Expression Analysis and Cloning of Cold-tolerant QTL qCTS3.3 Candidate Gene LOC_Os03g53670-DX in Dongxiang Wild Rice at Seedling Stage under Low Temperature Stress

PurposeIn order to study the functional mechanism of cold-tolerant QTL qCTS3.3 candidate gene LOC_Os03g53670-DX in Dongxiang wild rice at seedling stage under cold stress.

MethodThe differentially expressed gene LOC_Os03g53670-DX located in qCTS3.3 QTL region was used as candidate gene for qCTS3.3 based on the analysis of transcription data. Fluorescence quantitative PCR was used to verify the expression of this gene in Dongxiang wild rice with different time lengths of cold stress based on the analysis of transcription data. The full length cDNA of LOC_Os03g53670-DX gene was cloned from Dongxiang wild rice by RT-PCR with the designed primers according to the corresponding locus sequence information of Nipponbare in the public database. The over-expression vector was then constructed successfully.

ResultSequence analysis showed that the full length cDNA of LOC_Os03g53670-DX was 1 986 bp and encoded a protein about 173 ku which was composed of 661 amino acids. Its corresponding theoretical isoelectric point (pI) was 4.92. It was predicted that the C-terminal amino acid sequence of LOC_Os03g53670-DX contained one highly conserved structure domain YTH using the online analysis software. It was suggested that the gene was a member of RNA binding protein gene family. Phylogenetic analysis results showed that the LOC_Os03g53670-DX had the closest relationship with Sorghum bicolor (XM_021447002.1) and Ananas comosus (XM_020225621.1). Then the over-expression vector of the gene LOC_Os03g53670-DX was transferred by Agrobacterium-mediated method, and 26 LOC_Os03g53670-DX transgenic rice plants were obtained after hygromycin selection and GUS histochemical staining detection.

ConclusionThis study laid an important material base for further study on the functional mechanism of gene LOC_Os03g53670-DX under cold stress.