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Temperate japonica rice plays a very important role in food security. In this study, a set of 191 temperate japonica accessions from 30 countries were planted in two sites in China, and 12 agronomic traits were measured. The results showed a wide range of variation for the traits measured. Most of the accessions are short; three accessions with large panicles and seven accessions with large grains were identified. Sixty-two quantitative trait loci (QTLs) were identified for 12 traits that were measured. Among them, twenty-one QTLs were identified in both experiments, and 41 QTLs were identified in only one site. Some known genes are located in the QTL regions identified in our study. SNP markers for grain size were identified and could be efficiently used for breeding selection. This study provided useful information for future gene validation and marker assisted selection for some important agronomic traits of temperate japonica rice.

Objective To construct a human ether-a-go-go-related gene (HERG) nonsense mutant L539fs/47-*558W into the autonomously fluorescent, eukaryotic expression vector pEGFP-C2, and to verify expression of the reconstruct in human embryonic kidney-293 (HEK293) cells. Methods The mutational fragment was subcloned into pEGFP-C2-HERG by double digestion of Sbf I, Eco91 I and rejoining of T4 ligase. After verification, the recombinant pEGFP-C2-L539fs/47-*558W and pEGFP-C2-HERG were respectively transfected into HEK293 cells for 48 h by the Lipofect method to observe the expression location of the fusion protein by laser confocal imaging scanning in vivo. pcDNA3 -L539fs/47-*558W and pcDNA3-HERG were transfected to observe the expression location of the HERG protein by immunofluoresceoce. The mutant protein size was determined by Western blotting. Results The about 1 kb-sized mutation region cDNA fragment from pcDNA3-L539fs/47-*558W and the about 7.2 kb-sized target vector fragment from pcDNA3-HERG were ligated after purification and gel recovery. pEGFP-C2-L539fs/47*-558W, approximately 8.2 kb, was demonstrated successfully been constructed under agarose gel electrophoresis and further sequencing. Laser confocal imaging showed that pEGFP-C2-HERG was mainly expressed in the membrane, whereas truncated mutant-type HERG in the pEGFP-C2 vector was partially located in the cytoplasm, the others were transported to the cell membrane in living HEK293 cells. The same as the immunofluoresceoce results after transfection of pcDNA3-HERG and pcDNA3-L539fs/47-558W. Wild-type HERG-GFP fusion protein expressed 160 and 180 kDa bands. The mutant and mutant-GFP fusion proteins were 70 and 100 kDa, respectively. Conclusion pEGFP-C2-L539fs/47-*558W was successfully constructed by double digestion method GFP had no effect on its protein expression and trafficking in HEK293 cells, which laid a foundation for the further study on L539fs/47-*558W.

Objective To reconstruct pEGFP-C2-L539fs/47, a HERG nonsense mutant in eukaryotic expression plasmid, and observe the fusion protein expressed in HEK293 cells (human embryo kidney cells). Methods After double digestion of pcDNA3-L539fs/47 and pEGFP-C2-HERG with sbf I and Eco91 I, the small product fragment, from pcDNA3-L539fs/47, was subcloned into the big fragment of pEGFP-C2-HERG under T4 ligase. pEGFP-C2-L539fs/47 was identified by agarose gel electrophoresis and sequencing. pcDNA3-L539fs/47 and pEGFP-C2-L539fs/47 were transiently transfected into HEK293 cells by Lipofect, respectively. The expression of fusion protein in HEK293 cells was detected through immunofluorescence, laser confocal imaging scanning in vivo , Western blot and PCR. Results Mutation region cDNA fragment (about 1 kb) and target vector fragment (about 7.2 kb) were ligated after purification and gel recovery. Agarose gel electrophoresis and sequencing successfully demonstrated eukaryotic expression plasmid pEGFP-C2-L539fs/47, constructed approximately 8.2 kb, sequencing consistent with template gene. The transfection efficiency of recombinant plasmid by fluorescence microscopy was more than 60%. Western blot analysis detected pcDNA3-L539fs/47 expression of the protein size 60 KD, the expression of pEGFP-C2 fusion protein size of approximately 90 KD. The L539fs/47 gene expression in HEK293 cells was significant by PCR analysis. Confocal laser imaging showed that pEGFP-C2-L539fs/47 protein was successfully expressed in cytoplasm and cytomembrane of HEK293 cells. Conclusions pEGFP-C2-L539fs/47 containing the HERG gene mutant was successfully constructed by double digestion method and expressed fusion protein in HEK293 cells, which laid a foundation for the further study on L539fs/47.