Study On Selection Of Reference Genes And A-class Floral Development Gene Expression Pattern From Cymbidium Faberi

Cymbidium faberi, has beautiful and fragrant flower, possessed high ornamental value to some extent. Using genetic engineering technology to cultivate new varieties of flowers, has been the world’s main research focus of ornamental horticultural plant breeding. So far, many ornamental plants floral developmental genes have been cloned and isolated, but few studies have been reported on floral developmental genes of Cymbidium faberi.In this study, by homologous cloning and RT-PCR, TUA, TUB, ACT, GAPDH,18S, rRNA, UBQ,EF1 alpha as the candidate reference genes were cloned from Cymbidium faberi. Reference genes were selected with the method of absolute quantification. Combined with RACE, the two flower development genes CfAPll, CfMADSl full lenth of cDNA were successfully separated from pedicels of Cymbidium faberi. Then we analyzed spatiotemporal expression of the two genes. Moreover, we constructed the sense and anti-sense plant expression vector of CfAPll, ransformed sense plant expression vector of CfAPll into tobacco by Agrobacterium-mediated of leaf disc method. PCR testing and southern blot showed that the sense fusion CfAPll gene had been integrated into tobacco genome. The main results are as follows:1.TUA, TUB, ACT, GAPDH,18S, rRNA, UBQ, EF1 alpha were selected as candidate internal reference gene and cloned. Expression levels were measured by real-time fluorescence quantitative RT-PCR and analyzed by GeNorm, Normfinder, then some of them were verified by the relative expression of functional gene. Analysis of internal reference gene selection showed that:for all organs in all stages, ACT, UBQ3 and GAPDH can be together selected as internal reference genes to detect the relative expression levels of other functional genes, and expression results can be relatively accurate; for the organs of vegetative stage, UBQ2 and UBQ3 can be together choosed to normalized gene expression analisis; for the bud stage, ACT and UBQ3 are together used to analyze gene expression; for full-blossom stage, ACT, UBQ3 and UBQ2 can together be introduced into relative gene expression analisis; for the vegetative organs, UBQ2 and ACT can together be used as reference genes; for the reproductive organs, ACT, UBQ3 and UBQ2 can be as reference for data processing; for the roots, two genes UBQ2 and ACT as internal reference; for leaves, two genes ACT and UBQ3 as the reference for data analysis; for pedicels, two genes ACT and UBQ2 as internal standard; for flowers, ACT and UBQ2 together as reference genes in different tissues of floral organs.2. According to conservative sequence segment of the known gene in NCBI, degenerate primers were desighned. Using RT-PCR and RACE, two floral development genes, named as CfAPll, CfMADS1, were cloned from Cymbidium faberi. GenBank accession number was respectively JQ031272 and KC 148540. Sequence analysis showed that the two genes, containing a MADS domain and K domain, belongs to MADS-box gene family. CfAP11 contains no C-terminal conservative motif, CfMADSl contains a C terminal conserved motif LPPWML. Bioinformatics analysis showed that, they are unstable hydrophilic proteins, and three-dimensional structures are similar.Expression levels of floral development gene CfAP11 and CfMADSl were measured by relative real-time fluorescence quantitative RT-PCR. The results showed that different gene has different spatiotemporal expression patterns and some tissue specificity.The expression level of CfAPll is higher in the leaves of vegetative stage, which is related to flower induction. It shows moderate expression in the leaves of bud stage and weak expression in the roots. Strikingly, it is highly accumulated in ovaries and pedicels of full-blossom, and expression levels are significantly increased. It is likely that CfAPll is closely related to fruit formation, it is accumulated in the ovaries through pedicels, and development of fruit is initiated.CfMADSl is expressed in the leaves and roots of vegetative stage, and the leaves of bud stage, which is related to flower induction, and initiation. It shows higher expression in the pedicels and buds of bud stage, which is likely related development and formation of floral organs, it is also likely expressed by pedicels. It shows high expression in the leaves and pedicels of full-blossom, and there are higher expression levels in full-blossom than in vegetative stage and bud stage. There is expression to some extent in the ovaries of full-blossom, which shows that CfMADS1 is involved in the formation of fruit to some mechanism.3. We constructed sense and anti-sense plant expression vectors of CfAPll. Then we transferred pRI101-CfAPll into Agrobacterium tumefacien EHA105. pRI101-CfAPll was transformed into tobacco by Agrobacterium-mediated of leaf disc method. PCR testing and southern blot showed that the sense fusion CfAPll gene had been integrated into tobacco genome.