Cloning and analysis of expression characteristics of CK2 in Dimocarpus longan Lour.

Abstract: 【Objective】CK2(casein kinase Ⅱ) is a highly conserved serine/threonine protein kinase present in eukaryotic cells, and usually consists of two catalytic subunits and two regulatory subunits.CK2 could phosphorylate over 300 substrate proteins involved in light signaling, translation, transcription and many other diverse pathways. Longan(Dimocarpus longan Lour.) is one of the most important subtropical fruit trees originated in China. Many regulatory pathways of secondary metabolites and many proteins in longan are regulated by phosphorylation. The objective of this study is to clone CK2-αgene in longan embryogenic callus, to investigate the gene structure and function, to reveal its expression characteristics.【Methods】Firstly, CK2 gene selected from the longan transcriptome database(SRA050205) was analyzed by BLAST in NCBI to find conserved region. Then the conserved region was cloned by PCR. The target band was purified by gel extraction after agarose electrophoresis detection. The purified product was cloned into TA vectors and then the recombinant vectors were transferred into E. coli. The positive clones were screened to confirm the sequence. To amplify the 3'-terminal nucleotide sequences, two upstream primers were designed based on CK2 conserved region sequence, and two universal primers, UPMLONG and UPMLSHORT were used as downstream primer. 3'RACE was used to obtain the 3'-terminal nucleotide sequences. In the first round of RACE, cDNA was used as template, but in the second round of RACE, PCR amplified product in the first round was diluted 100 times and then was used as template. The conserved sequence and 3'-terminal nucleotide sequences obtained through 3'RACE were assembled together by DNAman, and open reading frame of this assembled sequence was also analyzed by this software. Then a pair of primers(CK2-F and CK2-R) were designed to confirm ORF and this full length ORF was cloned by PCR. Bioinformatics and subcellular localization analysis were based on the cloning results. ExPASy was used to analyze phyphysicochemical properties of DlCK2-α; SOPMA was used to predict secondary structure; SWISS-MODEL was used to predict tertiary structure; TMHMM was used to analyze transmembrane regions; ExPASy-ProtScale was used to analyze hydrophilicity; SignalP4.1 Server was used to predict signal petide; NCBI-Conserved Domain Search Service(CD Search) was used to analyze conserved domain; NetPhos 3.1 Server was used to predict phosphorylation site; MEGA6 was used to construct phylogenetic trees; Wolf Psort was used to predict subcellular localization; psRNATarget and Plantcare were used to predict miRNA and cis-acting element. Fusion expression vector pCAMBIA1302-35 S-GFP-CK2 was constructed and be used in subcellular localization of DlCK2-α. The recombinant pCAMBIA1302-35 S-GFP-CK2 was introduced into EHA105 agrobacterium and then was sent to BGI for sequencing. The onion epidermis cells were transformed with EHA105 agrobacterium carrying recombinant pCAMBIA1302-35 S-GFPCK2 and were observed by laser scanning confocal microscope(OLYMPUS FV1000) 3 days later. Realtime quantitative PCR(qPCR) was used to analyze the differential expression characteristics of DlCK2-α under different light qualities treatment(including red light, blue light, green light, white light and dark environment) and at different stages of somatic embryogenesis(including loose type embryogenic callus, incomplete embryo compact structure, incomplete embryo compact structure globular embryo and globular embryo).【Results】The results of cloning showed that: the DlCK2-α gene contained an open reading frame of 1 002 bp, encoding 333 amino acids(GenBank accession number: MG181952).Bioinformatics analysis results showed that DlCK2-α was a hydrophilic protein and has a typical conserved domain; Prediction of subcellular localization indicated that it was located in cytoplasm. The Phylogenetic tree analysis results showed that the DlCK2-α was in the same branch as the Citrus clementina CK2-α sequence, indicating DlCK2-α may might have the closest relationship with Citrus clementina from an evolutionary perspective. The miRNA prediction results showed that miR172 a and other9 miRNAs would regulate DlCK2-α gene expression. Cis-acting element prediction results showed that the 5'non-coding region of DlCK2-α contained many cis-acting elements, including light responsive element, defense and stress responsiveness element and so on. Subcellular localization of DlCK2-α indicated that DlCK2-α was located in cytoplasm. The QRT-PCR results showed that DlCK2-α expression level in dark environment was much higher than that under light treatment; Among light treatment,DlCK2-α expression level was the lowest under blue light, and the highest under green light. At different stages of somatic embryogenesis, DlCK2-α had the lowest expression level in incomplete embryo compact structure globular embryo stage and had the highest level in globular embryos stage.【Conclusion】Results suggested that DlCK2-α gene in longan belong to CK2 gene family. There were significant differences between the expression level of DlCK2-α in longan embryogenic callus under light treatment and under dark condition, which indicated that DlCK2-α might play an important role in light signal transduction. QRT-PCR results also showed that DlCK2-α might regulate somatic embryogenesis in longan. The prediction of miRNA and cis-acting element showed that DlCK2-α might be regulated by stress responsiveness elements and stress response related miRNA, which indicated that DlCK2-α might be involved in stress responses processes in longan.