基因组学研究和遗传育种还很落后,这大大制约了我们对人参功能基因,特别是控制人参主要药用活性成分-人参皂苷生物合成相关基因的克隆、分析、开发和利用,制约着我国人参产业的现代化。研究证明,大片段DNA重组技术及大片段DNA BIBAC基因文库已成为现代基因组学研究和现代分子遗传育种核心技术之一和重要的研究平台,广泛用于基因组学和分子遗传育种研究各个方面,但大片段DNA重组技术及大片段DNA BIBAC基因文库在我国人参研究中尚属空白。
本研究成功地在我国吉林人参中建立了数以兆级的大片段核DNA分离、纯化技术体系。人参富含代谢产物,特别是多酚、多糖和淀粉,常常限制大片段DNA酶解和克隆。至今,植物数以兆级的大片段核DNA分离、纯化多以幼苗或嫩叶为材料以减少这些代谢产物对大片段DNA酶解和克隆的影响。本研究首次选用人参须根为材料,并适当增加大片段核DNA分离、纯化程序中细胞核的离心与洗涤次数,从吉林人参须根中分离、纯化获得了大片段核DNA。分析表明,用此优化的方法提取的核DNA,不但片段大小数以兆级,而且容易酶解和克隆。因此,在我国吉林人参中建立了简单、有效的大片段核DNA提取和重组的技术体系。
利用在吉林人参中建立的大片段DNA提取和重组技术体系,本研究以4年生吉林人参-大马牙须根为材料成功地构建了世界上第一个人参BIBAC基因文库。该基因文库包含141,312个克隆,有序存放于368个384孔平板中,在-80℃冰箱中长期保存。该基因文库克隆插入片段大小为45-200kb,平均为110kb,每个克隆可能含有几个至几十个基因。由于人参基因组约3,300Mb/1C,该基因文库相当于吉林人参基因组的4.8倍,用单考贝探针从中获得至少一个阳性克隆的机率在99%以上。更重要的是,这个基因文库是构建在细菌和植物双元载体上,可通过农杆菌或基因枪在不同植物中直接进行遗传转化,因此,适合于人参功能基因克隆、分析,多基因遗传转化、分子育种和分子农业。
为进一步确认该基因文库的质量及其在功能基因克隆和分子育种上的应用,本研究将整个基因文库所有的克隆都点样、印记在尼龙膜上,并通过Southern杂交从中筛选出含有9个人参皂苷生物合成相关基因的36个阳性克隆,每个基因探针获得1-7个阳性克隆,平均为4.0个阳性克隆。这充分证明了该基因文库质量较高,适于人参功能基因组不同方面的研究。同时,本研究还发现DS(达玛烯二醇合成酶家族)和OSC(环氧角鲨烯环化酶家族)两个基因家族,共同杂交到5个阳性克隆上,说明人参皂苷生物合成有些基因在人参基因组中是群集的,这样有可能通过BIBAC将群集的不同基因同时转移到不同的物种中,用以探讨人参皂苷合成的分子农业。
研究证明,大片段DNA BIBAC在植物中遗传转化成功的关键是大片段DNA BIBAC在农杆菌中遗传的稳定性。因此,本研究从吉林人参BIBAC基因文库中随机选择了两个插入片段大小分别为100kb和150kb的克隆(Pg100和Pg150)和上述9个人参皂苷生物合成相关基因的阳性BIBAC,利用电击法转化农杆菌菌株C1C58和COR308。结果表明,这些克隆在农杆菌中生长100多代后,经过限制性内切酶NotI酶切、琼脂糖凝胶电泳分析表明,都含有完整的原始BIBAC DNA。这些结果表明,人参大片段BIBAC可通过电击法完整地转化到农杆菌中,而且能稳定遗传。这为人参大片段BIBAC在不同植物中进行遗传转化提供了技术支撑。
本研究在吉林人参中建立的大片段重组技术、构建的吉林人参BIBAC基因文库和相关研究结果,为吉林人参功能基因组研究,特别是对人参皂苷生物合成相关基因克隆、分析、开发和利用提供了技术体系、奠定了基础、建立了必需的平台,同时为吉林人参基因组测序后的组装提供了必要的工具。这些研究结果将大大促进人参功能基因的开发利用、人参产业现代化的发展。
Ginseng (Panax ginseng C. A. Mey.) has been historically well-known as a medicinal herb in China. Because of its roles in health recovery, immune improvement, anti-aging, anti-cancer, calming and intelligent benefits, type2diabetes adjustment and sexuality improvement, ginseng has been widely used in a variety of industries, including medicine, health care, cosmetics, foods and beverages. It has become a unique and rapidly developing industry in China. However, ginseng biological research, especially modern genomics research and genetic breeding, is far behind those of many crop plants. This situation is significantly restricting the identification, characterization and applications of its economically important genes, particularly those controlling the biosynthesis of its major medically active components-ginsenosides, thus restricting ginseng production and industry modernization in China. Studies have documented that high-molecular-weight (HMW) recombinant DNA technology and large-insert BIBAC libraries are important platforms, resources and essential tools for modern genomics research and molecular breeding, and have been widely used in different aspects of the research fields. Nevertheless, no such technology and large-insert BIBAC library have been available in Chinese ginseng.
