| The development of Dendrobium spp.industry had been greatly limited because of the contradiction between its natural flowering time and the market demand.Therefore,it is necessary for Dendrobium spp.to clarify the molecular mechanism of flowering regulation.In this experiment,the four SOC1 genes DenSOC1-1,DenSOC1-2,DenSOC1-3,and DenSOC1-4 of Dendrobium 'Sonia Hiasakul' were cloned and their expression patterns were studied.(1)A 1596 bp cDNA of Denactin was cloned by RT-PCR which including 1134 bp open reading frame encoding 377 amino acids.Bioinformatics analysis showed that it was highly homologous to Dendrobium.The expression atability analysis showed that Denactin was stably expressed in seeding leaves and stem tips,roots,leaves of medium plants,stems,roots,leaves,peduncle,Flower with a diameter of 1 mm,3mm,5mm,9mm of mature plants,this result indicated that Denactin could be used as reference gene for DenSOCIs.The discovery of an intron sequence in Denactin can effectively eliminate the possibility of template DNA contamination during qRT-PCR and improve the quality of the reference gene.(2)Four genes including DenSOC1-1,DenSOC1-2,DenSOC1-3 and DenSOC1-4 were cloned by RT-PCR technique.The length of DenSOC1-1 is 1084 bp involveing 633 bp open reading frame and encoding 210 amino acids.DenSOC1-2 gene is 1188 bp in length with an open reading frame of 645 bp which encodes 214 amino acids.DenSOC1-3 gene is 1143 bp in length including the 609 bp open reading frame which encodes 202 amino acids.DenSOC1-4 is 930 bp in length including 672 bp open reading frame,encoding 223 amino acids.The four DenSOC1s all belonging to the SOC1 gene because of having the common domain of the SOC1s homologous genes include a MADS region,an I region,a K region,a C region,and an SOC1-motif domain.Bioinformatics analysis showed that all four genes have high homology with Orchidaceae plants.(3)The expression pattern of DenSOC1s was studied by qRT-PCR.First of all,the result of spatio-temporal expression showed that DenSOC1-1 had high expression in medium seedling stem tips,mature stems,pedicels,stalks and open full flowers,while DenSOC1-2 mainly expressed in stem tips of medium seedling,mature stems,pedicels and stalks,DenSOC1-3 highly expressed in mature stems and stalks,DenSOC1-4 highly expressed medium seedling stem tips,mature stems,pedicels,stalks.The expression of DenSOC1-1 decreased gradually in floral buds with the growth of plant and highly expressed in early development of flower while DenSOC1-2,DenSOC1-3 and DenSOC1-4 remained basically stable.DenSOC1s was significantly expressed in petals and labellum of the full open flower in the three development phases.Then,the expression pattern of daily cycle showed that DenSOC1-1 mainly expressed during the day while DenSOC1-2,DenSOC1-3 and DenSOC1-4 were at night.In addition,the expression pattern of hormone treatment showed that the expression of DenSOC1s was increased by hormone regulation after 240 h treatment.What's more,the expression pattern of temperature treatment showed that the expression pattern of DenSOC1-2 and DenSOC1-3 were analogous in two different temperatures.The expression of DenSOC1-1 and DenSOCI-4 did not varies to low temperature,but they were up-regulated in response to high temperature treatment.Last but not least,photoperiodic processing showed that the expression of DenSOC1-2 and DenSOCl-3 were affected by the photoperiod rhythm while DenSOC1-1 and DenSOC1-4 were not.The expression of DenSOCls under short-day and full-dark conditions were higher than that of full-light and long-day conditions.(4)The plant overexpression vectors DenSOC1-1-pCAMBIA2300,DenSOC1-2-pCAMBIA2300,and DenSOC1-4-pCAMBIA2300 were successfully constructed and transferred into Agrobacterium GV3101,which laid the foundation for the next step of genetic transformation to verify gene function. |
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