Cold stress is an important factor that limits apple production.  In this study, we examined the tissue-cultured plantlets of apple rootstocks ‘M9T337’ and ‘60-160’, which are resistant and sensitive to cold stress, respectively.  The enriched pathways of differentially expressed genes (DEGs) and physiological changes in ‘M9T337’ and ‘60-160’ plantlets were clearly different after cold stress (1°C) treatment for 48 h, suggesting that they have differential responses to cold stress.  The differential expression of WRKY transcription factors in the two plantlets showed that MdWRKY40is and MdWRKY48 are potential regulators of cold tolerance.  When we overexpressed MdWRKY40is and MdWRKY48 in apple calli, the overexpression of MdWRKY48 had no significant effect on the callus, while MdWRKY40is overexpression promoted anthocyanin accumulation, increased callus cold tolerance, and promoted the expression of anthocyanin structural gene MdDFR and cold-signaling core gene MdCBF2.  Yeast one-hybrid screening and electrophoretic mobility shift assays showed that MdWRKY40is could only bind to the MdDFR promoter.  Yeast two-hybrid screening and bimolecular fluorescence complementation showed that MdWRKY40is interacts with the CBF2 inhibitor MdMYB15L through the leucine zipper (LZ).  When the LZ of MdWRMY40is was knocked out, MdWRKY40is overexpression in the callus did not affect MdCBF2 expression or callus cold tolerance, indicating that MdWRKY40is acts in the cold signaling pathway by interacting with MdMYB15L.  In summary, MdWRKY40is can directly bind to the MdDFR promoter in order to promote anthocyanin accumulation, and it can also interact with MdMYB15L to interfere with its inhibitory effect on MdCBF2, indirectly promoting MdCBF2 expression, and thereby improving cold tolerance.  These results provide a new perspective for the cold-resistance mechanism of apple rootstocks and a molecular basis for the screening of cold-resistant rootstocks.

The highbush blueberry (Vaccinium corymbosum), Duke, was used to construct a de novo transcriptome sequence library and to perform data statistical analysis.  Mega 4, CLC Sequence Viewer 6 software, and quantitative PCR were employed for bioinformatics and expression analyses of the basic helix-loop-helix (BHLH) transcription factors of the sequencing library.  The results showed that 28.38 gigabytes of valid data were obtained from transcriptome sequencing and were assembled into 108 033 unigenes.  Functional annotation showed that 32 244 unigenes were annotated into Clusters of Orthologous Groups (COG) and Gene Ontology (GO) databases, whereas the rest of the 75 789 unigenes had no matching information.  By using COG and GO classification tools, sequences with annotation information were divided into 25 and 52 categories, respectively, which involved transport and metabolism, transcriptional regulation, and signal transduction.  Analysis of the transcriptome library identified a total of 59 BHLH genes.  Sequence analysis revealed that 55 genes of that contained a complete BHLH domain.  Furthermore, phylogenetic analysis showed that BHLH genes of blueberry (Duke) could be divided into 13 sub-groups.  PCR results showed that 45 genes were expressed at various developmental stages of buds, stems, leaves, flowers, and fruits, suggesting that the function of BHLH was associated with the development of different tissues and organs of blueberry, Duke.  The present study would provided a foundation for further investigations on the classification and functions of the blueberry BHLH family.

    ω-Secalin was an important factor influencing processing quality of wheat 1BL/1RS translocations. On the basis of four ω-secalin gene sequences cloned from Lankao 906 (a wheat cultivar with 1BL/1RS translocation) with putative transcription activity, a pair of primers with suitable restriction endonucleases added at their 5´ ends were designed to amplify the mature protein-coding regions of the four genes. After digestion with restriction endonucleases, the amplified products were ligated into the prokaryotic expression vector pET30a(+). The prokaryotically expressed recombinant proteins and gliadin isolated from the Lankao 906 seed were analyzed on the same acid polyacrylamide gel electrophoresis. All four prokaryotically expressed secalin bands had corresponding seed-expressed gliadin bands. The four corresponding gliadin bands were confirmed to be the expression products of the four ω-secalin genes by liquid chromatography tandem mass spectrometry (LC-MS/MS). This conclusion was further confirmed because the four ω-secalin bands could be detected in all 14 wheat 1BL/1RS translocation cultivars used in the study, although there was some interference for the detection of one ω-secalin band from nearby wheat gliadin bands. The sequence information of ω-secalin genes with expression activity will be helpful for improving the processing quality of wheat with 1BL/1RS translocations by using RNA interference method to silence the expression of the ω-secalin genes.