This study has successfully developed a technical system of megabase-sized nuclear DNA preparation, purification and manipulation in Jilin ginseng. Ginseng is abundant in polyphenolics, polysaccharides and starches that often significantly limit HMW DNA digestion and cloning; therefore, it is challenging to develop such a system. Currently, seedlings or young leaves are used as materials for preparation of megabase-sized DNA from plants in order to minimize these metabolites whereas they are abundant in such tissues of ginseng. In this study, fibrous roots were, for the first time, used as materials for megabase-sized DNA preparation from ginseng. In addition, additional centrifugations and washes were applied to purify nuclei from those cytoplasm contents. Analysis showed that the DNA prepared using this optimized method was not only megabase-sized, but also readily digestible and readily clonable. Therefore, we have established a simple and efficient system for preparation of high-quality megabase-sized nuclear DNA for Jilin ginseng.
Moreover, a large-insert BIBAC library has been successfully constructed for Jilin ginseng from nuclear DNA of four-year old fibrous roots in this study using the technical system of HMW recombinant DNA that we developed in ginseng. This library represents the first large-insert BIBAC library of ginseng worldwide. It consists of141,312clones that were individually arrayed in368384-well microplates and stored at-80℃for long-term uses. The clones of the library have insert sizes arranging from45-200kb, with an average insert size of110kb, possibly each clone containing from a few to dozens of genes. Since ginseng has a genome size of approximately3,300Mb/1C, the library has a genome coverage equivalent to4.8x ginseng haploid genomes, with a probability of greater than99%of obtaining at least one positive clone using a single-copy probe. Importantly, this BIB AC library was constructed in a binary vector (pCLD04541) that can be directly transformed in a variety of plant species by either the Agrobacterium-mediated or bombardment method. Therefore, the BIBAC library can be widely used for cloning and analysis of functional genes in ginseng, multiple gene genetic transformation, molecular breeding and molecular farming.
To further validate the quality and demonstrate applications of the Jilin ginseng BIBAC library in gene cloning and characterization, and molecular breeding, all the clones of the library were double-printed on Southern membranes and hybridized with the overgo probes designed from nine genes likely involved in the process of ginsenoside biosynthesis. A total of36positive clones were obtained, each probe resulting in1-7positive clones, with an average of4.0positive clones per probe. These results strongly indicate that the library is high in quality and well-suited for different aspects of ginseng functional genomics research. It was also discovered that the probes derived from DS and OSC genes were co-hybridized to five of the36positive clones, suggesting that at least these two gene families of genes involved in ginsenoside biosynthesis are physically clustered in the ginseng genome. This result has provided a strong support for genetically engineering the ginsenoside biosynthesis pathway via BIBAC transformation into different plant species for ginsenoside biosynthesis through molecular farming.
Previous studies have documented that the key to the successful transformation of large-insert BIBACs in plants is their transformation and stability in the Agrobacterium strain mediator. Therefore, two large-insert BIBACs with insert sizes of100kb and150kb (named Pg100and Pg150), respectively, were randomly selected. These two clones and the36positive clones of the nine genes likely involved in ginsenoside biosynthesis were individually transformed into Agrobacterium strains C1C58and COR308by electroporation. Analysis of random transformants by their DNA digestion with Not I, followed by pulsed-field gel electrophoresis, showed that the BIBACs transformed were intact and stable in the Agrobacterium strains after they grew in the strains for over100generations. These results suggest that large-insert ginseng DNA BIBACs can be readily transformed by electroporation and stably maintained in Agrobacterium strains, thus having provided a technical support for large-insert ginseng BIBAC transformation in plants.
In conclusion, a technical system for megabase-sized nuclear DNA isolation and manipulation has been successfully established in Jilin ginseng, a high-quality large-insert arrayed BIBAC library has been successfully constructed for Jilin ginseng, and large-insert ginseng BIBACs containing nine genes likely involved in ginsenoside biosynthesis have been isolated and stably transformed into Agrobacterium in this study. These results provide a technical system, a foundation and a essential platform for different aspects of ginseng functional genome research, especially cloning, characterization and uses of the genes involved in ginsenoside synthesis. The BIBAC library has also provided a tool necessary for sequencing and accurate assembly of the ginseng genome. These results will significantly promote research and application of genes economically important in ginseng, thus accelerating the modernization of ginseng production and industry.
