Construction Of SSH CDNA Library Of Cabbage Induced By High Temperatures And It's ESTs Analysis

Cabbage (Brassica oleracea var. capitata) is originated in the Mediterranean ,introduced into China during the 16th century. Because of its strong resistance and board adaptability ,Cabbage now is cultivated throughout the world ,and has been widely planted all over the country as an important vegetable. Cabbage is a kind of cold~resistant vegetable crops. Its optimum temperature is 15~20℃balls , adapting to the temperature of 7~25℃.When it is more than 25℃,cabbage assimilation decreases,breath strengthen,leading to lower quality and yield. Therefore ,high temperature has become a limiting factor for cabbage production,the high~temperature damage urgent to be address. Nevertheless , only in~depth study of the heat~resistant mechanism of cabbage can fundamentally solve the problem. This study use the suppression subtractive hybridization (SSH) technology to build cDNA library , and use EST technology combined with bioinformatics technology to study the heat~resistant mechanism of cabbage from the genetic level according to expression analysis of related heat genes.In this experiment ,heat-resistant cabbage inbred lines ,"Xia Huang 6-105" for materials,built to high temperatures induced by 0.25 h ,1 h ,3 h ,6 h ,12 h after the cabbage leaves mixed SSH library.1010 single colonies were obtained ,of which white spots 839. Were randomly picked 300 positive clones were verified by reverse Northern hybridization of the 107 clones of extracting plasmid and sequenced.Received a total of 105 high-quality ESTs , CAP3 software clustering obtained a total of 45 Unigenes , including 7 Contigs and 38 Singlets. The use of BLASTx ,BLASTn in the Genbank database sequences homologous to these 45 Unigenes ,10 Unigenes is the heat shock protein , species are divided into ATHSP101 ,HSP70 , HSP81.4 ,HSP70b , HSP22 , HSP21, HSP18,HSP23.6.Others ,there are 5 Unigenes with primary metabolism and energy metabolism-related genes; 2 Unigenes with transit-related;2 Unigenes associated with signal transduction;9 Unigenes with protein modification processing phase; 2 Unigenes with transcription,7 Unigenes'function unknown;the four Unigenes'homology is very low; 4 Unigenes in the database did not find any homology.Obtained Unigenes further analysis found:Calcium signal in the process of cabbage heat played a certain role and with the outside world the existence of a non-biological stress the same mechanism, but the exact mechanism is not clear;Ubiquitin-proteasome pathway play an important role in the process of denatured protein degradation(Heat Shock Response of plants produced); Speculated that NAC transcription factors in plants have to respond to the role of high-temperature environmental stimuli, Or to high-temperature stimuli-responsive Cabbage with specificity,there are to be studied.