Expressional And Functional Analysis Of Several Genes For Floral Organ Development From Cymbidium Ensifolium

Research of the molecular mechanism of floral organ development is very significant in theory.It lays the foundation for breeding of new orchid varieties by using of genetic engineering.Orchids are among the most important plants in the flower market around the world,and have very high ornamental and economic values.The nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them very special plant species for study of floral organ development.The molecular mechanism of floral organ development of orchids is still remains to be clarified.A more extensive and detailed analysis of the orchid MADS-box genes belonging to classes A,B,C,D and E will allow the proposal of a more precise model.The unique floral patterning and many natural mutants make Cymbidium ensifolium ideal for study of floral organ development.A normalized cDNA enriched in full-length sequences was constructed from flowers of Cymbidium ensifolium using DSN(duplex-specific nuclease)normalization method combined with SMART(switching mechanism at 5'end of the RNA transcript)technique.Seven MADS-box genes were isolated from the cDNA library.Another MADS-box gene of Cymbidium ensifolium was amplified by RACE method.The expression,function and relationship between the interactions of eight MADS-box genes were analyzed.The main results are as follows:1.A full-length normalized cDNA library of Cymbidium ensifolium was constructed using DSN-normalization method combined with SMART technique.The titer of primary library was 1.6×106cfu·mL-1.The average size of cDNA inserts was 1.1 kb and with a recombination rate of 97%.These results showed that this library was high quality.2.Seven full-length cDNA clones encoded MADS,CeAP3,CeAP32,CeP11,CeAG1,CeSTK,CeSEP3 and CeAGL6,were isolated from Cymbidium ensifolium normalized cDNA library based on pooling strategy combined with PCR-based screening method.CeAPl and Actin of Cymmbidium ensifolium were amplified by RACE method.A highly conserved MADS domain and K domain of MADS-box family were found in both MADS-box proteins.Each MADS-box gene has typical motifs of its gene family.3.The A-class CeAPl was mainly expressed in pedicel,leaf,root and column.It showed a relatively higher expression level in pedicel than in leaf,root and column,hardly expressed in sepal,petal and lip.CeAP1 was expressed more in the initial period of flower development than in the subsequent stages of flower development.Ectopic expression of CeAP1 in transgenic tobacco plants showed novel phenotypes by flowering early and axillary branching.35S::CeAP1 plants flowered significantly earlier than wild-type plants,even in vitro flowering.Yeast two-hybrid analysis indicated that CeAP1 formed homodimers and heterodimers with CeAP31,CeAP32,CePIl,CeSTK,CeSEP3 and CeAGL6,but was not able to interact with CeAG1.CeAP1 may be a floral meristem identity gene and can cause flowering early.4.The expression patterns of class B gene,CeAP31,CeAP32 and CePI1,were conformed to the model of "orchid code".CeAP31 and CeAP32 were flower specific and absent in leaf,root and pedicel.CeAP31 was expressed in sepal,petal,lip and column.CeAP32 was expressed in petal,lip and column.CeAP32 mRNA was highly expressed in lip was undetectable in sepal.CePI1 was expressed in all floral organs and pedicel and was lowerly expressed in pedicel,was undetectable in leaf and root.Three B-class genes were expressed all stages of flower development and their expression patterns were similar.The expressions of three B-class genes were detected in all the developmental stages of flower buds with the highest expression level in the floral bud more than 2 cm,and to a less extent in the 1-2 cm floral bud,and the lowest expression level in wilting flower.The expressions of CeAP31 and CeAP32 in multitepal mutant were studied by real-time PCR.The results showed that the mRNA for CeAP31 and CeAP32 were significantly up-regulated in double flower and petaloid column of Cymbidium ensifolium.Our data suggest a revised hypothesis where high levels of CeAP31 and CeAP32 expression specify double flower and petaloid column.Transgenic tobaccos with overexpressed CeAP31 or CeAP32 did not show altered flower morphology.CeAP31 was able to form heterodimers with CeAP1,CePI1,CeAG1 and CeAGL6.CeAP31 was not able to interact with CeAP32 and CeSTK,and was not able to form homodimers.CeAP32 was able to form heterodimers with CeAP1.CePI1,CeAGl and CeAGL6.CeAP32 was not able to interact with CeAP31 and CeSTK,and was not able to form homodimers.CePI1 was able to form heterodimers with CeAP1,CeAP31,CeAP32,CeAG1 and CeAGL6,whereas CePI1 was not able to interact with CeSTK,and was not able to form homodimers.5.Both C-class CeAGl and D-class CeSTK involved in the development of column and ovule of Cymbidium ensifolium.CeAG1 played an important role in the formation of double flower and petaloid column of Cymbidium ensifolium.CeSTK was mainly expressed in column and pedicel.The expression of CeSTK was sharply increased up to 16 days after pollination.Analysis of CeSTK expression profile revealed high levels of mRNA in the initial period of floral development,then decreased and stabilized in the floral buds more than 0.5 cm.CeSTK has the lowest expression in wilting flower.The expressions of CeAG1 and CeSTK in multitepal mutant were studied by real-time PCR.In the initial period of floral development,the expressions of CeAG1 and CeSTK in multitepal mutant were lower than the wild plant.The mRNA for CeAG1 and CeSTK were significantly down-regulated in petaloid column of multitepal mutant.Transgenic tobaccos with overexpressed CeAGl did not show altered flower morphology.Transgenic phenotypes of overexpressed CeSTK showed dehiscent petal,abnormal elonged stigma,reduced size of sepals,increased seed sterility,splitedstyle,a small pistil attached to stamen.Protein-protein interaction analyses revealed that CeAGl could form homodimers and heterodimers with CeAP31,CeAP32,CePIl,CeSTK,CeSEP3 and CeAGL6,but was not able to interact with CeAP1.CeSTK formed heterodimers with CeAP1,CeAG1 and CeSEP3,could not form homodimers and heterodimers with CeAP31,CeAP32,CePIl and CeAGL6.6.According to real time PCR analysis,CeSEP3 was expressed in all floral organs and pedicel,absent in leaf and root.CeSEP3 was expressed in all developmental stages of floral development.Overexpression of CeSEP3 caused novelty phenotypes of flowering early,four-petal flower and petaloid stamens.Yeast two-hybrid analysis indicated that CeSEP3 formed homodimers and heterodimers with CeAP1,CeAGl,CeSTK and CeAGL6.7.CeAGL6 mRNA was detected in sepal,petal,lip,column,pedicel and was highly expressed in sepal.Overexpression of CeAGL6 caused novelty phenotypes of four-petal flower,six-petal flower,petaloid stamens and a variety of irregular five-petal flower.Apart from CeSTK,CeAGL6 was able to form heterodimers with CeAP1,CeAP31,CeAP32,CePI1,CeAGl and CeSEP3,but was not able to form homodimers.The results showed that CeAGL6 may be involved in the development of sepal and petal of Cymbidium ensifolium.8.A 2632 bp upstream region of CeAGL6 was cloned by genomic walking PCR.In this region,many potential cis-acting elements were predicted by PlantCARE and PLACE programs.The promoter of CeAGL6 was predicted to contain important regulatory elements including typical TATA box,CAAT box and stress response elements including wound,drought,heat,and hormone response elements including auxin,abscisic acid and gibberellin,endosperm expression element,light regulation element,meristem expression regulation element,circadian control regulation element.Eight potential binding sites for MADS box proteins known as CArG boxes(C-A/T-rich-G)were found in the promoter of CeAGL6.