The Establishment Of A Robust Ordered MRNA Differential Display Method And Its Application

The global gene expression profiling plays a pivotal role in biological research in post-genomic era and therefore the development of cost-effective and universal methods for global gene expression profiling is of great necessity. The current global gene expression profiling tools, principally, may fall into three categories:sequencing-based, hybridization-based and gel-based. The sequencing-based methods offer the sequence information and its abaundance. But the fl-cDNA sequencing suffers from formidable costs, while the tag sequencing from ambiguous tag mapping. Microarray is the leading method of hybridization-based category that has been widely used. Although great success, it is only a "close" system that is limited to species with available genome or transcriptome sequence. In addition, both sequencing and microarray need sophisticated platforms. In comparison, the gel-based category has many advantages:it needs only routine technologies that consists of RT-PCR, DNA sequencing gel electrophoresis and cDNA cloning, which can be readily performed with conventional instruments and reagents; it is applicable to any eukaryotic species and transcripts; the input RNA can be detected by PCR as sensitive as 20 ng; high-throughput can be achieved by a limited number of primer combinations; polymorphisms of many samples can be visualized and compared side by side in the same gel; the transcript-derived fragments (TDFs) of interest can be cloned and sequenced for further analyses.(1) We have established a relatively cheap method to profile global gene expression. It incorporates the advantages of elaborately designed primers, poly(dT:A) replacement, bead-based isolation and a 4-bp-site-dependent coverage solution, combining the virtues of two popular gel-based GGEP methods, mRNA differential display (DD) and complementary DNA amplified fragment length polymorphisms (cDNA-AFLP). Briefly, ds-cDNA was synthesized using a 5'-biotin-MlyI-AcuI-T16-V primer (bmaT16V) and total RNA of the leaves and roots of rice (Oryza sativa), followed by digestion using Mspl and ligation to the MspI adapters. The biotinylated restriction fragments were trapped by streptavidin-coated magnetic beads and digested using Acul. Newly-generated biotinylated fragments were arrested by the magnetic beads while the released non-biotinylated restriction fragments were isolated and ligated to the pseudo adapters, which served as templates for pre-amplification, followed by selective amplification, DNA sequencing gel electrophoresis and cDNA cloning. It anchors the 3'-UTR of mRNA just like DD. However, its procedures are identical to those of bead-based cDNA-AFLP. Therefore, it is robust, holding the virtues of high polymorphism, high PCR reliability, less false positives, low redundancy and low cost. Furthermore, it has higher coverage because of the dependence on the orderly presence of only one 4-bp restriction site on each cDNA. Thus, this method was named "Robust Ordered mRNA Differential Display (RoDD)".(2) To evaluate the coverage of RoDD, a draft mathematical modeling, an in silico simulation were performed, and a simple RoDD validation experiment was carried out using the leaves and roots of one-week-old seedlings of rice (Oryza sativa spp. japonica cv. Nipponbare). The results of the mathematical modelling and the in silico simulation showed the RoDD method has coverage of 92.79% and 82.13% on the published human transcripts, and 92.75% and 84.56% on the published rice transcripts, respectively, higher than any methods of the same categories.1,650 (containing 552 TDF) and 1,576 (containing 478 TDF) bands were obtained in the rice leaves and roots by PCRs using 32 PCs for the PTT adapters, respectively. Summing up,2,128 transcripts were covered. Assuming that 196 PCs for the PTT adapters were completely used, an estimated 10,106 and 9,653 transcripts of the rice leaves and roots could be covered, respectively, summing up to 13,000.1,274(containing 368 TDF) and 1,260(containing 354 TDF) bands were obtained in the rice leaves and roots by PCRs using 30 PCs for the PNN adapters, respectively. Summing up,1,628 transcripts were covered, Assuming 240 PCs for the PNN adapters were completely used, an estimated 7,803 and 7,717 transcripts of the rice leaves and roots could be covered, respectively, summing up to 13,000. In all,17,909 and 17,370 (summing up to 26,000) transcripts could be covered in the leaves and roots of rice if a total number of 432 PCs were completely used. These results indicated high coverage of the RoDD method. Forty TDFs were randomly picked for sequencing and BLAST analysis, of which 39 TDFs were well mapped on O. sativa (japonica cultivar-group) cDNA, and 34 TDFs were confirmed to be true positives, indicating a high reliability. It was interesting to note that the only TDF with no significant similarity found was confirmed to be true positive in many repeated semi-quantitative RT-PCR assays using newly-prepared root samples, indicating a new candidate transcript. In addition to low cost and high coverage, experimental data also showed that the RoDD method has the characteristics of low redundancy, less false positives and high efficiency of polymerase chain reactions (PCRs). A primer efficiency comparason assay and a RoDD miniature assay were performed, which showed the advantages of the high primer efficiency and reliability of RoDD. Therefore, the RoDD method is a cost-effective and universal alternative for the state-of-the-art GGEP methods, sequencing and microarray.(3) The application of the RoDD method. The cytoplasmic male sterile line of Yunnan purple rice A (CMS-P) represents a new cytoplasm source, but it lacks the details of the cytoplasmic effect and mechanism of male sterility and fertility restoration. For it is one of the most effective ways to use the isogenic alloplasmic CMS lines to explore the cytoplasmic characteristics, we constructed 6 isogenic alloplasmic CMS lines (each contains the cytoplasm of CMS-WA, CMS-K17A, CMS-D, CMS-G, CMS-ID, CMS-MA, respectively) of Meixang A, a CMS line of CMS-P type, though hybridization and subsequently recurrent backcrossings to Meixiang B, the isogenic alloplasmic maintainer of Meixiang A. The RoDD method was used to comparatively profiling the global gene expression of seven near-isogenic alloplasmic CMS lines of rice. Moreover, the RoDD patterns of the F1, BC1F1, BC2F1 of Zhenshan 97A/Meixiang B and K17A/Meixiang B were compared so as to monitor the dynamic alterations during the nuclear replacement. However, results showed little polymerphysm. We speculated that it is due that they belong to sporophytic CMS system, which have the same origin and coordinated evolution process. Several characteristic bands of Meixiang A, B or both were found. With further study, these bands may provide some molecular evidence for the "unique" cytoplasm of Yunnan purple rice.