| 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. |
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