本研究成功地在我国吉林人参中建立了数以兆级的大片段核DNA分离、纯化技术体系。人参富含代谢产物,特别是多酚、多糖和淀粉,常常限制大片段DNA酶解和克隆。至今,植物数以兆级的大片段核DNA分离、纯化多以幼苗或嫩叶为材料以减少这些代谢产物对大片段DNA酶解和克隆的影响。本研究首次选用人参须根为材料,并适当增加大片段核DNA分离、纯化程序中细胞核的离心与洗涤次数,从吉林人参须根中分离、纯化获得了大片段核DNA。分析表明,用此优化的方法提取的核DNA,不但片段大小数以兆级,而且容易酶解和克隆。因此,在我国吉林人参中建立了简单、有效的大片段核DNA提取和重组的技术体系。
利用在吉林人参中建立的大片段DNA提取和重组技术体系,本研究以4年生吉林人参-大马牙须根为材料成功地构建了世界上第一个人参BIBAC基因文库。该基因文库包含141,312个克隆,有序存放于368个384孔平板中,在-80℃冰箱中长期保存。该基因文库克隆插入片段大小为45-200kb,平均为110kb,每个克隆可能含有几个至几十个基因。由于人参基因组约3,300Mb/1C,该基因文库相当于吉林人参基因组的4.8倍,用单考贝探针从中获得至少一个阳性克隆的机率在99%以上。更重要的是,这个基因文库是构建在细菌和植物双元载体上,可通过农杆菌或基因枪在不同植物中直接进行遗传转化,因此,适合于人参功能基因克隆、分析,多基因遗传转化、分子育种和分子农业。
为进一步确认该基因文库的质量及其在功能基因克隆和分子育种上的应用,本研究将整个基因文库所有的克隆都点样、印记在尼龙膜上,并通过Southern杂交从中筛选出含有9个人参皂苷生物合成相关基因的36个阳性克隆,每个基因探针获得1-7个阳性克隆,平均为4.0个阳性克隆。这充分证明了该基因文库质量较高,适于人参功能基因组不同方面的研究。同时,本研究还发现DS(达玛烯二醇合成酶家族)和OSC(环氧角鲨烯环化酶家族)两个基因家族,共同杂交到5个阳性克隆上,说明人参皂苷生物合成有些基因在人参基因组中是群集的,这样有可能通过BIBAC将群集的不同基因同时转移到不同的物种中,用以探讨人参皂苷合成的分子农业。
研究证明,大片段DNA BIBAC在植物中遗传转化成功的关键是大片段DNA BIBAC在农杆菌中遗传的稳定性。因此,本研究从吉林人参BIBAC基因文库中随机选择了两个插入片段大小分别为100kb和150kb的克隆(Pg100和Pg150)和上述9个人参皂苷生物合成相关基因的阳性BIBAC,利用电击法转化农杆菌菌株C1C58和COR308。结果表明,这些克隆在农杆菌中生长100多代后,经过限制性内切酶NotI酶切、琼脂糖凝胶电泳分析表明,都含有完整的原始BIBAC DNA。这些结果表明,人参大片段BIBAC可通过电击法完整地转化到农杆菌中,而且能稳定遗传。这为人参大片段BIBAC在不同植物中进行遗传转化提供了技术支撑。
本研究在吉林人参中建立的大片段重组技术、构建的吉林人参BIBAC基因文库和相关研究结果,为吉林人参功能基因组研究,特别是对人参皂苷生物合成相关基因克隆、分析、开发和利用提供了技术体系、奠定了基础、建立了必需的平台,同时为吉林人参基因组测序后的组装提供了必要的工具。这些研究结果将大大促进人参功能基因的开发利用、人参产业现代化的发展。
Ginseng (Panax ginseng C. A. Mey.) has been historically well-known as a medicinal herb in China. Because of its roles in health recovery, immune improvement, anti-aging, anti-cancer, calming and intelligent benefits, type2diabetes adjustment and sexuality improvement, ginseng has been widely used in a variety of industries, including medicine, health care, cosmetics, foods and beverages. It has become a unique and rapidly developing industry in China. However, ginseng biological research, especially modern genomics research and genetic breeding, is far behind those of many crop plants. This situation is significantly restricting the identification, characterization and applications of its economically important genes, particularly those controlling the biosynthesis of its major medically active components-ginsenosides, thus restricting ginseng production and industry modernization in China. Studies have documented that high-molecular-weight (HMW) recombinant DNA technology and large-insert BIBAC libraries are important platforms, resources and essential tools for modern genomics research and molecular breeding, and have been widely used in different aspects of the research fields. Nevertheless, no such technology and large-insert BIBAC library have been available in Chinese ginseng.
