Cloning And Expression Analysis Of DORCA Gene From Dendrobium Officinale

Dendrobium officinale Kimura et Migo is a kind of traditional Chinese orchid herb that has both ornamental value and a broad range of therapeutic effects.It is mainly distributed in Guangxi,Zhejiang,Yunnan,Sichuan,Fujian,Anhui,Henan and other regions.To date,several bioactive constituents,such as polysaccharides,alkaloids and phenanthrene compounds,have been identified and validated to make great contributions to the excellent tonic efficacy of D.officinale,which makes them valuable as traditional medicines used for relieving stomach upsets,promoting body fluid production,nourishing ‘‘yin’’ and antipyresis.In recent years,technology of molecular biology has been widely used in photosynthetic mechanism researches,and the photosynthesis-related genes and their function have been a hot spot of current research.In this study,we isolated a full-length c DNA of RCA from D.officinale by the method of RT-PCR and RACE using degenerate primers designed according to the conserved region of RCA gene.The full-length c DNA of DORCA was also bioinformatics analyzed.The temporal and spatial expression characterizations were confirmed via real-time quantitative PCR,and the plant expression vector of DORCA gene was constructed.The photosynthetic characteristics were studied in leaves of D.officinale using a portable infrared gas analyzer(Li-6400,Li-Cor,Lincoln,NE,USA).The main research results were as followed:1.Cloning of the full c DNA sequence of DORCA and bioinformatics analysisTotal RNA were extracted from the leaves of D.officinale,and a full-length 1724 bp c DNA of RCA was obtained through RT-PCR and RACE amplification.The gene consisted of 1317 bp ORF which encoded a protein of 438 amino acids,was designated as DORCA(accession number: KT205841).The secondary structure of protein was analysised,result showed that the protein was hydrophilic,and it contained 31.96% of α-helix,24.20 % of β-sheet and 43.84 % of random coil.The protein sequence alignment and cluster were analysised,result showed that the DORCA was close to Za RCA.It was predicted that the 3D structure of the protein which was expressed by the DORCA gene.2.Construction of the plant expression vector p CAMBIA1300-DORCAThe cloned DORCA gene was connected with p GEM-T Easy Vector to construct the cloning vector p GEM-DORCA.Double digest the plasmid p GEM-DORCA and plasmid p CAMBIA1300 by restriction enzymes Xba I/ Sal I,then connected by T4 DNA ligase to construct the expression vector.The plant expression vector p CAMBIA1300-DORCA of D.officinale has been constructed successfully by a series of verification.It will usefull for genetic transformation of the next experiments.3.Expression patterns of DORCA Gene from Dendrobium OfficinaleThe temporal and spatial expression characterizations were confirmed via real-time quantitative PCR.The results showed that DORCA was only detected in green tissues.In the 12 h dark and 12 h natural light photoperiods,maximal and minimal DORCA m RNA expression levels were detected at 8:30 and 20:30,respectively,which indicated that DORCA has the obvious characteristics of light-induced expression.4.A Preliminary Study on Photosynthesis in Dendrobium officinaleThe photosynthetic characteristics were studied in leaves of D.officinale using a portable infrared gas analyzer(Li-6400,Li-Cor,Lincoln,NE,USA).The result showed that CO2 uptake appeared only in daytime,and the exchange pattern was typical of C3 photosynthesis.