Sequence Analysis And Expression Characterization Of SOC1 Gene From Phalaenopsis Aphrodite

Phalaenopsis aphrodite is a species of the family epiphytic Orchidaceae.It has a very wide distribution in the world,and possesses the high ornamental and economic value because of its graceful color and charming appearance.With the development of science technology,the molecular biology becomes widely available in the flower studies,which leads that many of genes and their expressions in Orchidaceae have also been investigated by the scientists.In this study,we have used the buds of Phalaenopsis to clone the conserved sequence of SOC1(SUPPRESSOR OF OVEREXPRESSION OF CO1)gene(included the ORF)by using the RT-PCR technology.Then the bioinformatics of this sequence were carried out by the online softwares.Moreover,the expression profiles of the gene in different tissues and development stages were validated by the quantitative real-time PCR;we also have constructed expression vector,and the SOC1 gene was introduced into Arabidopsis thaliana and Phalaenopsis protocorm by Agrobacterium-mediated method.The main results are as follows: 1 Clone and analysis of the gene of SOC1(SUPPRESSOR OF OVEREXPRESSION OF CO1)in PhalaenopsisTotal RNA was extracted from the bub of Phalaenopsis in the period of flowering by using plant total RNA extraction kit.The conserved sequence of SOC1(682 bp,219 amino acids)gene have cloned by using the RT-PCR technology.The physicochemical properties suggested that it was belong to the hydrophilic protein,wes made up of 20 kinds of amino acids.And the structures of it include α-helix(51.60%),β-sheet(16.89%),and random coil(31.51%).Through the homology and phylogenetic tree analysis,we found that the genetic evolution of SOC1 gene was consistent with the former speculation.Furthermore,the tertiary protein structure has been predicted that will help to build the foundation for protein research in the future.2 Construction and transformation of the plant expression vector p CAMBIA1300-SOC1 of SOC1 gene in PhalaenopsisThe plasmid p CAMBIA1300 and p GEM19-SOC1 were digested by restriction enzymes Sma I/Xba I,respectively and then connected by T4 DNA ligase to construct the expression vector plasmid p CAMBIA1300-SOC1.After positive colony screened,PCR detection and double enzyme digestion,the plant recombinant expression vector p CAMBIA1300-SOC1 of SOC1 gene in Phalaenopsis has been build successfully.3 Analysis about spatiotemporal expression of SOC1 gene in PhalaenopsisRNAs were extracted from petals of Phalaenopsis in different periods and different parts,and then the relative expression of the target gene was analyzed by real-time PCR and determined according to Rel.Exp = 2-ΔΔCt formula.The results showed that expression quantity of SOC1 gene in Phalaenopsis varied in all the organizations.In roots,the expression level of vegetative growth stage was the highest,which in the reproductive growth stage,the flowering stage and the overy development stage was continuous declination.In leaves,all of the expression level of vegetative growth stage,reproductive growth stage and flowering stage were high,especially the reproductive growth stage was the highest,while there was only a little in the overy development stage.In the scape,the expression level in the reproductive growth stage,flowering stage,and the overy development stage were commendable,and the difference of them was insignificant.In the floral organs,the expression level of gynostemium was the highest,followed by the overy,and were very little in the calyx,petal and labiate.It is speculated that SOC1 gene not only regulates the vegetative growth and reproductive growth in Phalaenopsis development,but also mainly participates in the growth in gynostemium and overy of floral organs..4 Genetic transformation of Phalaenopsis protocorm-like-bodiesThe suitable conditions of the agrobacterium mediated transformation of Phalaenopsis protocorm-like-bodies was explored,and the optimal filtering of Phalaenopsis was ultimately determined,which will lay the foundation for establishing the optimal genetic transformation system.5 Genetic transformation of Arabidopsis thalianaThe positive expression vector was transformed into the model plant Arabidopsis thaliana.The transgenetic plants were selected by used the hygromycin and then were detected with PCR to determine whether the gene was transformed successfully.Compared the F1 generation with the wild type,it was observed that the positive plants were shorter than the wild type,the F2 generation is currently being identified.