Study On The Selection Of Reliable Refefence Genes And Expression Anaiysis Of Transcription Factors DREB In Cymbidium Kanran Mak.

Cymbidium kanran is an important member of Chinese orchid as leggy plant type, delicate fragrance, abundant colour and long florescence. In addition, the labellum, which is the focus in evaluation of orchid, exhibit diverse morphological variation. Thus, this plant possesses high economic value as ornamental plants. Due to long juvenile period, breeding in Cymbidium kanran falls far behind other ornamental plants, and molecular breeding has become an important direction as shortening breeding period and enhancing selecting efficiency. Thus, here, we set out to test the stability of nine commonly employed plant genes in Cymbidium kanran, and then expression analysis of DREB in Cymbidium kanran was performed, the results are as follows:1. Selection reference gene in Cymbidium kanran MakCymbidium kanran Mak as research materials, analysis nine biological common housekeeping gene expression by RT-PCR technique, then use biology software (geNorm, NormFinder) of nine candidate housekeeping genes in different tissues, different hormones and different expression result of abiotic stress conditions are analyzed. Abiotic stresses including drought, salt, low temperature, high temperature, heavy metal stress, different organizations including the sepals, petals, column, labellum, root, stem and leaf, hormone treatment including ABA. GA, Paclobutrazol, NAA, TDZ,6-BA,1BA etc. We will get the Ct values were analyzed by geNorm and NormFinder respectively, so as to select the appropriate internal genes. Compare geNorm and NormFinder two software analysis, found that two software analysis results are basically the same, just in different treatment root, there have been some inconsistency. Further analysis indicated that, this is due to pasB and TUB gene regulation. Comprehensive analysis of results of two software that, to a plurality of processing and tissue and organ involved in the expression of reference genes, Actin and TUB is the most stable; in different tissues, RPS3and rpoB the most appropriate reference genes; Actin and TUB in different treatment of root was the most stable; while the leaves in different treatments. Actin and28S as the most reliable reference genes. 2. Gene expression analysis of transcription factor DREBBy the RT-PCR analysis application under high temperature, high salt and waterlogging of treatment of24, has been clear about the DREB gene expression in different strains, to determine the Cymbidium kanran Mak of stress induced stress ability. Analysis shows that DREB gene expression level can strongly by high temperature, high salt, and waterlogged stress effect. Under the condition of heat treatment, DREB gene expression began to rise rapidly, peaked at1h after slow down, and again in24h a sharp increase in the peak, and then express the amount will gradually decline. In high salt treatment, DREB gene expression in30min will gradually decline, and then a sharp increase in peaked at2hours and after that, the gene expression of the quantity will reduce gradually, after24h will achieve high again and gradually reduce. In waterlogged stress processing, DREB gene expression quantity will increase gradually, and reached its peak at2h, then DREB gene expression quantity will be reduced sharply, at the time of24h, the expression of the gene will gradually increase, and then decrease again. So, we found that the expression of DREB gene in Cymbidium kanran Mak. in vivo is very complex, within48h of fluctuations is bigger, change more quickly.The results show that DREB gene expression pattern is complicated, cymbidium provides important information about the molecular mechanism of adversity stress.