Large-scaled Gene Cloning Based On EST Analysis And The Study On Gene Transformation Of Salvia Miltiorrhiza Bunge

Salvia miltiorrhiza Bunge, the roots of which have been formulated and used clinically for many years in China for the treatment of various diseases, has come to be drawn much emphasis upon its molecular biology studies because of its fewer numbers of chromosomes(2n=14), abundant secondary metabolism and national popularity. Obtaining abundant genes and establishing high-efficient gene transformation system are basilic groundwork for S. miltiorrhiza' functional genome research. In this paper, we try to make some gene cloning works based on the expressed sequence tags (ESTs) and establish a high-efficient gene transformation system. The main results are as follows:1. An efficient, practical method for isolation of high quality total RNA from materials of S. miltiorrhiza Bunge was described by comparing with Urea, CTAB, Phenol, Modified Hot Borate and Guanidine Thiocyanate methods. The proteins, genomic DNA and secondary metabolites in the extract were totally removed by precipitation with pre-cooled sodium acetate, 10-20% pre-cooled alcohol and repeated phenol/chloroform extractions. The RNA was recovered by ethanol precipitation. The OD260/280 is 2.03(±0.04) and the yield is 100 microgram per gram fresh materials. The method which results in high quality RNA is suitable for RT-PCR, cDNA library construction and northern blot experiments.2. The cDNA plasmid library was constructed by pBluescript IIXR cDNA Library Construction Kit made in Invitrogen Company. It contains about 1.2 x105 clones and covers estimably at least 10 folders of genes expressed at the same time. This library was suitable for gene expression profiles analysis and EST analysis.3. About 12,056 clones of the library have been randomly selected, subjected to single-pass sequencing from the 5' end of the vector and the batting average of sequencing is 89.5%. We generated 9,664 ESTs (valid size ≥100bp) by mass editing (http://www.mrc-lmb. cam.ac.uk/punseq/manual/pregap4_unix_toc.html) for vector and adaptor sequence clipping and elimination of low-quality or short sequences and the average reads size is 411bp.4. All sequences were assembled and clustered into 1,492 contigs and 3,545 singletons by the EST Analysis Pipeline System? Program and total 5,037 Unigenes were obtained, among which 73.9% are singletons.5. Based upon blastn search similarity cutoff E^IO"15 (alignment^30%) and blastx search similarity cutoff of E^ 10"10 (alignment^25%) in NCBI, blastX search similarity cutoff of E^IO"10 (alignment^25%) in Swiss-prot, differently 1,055, 3,428 and 1,615 unigenes were noted. Compared these notations, 31.6% of all contigs and singletons were orphans while 68.4% showed similarity to sequences in all the databases.6. The EST Analysis Pipeline System? Program was used to do the gene ontology and all the notational genes were classified into cellular component ontology, molecular function ontology and biological process ontology. There are 3,130 genes classified into cellular component ontology, 3,428 genes into molecular function ontology and 4,229 genes into biological process ontology.7. The EST Analysis Pipeline System? Program was also used to do the pathway classification. Total 2,689 genes, which represent 607 metabolic-related enzymes, were analyzed and located in metabolic maps. All the genes and enzymes could be classified into 11 different pathways and among which the genes involved in Carbohydrate Metabolism, Amino Acid Metabolism, Cofactors and Vitamins Metabolism and Biodegradation of Xenobiotics showed much abundant expression level. This is probably because the materials we chose for this experiment were at heterotrophic state.8. There are two types of genes highly abundant expressed, one is growth and development related such as stem-specific protein, auxin induced protein, etc. and the other is response to many kinds of stress such as water channel protein, late embryogenesis abundant protein, heat shock protein and so on according to the digital northern analysis. Of all ESTs, there are 9.77% took on these two types of characters and this showed altitudinal accordance with the materials' characters.9. The factors affecting transformation efficiency were studied and optimized. Explants coming from the cutting buds cultivated on MS basal medium within 10 days, were cut into 0.5 cm * 0.5 cm clumps and pre-cultivated for one day as the infected materials. The bacterial stain EHA105 harboring plasmid pCAMBIA2301 cultured on LB liquid medium with OD6oo 0.8-1.0 were collected by centrifugation and resuspended...