Construction And Sequence Analysis Of SMART CDNA Library From Ammopiptanthus Mongolicus Under Drought Stress

As an only evergreen broad-leaf shrub in the northwest desert of China, Ammopiptanthus mongolicus shows very strong resistance to both drought and cold stresses. At present, studies on the sterss-resistant molecular mechanisms and the involved genes in the plant are limited to its cold resistance only, and report about the isolation and characterization of the drought-resistant genes from the plant has not been found as yet. In the present study, a drought-induced full-length cDNA library of A. mongolicus was constructed by using SMART (switching mechanism at 5'-end of RNA transcript) cDNA library construction method. In addition, 5'-sequencing and the seqnence analyses of some clones from the library were carried out. The work laid a foundation for both the expression profile analysis and the cloning and characterization of drought-resistant genes from the plant in the future.Firstly, the classical hot phenol method was improved and the contamination problems of total RNA by polyphenols and polysaccharides during its extraction from A. mongolicus were solved, and as a result, high qulity total RNA meeted the requirements for SMART cDNA library construction were extracted from the plant. What's more, the PolyATtract? mRNA Isolation Systems produced by Promega company was proved more suitable for the mRNA isolation from total RNA of A. mongolicus.Subsequently, using mixed mRNA isolated from different A. mongolicus samples treated under different drought conditions as template for cDNA synthesis, we constructed a full-length cDNA library with SMART cDNA library construction method. The titer of the unamplified library is 1.88×107; the recombination percentage of the library reaches up to 97.87%; the titer of the amplified library is 4.32×1010 pfu/ml; the sizes of the insert cDNA fragments ligated toλTriplEx2 vector ranges from 0.5 to 2.5kb. All the results suggest that the library has a higher qulity.Furthermore, part of the unamplifiedλTriplEx2 library was converted to pTriplEx2 library, and 3500 positive clones were selected from the plasmid library by PCR. Up to date, 960 positive clones have been sequenced from their 5'-end, and 875 useful EST sequences were obtained. After removing the vetor sequences and clustering them, we finally obtained 97 contig and 509 singlet, summing into 606 Unigene. All these Unigene can be classified into 19 clusters. Of which, the genes related to synthesis, modification, transport and degradation of proteins are the most, and the genes with genaral functions and the genes associated with the transport or metabolism of small molleculars and irons rank second. The genes associated with energy metabolism, transcription, cell wall and membrane formation, signal transduction, cell cycle, intracellular transportation, RNA processing and modification, and so on, respectively are also involved in the Unigene. Finally, there exist 2.65 percent Unigene which have not been functionally annotated as yet.