| Dendrobium officinale Kimura et Migo, belonging to the genus of Dendrobium,Orchidaceae,is a traditional herb in China. The active component of D. officinale contains polysaccharides, alkaloids, phenanthrenes and other medicinal ingredients.Therefore, it can be used for maintaining gastric tonicity, enhancing the production of bodily fluids, enhancing immunity and so on. Research on function of key genes is helpful for the effective utilization of D. officinale, and gene expression analysis is an important aspect of gene function research. At present, gene expression is generally quantified by relative quantitative PCR method,and the selection of suitable internal reference gene is a guarantee of accurate detection. In order to investgate the functional gene expression in D. officinale, screening the best reference genes under specific conditions is necessary.In this study, seven reference control genes (Actin, 18S rRNA, GAPDH, EF-1?,TUA, TUB, UBQ) of D. officinale were detected in different tissues (PLB, root, stem,leaf, flower, seed), different developmental stages and abiotic stress conditions (heat stess, cold stess and drought stess). Afterwards, the expression stability of these genes were analysed by three softwares of geNorm,NormFinder and BestKeeper and got the most appropriate reference genes.In the different tissues of D. officinale, expression stability of candidate reference genes analyzed by geNorm and NormFinder was similar,while the result analyzed of Bestkeeper was different. The analysis results of geNorm and NormFinder showed that the 18S rRNA was the worst and GAPDH was the best. The results of Bestkeeper software showed that the TUA was the worst and 18S rRNA was the best In addition,the optimal number of reference genes required for this test was 3, according to the geNorm software matching differential value Vn/n+1. In general, the most stable expression should be EF-1?+ Actin + GAPDH. Expression stability of candidate genes candidates was not completely consistent that analyzed by three softwares in five developmental stages stages of D. officinale.The results of three software showed that the TUB was the worst In this study, analysis by geNorm demonstrated that 5 genes could be required for accurate normalization. However, the number of reference genes needed balancing between accuracy and practical consideration. In general, the most stable expression should be UBQ + GAPDH.In the heat stress (40 ?) and cold stress (4?) of D. officinale, expression stability of candidate reference genes analyzed by geNorm and Bestkeeper was similar,while the result analyzed by NormFinde was different. The analysis results of geNorm and Bestkeeper showed that the TUA was the worst and 18S rRNA was the best. The results of NormFinder software showed that the GAPDH was the worst and Actin was the best.In addition, the optimal number of reference genes required for this test was 5,according to the geNorm software. However, the number of reference genes needed balancing between accuracy and practical consideration. In general, the most stable expression should be TUB+18S rRNA. In the drought stress of D. officinale,expression stability of candidate reference genes analyzed by three programs was similar The results of three software showed that the TUA was the worst. In addition,the optimal number of reference genes required for this test was 2. In general, the most stable expression should be 18S rRNA Actin.Farnesyl phosphate synthase (FPS) plays an important role in plant sesquiterpenoids synthesis. The expression of FPS was analyzed by relative quantitative methods . The expression of FPS was specific in different tissues. PLB and seeds were the highest, however the leaf was the lowest. The expression of FPS is differencent in different growth stages, showing decreases after the first increase trend.Therefore, the expression of FPS was analyzed by absolute quantitative methods that verified the accuracy of the reference genes. At the same time, the expression profile could be revealed in different tissuese and different developmental stages of D.officinale.A total of 5 classes MADS - box genes (FUL, PI, AP3, AG1, AG2, SEP3, API)were cloned based on D. officinale genome. Expressions of these genes were also analyzed in the process of flowering in flower buds, normal flowers and malformed flowers. The results showed that 6 flowering-related genes were expressed in the three kinds of materials,but the expression patterns were not the same. The expressions of flowering related genes were significantly increased during flowering, and the expressions were significantly decreased in the malformed flowers except AP1. |
PDF Full Text Request