| Tea plant (Camellia sinensis) is an important economic crop and tea is one of the most popular beverages in the world. In order to better understand the gene expression profile of tea tender shoots and obtain the interested genes, two tender shoots cDNA libraries of Longjing 43 and Anji Baicha were constructed, and Longjing 43 library was subjected to high-throughout EST sequencing and bioinformatical analysis. The main results were as follow:1. Total RNA was isolated from tender tea shoots using Trizol single-step method, mRNA was separated and then the double-strand cDNA was amplified by LD-PCR. After size fractionation, the ds-cDNA was ligated to XTriplEx2 and recombinant bacteriophages were packaged to construct cDNA library. The liters of Longjing 43 and Anji Baicha cDNA libraries were 6.8×105 and 4×105 pfu/mL, respectively, matching the requirement of high quality library. The recombinant rates were 98.05% and 96.15%, respectively, and the inserts were about 1.0-1.5kb. There were 3.5×105 and 2.1×105 clones in total, respectively. The results indicated that the two libraries were integrated and efficacious.2. A new vector SfiI-pUC was constructed in order to obtain high EST sequencing quality based on the original vector pTriplEx2. Two new restriction sites of enzyme SfiI were introduced into the new vector.3. Totally 4320 clones were randomly selected from Longjing 43 cDNA library and partially sequenced by MegaBace 1000 capillary sequence machine, 2963 readable sequences that are more than 200 bp were produced from them, which was 68.60% of all the sequences. From the 2963 useful sequences, 1684 ESTs were identified by redundancy analysis with DNATool software. The redundant ratio was about 33.88%, and the average length of the 1684 ESTs was 478 bp.4. The 1684 ESTs were blasted against the non-redundant nucleotide and protein database of NCBI and the databases of tea plant ESTs were constructed. On the basis of NCBI database searches, 36.0% of the total 1684 ESTs showed significant homology with the nucleotide database and 71.3% with the protein database.5. BlastX analysis of significant homology with the non-redundant protein database suggested that about 10% of the total sequences were high expression abundance genes (expression frequency>5), 30% were moderate abundance genes (expression frequency scope 2-5), and 60% were low abundance genes (expression frequency<2). The results indicated that most genes in tea tender shoots were low or moderate expression abundance.6. Genes with function annotation or putative function annotation were assigned to 12 categories according to the classification of Arabidopsis genes. They were genes related to energy (corresponding 26.39% of total genes), protein synthesis and storage (17.86%), cell structure (10.71%), cell disease and defence (9.23%), metabolism (8.33%), cellular signal transduction (5.36%), cellular transporters (4.66%), transcription (3.87%), cell growth anddivision (3.77%), secondary metabolism (3.47%), protein destination and storage (1.69%), unclear classification and unclassified(4.66%). |
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