Construction Of Suppression Subtractive Hybridization Library Of Kenaf Anthers And Petals And EST Analysis

Kenaf belongs to the Malvaceae Hibiscus (Hibiscus), annual bast fiber crops, with a scientific name Hibiscus cannabinus L., or called as ambary, ambari, which is the important raw materials for paper making. In this study, anthers and petals of kenaf P3B which have been bred by Zhou Ruiyang were imployed as materials. Kenaf anthers were used as the Tester, petals as the Driver for construction of suppression subtractive (SSH) library. As a consequence, we could get the following results:(1) The high quality total RNA were successfully extracted from kenaf anthers and petals.(2) mRNA was purified from high quality and pollution-free total RNA.2ug mRNA was used as the starting materials for successfully constructing suppression subtractive hybridization library. The hybridization efficiency analysis proved that the subtractive effect is good. After the secondary PCR products were collected and purified, the cDNA was connected to the T-vector and converted into Escherichia coli. Randomly picked1440clones respectively from forward subtracted library and reverse subtracted library into96-holes bacterial culture plates to culture for16-24h under37℃. Through the detection of bacterium liquid PCR, the size of these clones mainly ranged from200bp to750bp, and then picked out the clones with a single band to sequence. Preliminarily selected500positive clones from forward subtracted library and25qualified clones from reverse subtracted library to sequence, and exclude the monoclonals and advanced structures, as a result, get442effective sequences for forward subtracted library and25sequences for reverse subtracted library. (3) Remove the T-vector, adaptors and the short sequences below100bp from effective sequences. Through clustering analysis,150clusters were obtained, then the overlapping EST sequences in a cluster were split joint into a long unigene. These ESTs were aligned with NCBI using Blastn and Blastx procedure. As a result, the number of known homologous genes were77, accounting for51.33%; the unknown sequences were49, accounting for32.67%, the predicted novel genes were24, accounting16%. Through Blastx, the number of functionally annotated sequences were78, accounting for52%,15unknown functional sequences accounted for10%, predicted functional protein sequences were57,accounting for38%.(4) According to the results of alignments, these EST sequences were carried out GO functional annotation and COG proteins classification. According to the cell component categories:cell(14unigenes), organelle(6unigenes), protein complex(4unigenes); According to the molecular function categories:the binding function(35unigenes), catalytic activity(59unigenes), nutrient reservoir activity(1unigene),structural molecule activity(2unigenes), transcription regulator activity(3unigenes), transporter activity(12unigenes); According to the biological process categories:cellular process(29unigenes),physiological process(56unigenes), regulation of biological process(3unigenes). According to the COG Library Classification of documents,these functions will be divided into11categories:Intracellular trafficking, secretion, and vesicular transport accounted for3.33%,Secondary metabolites biosynthesis, transport and catabolism accounted for10%,Translation, ribosomal structure and biogenesis accounted for6.67%, modified, and Posttranslational modification, protein turnover, chaperones accounted for6.67%, Energy production and conversion accounted for16.67%; Chromatin structure and dynamics accounted for3.33%; Carbohydrate transport and metabolism accounted for10%;and the Inorganic ion transport and metabolism accounted for3.33%; General function prediction only accounted for6.67%; Lipid transport and metabolism accounted for30%and the sequences associated with transcription response accounted for3.33%.(5) By comparative analysis of the functional annotation, these EST sequences coded the gene products which were mainly associated with metabolism, such as some hydrolytic enzymes, protein kinase, oxidoreductase system and so on. Than, electron carriers, ion-binding protein, water channel protein and Ca2+-binding protein accounted for the majority. These proteins were closely related to respiratory, transmembrane transport and signal transduction. All the results reveal that anthers were doing active metabolism, which is associated with the development period of anthers.(6) Based on the functional annotation for protein, some EST sequences were predicted which are involved in tissue-specific expressive genes in anthers. For instance, the EST sequences contig6, contigl6, contig24and Z7-F5are related to chalcone synthase, and the EST sequences Z1-G9and Z7-F2is associated with MADS-box transcription factor.(7) Eight Unigenes were selected for semi-quantitative analysis, the result showed that6Unigenes expressed higher in the anther than that in petals, while the other2Unigenes showed no significant differences.