Construction Of CDNA Library And Analysis On Expressed Sequence Tags Of Genus Of Lycoris Longituba

There are various species of Lycoris plants in our country, which have rich variations in colors and shapes of flower. Hereinto, the Lycoris longituba has much more ineraspecific variations than others.The generation of expressed sequence tags (ESTs) has been proven to be a rapid and efficient approach by which to identify the novel genes and analyze the function of genes. In order to identify and isolate correlative genes of colors and shapes of flower, a petals cDNA library of Lycoris longituba was constructed, and it was subjected to high-throughout 5'EST sequencing and bioinformatical analysis. The main results were as follow:1. Total RNA was isolated from petals of Lycoris longituba using improved method of guanidine isothiocyanate, total RNA was separated and further purified to obtain mRNA( messenger RNA),the latter was synthesized to the first strand of cDNA with RT (Reverse Transcriptase).Further changed to double-cDNA strands was realized by using"nick-translate"method. The cDNA was then fractionated by electrophoresis and the fragments longer than 500bp were renovated by gel-purification and ligated to the vector to transform the host by electroporator. Finally the petals primery cDNA library of Lycoris longituba was constructed. The sink size of the primary cDNA is 1.112×106, in which 96.8% phages were recombinant, insert sizes ranging from 0.8～3.0kb were obtained. The results indicated that the library was integrated and efficacious,which is suited for ESTs sequencing.2. Totally 3676 clones were randomly selected from cDNA library of Lycoris longituba and partially sequenced by ABI Prism?3100 capillary sequence machine, 3584 readable sequences that are more than 100bp were produced by analysis with Phred software .And the average length of the 3584 ESTs was 449bp, the average quality is 37.06; the GC content is 45.28%.3. Using the Phrap software, the 3584 effective ESTs of Lycoris longituba petals has been cluster analyzed and assembleed into 2764 UniGene including 431 contigs and 2333 contigs were composed of only one EST which was called singlet.4. B1astX analysis of significant homology with the non-redundant protein database suggested that about 9.039% of the total sequences were high expression abundance genes (expression frequency≥5), 25.871% were moderate abundance genes (expression frequency scope 2～5), and 65.09% were low abundance genes (expression frequency<2). The results indicated that most genes in Lycoris longetuba were low or moderate expression abundance.5. The 3584 ESTs were blasted against the non-redundant nucleotide and protein database of NCBI. On the basis of NCBI database searches, 28.99% of the total 1684 ESTs showed...