Construction Of CDNA Subtractive Library Of Kidney Yin Deficiency Syndrome Of Diabetic Mellitus

BACKGROUND:Treat Base on Syndrome is the core of Traditional Chinese Medicine (TCM) theory, and is the most distinctive science essence of TCM. The Syndrome is the basic of treatment, and is the bridge between the TCM theory and the clinic treat system. In the past several decades, the researchers all around the world were trying to reveal the essence of Syndrome. Using the modern technique, they have found some modern pathophysiolgic element. But with the deeping in the research, some puzzle and problem are also emerged. The Chinese medicine "syndrome" is the recapitulate of pathology and physiology of the different phases of different diseases. Since there are some common clinic manifestation and pathomechanism for syndrome, there must be some common material basis. According to the life central dogma, the genetype (DNA or gene) is the most primary and most essential matter of the life, mastering and confining the phenotype program (protein to cell to organism). Thus, the life phenomena, diseases, Chinese medicine syndrome can all have the most essential trace on the DNA or gene. TCM considers that the kidney is the congenital foundation, stores the spirit. The spirit in the kidney comes from the essential substance of reproduction of the parents, and is the primary substance for embryonic development. So, the kidney sprite serves the common identity with the genome DNA. The material basis of kidney deficiency syndrome is the kidney spirit insufficiency, and many domestic professors think that there are a lot of similarities between the methodology of human genomic study and traditional Chinese medicine's whole discrimination, and the functional genomics can respond thechanges of the Chinese medicine "syndrome" exactly, and the functional genomics would be a great breakthrough of the research on kidney deficiency syndrome. Therefore, to research the molecular mechanisms of kidney deficiency syndrome on the aspect of functional genome has a great significance.The development of modern life science can provide essential technique platform for modernization research of TCM theory. It is very necessary, realistic, and feasible to elucidate the essence of TCM with modern theory of medicine. It have been discovered by modern study that 2 -type diabetes mellitus have visible predisposing genes. It is a polygenic inheritance disease that is correlated with environment and heredity factors. In the past few years, the finding research by our research team on diabetes mellitus have indicated that renal deficiency may run through whole process with genesis and growth on diabetes mellitus. Therefore, We think it would be very perfect to select diabetes mellitus to construct of cDNA Subtractive Library of kidney yin deficiency syndrome of diabetic mellitus,and then research the related genes of the kidney yin deficiency syndrome. OBJECTIVE:Construct the cDNA Subtractive Library of kidney yin deficiency syndrome of diabetic mellitusMETHODS:1. Total RNA were isolated from equal numbers of cells using a commercial reagent RNA TRIzol, according to the manufacturer's instructions. Microamount RNA were amplificated by switching mechanism at 5'-end of RNA transcript technique (SMART). Purification of the amplificated cDNAs were used by spin-1000 purification collumn. The cDNAs were digested with Rsa I or Hae III restriction enzyme.2. Establishment of suppression subtractive hybridization(SSH). 4(j,g cDN As of normal group were divided into two, in which a 2ug cDNA was Driver, but another adding up 0.2 ng (pX174 was Tester. After Hae III digested, Tester cDNA was divided two groups to ligated to the specific adaptor 1 and adaptor 2. Then Tester cDNA was hybridized with Driver cDNA twice and underwent nested PCR twice. This method is based on the following difference between target and background cDNAs: each kind of background molecular has only one orientation with respect to the two different flanking adapter sequences used in SSH, while truly differentially expressed targetcDNA fragments are represented by both sequence orientations. The background moleculars were substantially decreased by mirror orientation selection (MOS) used in SSH, in order to raise the quality of cDNA in the subtracted libraries.3. This study successfully constructed the cDNA subtractive library of the kidney yin deficiency syndrome of DM of Chinese Han by SSH. After PCR amplification of cDNA subtractive library, the PCR product was ligated with T plasmid vectors, transformed into E. coli ToplO, screened through the blue-white screening system, and then positive recombinant clones were confirmed by PCR method. After that, we farther screened the positive clones using the dot blot technology. The positive clones from the subtractive library were sequenced, and carryed out homology analysis by http://www.ncbi.nlm.nih.gov/blast/.RESULTS:1. The isaloted RNA is not degraded. The purity of the isolated RNA is quite satisfied (its 2.10>OD26onm/OD28onm>1.90).The good quality of ds cDNA was obtained from microamount total RNA by switching mechanism at 5'-end of RNA transcript technique (SMART) . Less than 500bp cDNA fragments were mostly removed by spin-1000 purification collumn. To compare the results side-by-side, electrophorese 2.5 ul of undigested ds cDNA and 5 ul of Rsa I-digested cDNA on a 1% agarose/EtBr gel in IX TAE buffer. The amplification cDNA derived from RNA appears as a smear from 0.5-10 kb. After Rsa I digestion, the average cDNA size is smaller (0.2-2 kb compared to 0.5-10 kb).2. After SSH, the subtractive products were amplification by PCR. The products of PCR was alike Hae III- digested cpX174, the molecular weight was only slightly big, maybe because of they were ligated adaptors. Mirror orientation selection (MOS) can substantially decrease the number of background molecules, which proves that it is necessary to remove false positive clone by MOS.This study successfully constructed the cDNA subtractive library of the kidney yin deficiency syndrome of Chinese Han DM. After amplification by PCR and screened out the positive clones which contained insert segments by blue-white screening method. There were 192 clones in the outcome of positive subtractive hybridization whereas 164 clones in the outcome of reverse subtractive hybridization.We took 48 clones random, and screened differential expression gene by opposite dot blot hybridization. 8 positive clones may be differential expressed genes in theforward subtractive library and 5 positive clones may be differential expressed genes in the reverse subtractive library.4. In the 8 clones which were screened preliminary in the forward subtractive library, 7 clones succeed by sequencing, 1 clones sequencing failed, because of there were two clones or the others. The other 7 clones were expressed sequence tag (EST). By homology analysis, 1 EST was the NCOA5 gene for nuclear receptor coactivator 5, 1 EST was Nef attachable protein, 3 ESTs were highly homology with partial sequences of different chromatosome, but non related genes. 2 ESTs were not found significant similarity. They might be new gene.5. 5 clones were screened preliminary in the reverse subtractive library, and all were succeeded in sequencing. 5 clones clones were EST. 1 EST was the LOXL4 gene for lysyloxidase -like 4. 4 ESTs were highly homology with partial sequences of different chromatosome, but there were not found significant similarity.CONCLUSION:1. This study had successfully amplified microamount RNA by SMART technique,and established and optimized S S H .2. This study had successfully constructed the cDNA subtractive library of kidney yin deficiency syndrome of DM by SSH, and initially researched the gene in the library. It is a foundation for researching related gene of kidney yin deficiency syndrome.