This study has successfully developed a technical system of megabase-sized nuclear DNA preparation, purification and manipulation in Jilin ginseng. Ginseng is abundant in polyphenolics, polysaccharides and starches that often significantly limit HMW DNA digestion and cloning; therefore, it is challenging to develop such a system. Currently, seedlings or young leaves are used as materials for preparation of megabase-sized DNA from plants in order to minimize these metabolites whereas they are abundant in such tissues of ginseng. In this study, fibrous roots were, for the first time, used as materials for megabase-sized DNA preparation from ginseng. In addition, additional centrifugations and washes were applied to purify nuclei from those cytoplasm contents. Analysis showed that the DNA prepared using this optimized method was not only megabase-sized, but also readily digestible and readily clonable. Therefore, we have established a simple and efficient system for preparation of high-quality megabase-sized nuclear DNA for Jilin ginseng.
Moreover, a large-insert BIBAC library has been successfully constructed for Jilin ginseng from nuclear DNA of four-year old fibrous roots in this study using the technical system of HMW recombinant DNA that we developed in ginseng. This library represents the first large-insert BIBAC library of ginseng worldwide. It consists of141,312clones that were individually arrayed in368384-well microplates and stored at-80℃for long-term uses. The clones of the library have insert sizes arranging from45-200kb, with an average insert size of110kb, possibly each clone containing from a few to dozens of genes. Since ginseng has a genome size of approximately3,300Mb/1C, the library has a genome coverage equivalent to4.8x ginseng haploid genomes, with a probability of greater than99%of obtaining at least one positive clone using a single-copy probe. Importantly, this BIB AC library was constructed in a binary vector (pCLD04541) that can be directly transformed in a variety of plant species by either the Agrobacterium-mediated or bombardment method. Therefore, the BIBAC library can be widely used for cloning and analysis of functional genes in ginseng, multiple gene genetic transformation, molecular breeding and molecular farming.
To further validate the quality and demonstrate applications of the Jilin ginseng BIBAC library in gene cloning and characterization, and molecular breeding, all the clones of the library were double-printed on Southern membranes and hybridized with the overgo probes designed from nine genes likely involved in the process of ginsenoside biosynthesis. A total of36positive clones were obtained, each probe resulting in1-7positive clones, with an average of4.0positive clones per probe. These results strongly indicate that the library is high in quality and well-suited for different aspects of ginseng functional genomics research. It was also discovered that the probes derived from DS and OSC genes were co-hybridized to five of the36positive clones, suggesting that at least these two gene families of genes involved in ginsenoside biosynthesis are physically clustered in the ginseng genome. This result has provided a strong support for genetically engineering the ginsenoside biosynthesis pathway via BIBAC transformation into different plant species for ginsenoside biosynthesis through molecular farming.
Previous studies have documented that the key to the successful transformation of large-insert BIBACs in plants is their transformation and stability in the Agrobacterium strain mediator. Therefore, two large-insert BIBACs with insert sizes of100kb and150kb (named Pg100and Pg150), respectively, were randomly selected. These two clones and the36positive clones of the nine genes likely involved in ginsenoside biosynthesis were individually transformed into Agrobacterium strains C1C58and COR308by electroporation. Analysis of random transformants by their DNA digestion with Not I, followed by pulsed-field gel electrophoresis, showed that the BIBACs transformed were intact and stable in the Agrobacterium strains after they grew in the strains for over100generations. These results suggest that large-insert ginseng DNA BIBACs can be readily transformed by electroporation and stably maintained in Agrobacterium strains, thus having provided a technical support for large-insert ginseng BIBAC transformation in plants.
In conclusion, a technical system for megabase-sized nuclear DNA isolation and manipulation has been successfully established in Jilin ginseng, a high-quality large-insert arrayed BIBAC library has been successfully constructed for Jilin ginseng, and large-insert ginseng BIBACs containing nine genes likely involved in ginsenoside biosynthesis have been isolated and stably transformed into Agrobacterium in this study. These results provide a technical system, a foundation and a essential platform for different aspects of ginseng functional genome research, especially cloning, characterization and uses of the genes involved in ginsenoside synthesis. The BIBAC library has also provided a tool necessary for sequencing and accurate assembly of the ginseng genome. These results will significantly promote research and application of genes economically important in ginseng, thus accelerating the modernization of ginseng production and industry.
引文
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