Construction Of Kenaf Leaves Full-length CDNA Library And ESTs Analysis

Kenaf which belongs to the Malvaceae, Hibiscus, is an annual herbaceous plant and an important industrial raw material crop. So far, molecular biology study and the function of kenaf genetic studies are lack, some areas are even completely blank.So it is difficult to use other plant to study the functional genes. Therefore, the constructing kenaf leaves full-length cDNA library, picking monoclonal bi-directional sequencing and analysing the ESTs can lay the foundation for related genes clone and molecular biology studies。In this study, the leaves of the Fuhong 992 was used as materials, using SMART technology building a kenaf leaf full-length cDNA library, and random selected part on cloning sequencing,then used bioinformatics processing analysis on sequencing results. The specific research results were as follows:1. In the leaves of kenaf there were tannins,phenols,carbohydrate, RNA extraction was difficulty,and the purity and integrity of RNA influenced the quality of cDNA library,in this study used six methods (Trizol method,proteinase K method,SDS method,CTAB method,the general plant kit and polysaccharide polyphenols kit) to extract kenaf leaves total RNA, by agarose gel electrophoresis and UV spectrophotometer detection, the results showed that SDS method and general plant kit can efficiently extract total RNA from leaf.2. The kenaf leaves full-length cDNA library was constructed successfully, and the library qualification evaluation showed:the titer of cDNA library was 1.34 X 106 (cfu/mL),accorded with the requirement of experiment.Randomly picked 24 monoclonal bacteria for bacteria liquid PCR and found that the percentage of recombination was about 100%., the insert size concentrated between 500bp-2000 bp, with an average length of approximately 700bp.3、Randomly picked 150 monoclonal bi-directional sequencing,after removed low-quality ESTs,147 EST sequences we get, the success rate of 98%. The effective sequence length of these 147 ESTs span from 148bp-1249bp, all effective sequence the total length of 78348.06bp, the average length of 532.98bp.60 unigenes of these 147 ESTs were spliced by using phrad software, including 12 contigs and 48 singlets.4、Using BlastX procedures, according to similar sequence of genetic structure and function description, compared with the non-redundant protein database, after the functional annotation, found that there are 35 unigenes successfully compared with the previous and similar sequences total from 17 species.5、GO classification showed that 17 genes have been classified in the molecular function, they were six carbohydrate transport and metabolism genes, four energy production and conversion genes, translation, two ribosomal structure and biogenesis genes, one amino acid transport and metabolism gene, one general function prediction only gene, one chromatin structure and dynamic gene, one transcription gene, one posttranslational modification, protein turnover and chaperones gene.