De Novo Characterization Of Hazelnut Floral Bud Transcriptome Using Solexa Sequencing And Expression Profiling Analysis Of Cold-regulated Genes

Temperature is one of the most important environmental factors limiting the geographical distribution of plants and accounts for significant reductions in the yield of agriculturally important crops. Low temperature damages many plant species, especially those adapted to tropical climates. In contrast, some species from temperate regions are able to develop freezing tolerance in response to low non-freezing temperature, an adaptive process named cold acclimation. Numerous molecular, biochemical and physiological changes occur during cold acclimation, most of them being associated with significant changes in gene expression. hazelnut is one of ecologically and economically important woody plant species. Corylus avellana is a diploid with a small haploid genome, Hazelnut could serve as a model for genomics studies in the family Betulaceae and the order Fagales. The European hazelnut, C. avellana, is the species of commerce and is genetically diverse as its large size, thinner shells. However, some highly desirable and economically important traits such as cold hardiness, resistance to eastern filbert blight do not exist within C. avellana. Corylus heterophylla Fisch. is widely distributed in Northern China. Compared with C. avellana L, the nut weight of C. heterophylla Fisch. is smaller, and the yield is poor, but it has very good winter hardiness. There are not sufficient transcriptomic and genomic data in public databases for understanding of the molecular mechanism underlying overwintering of hazelnut. In this article, we report a transcriptome sequence of cold-hardy corylus species, C. heterophylla Fisch, derived by high-throughput Solexa sequencing. Transcript changes of 20 genes under the field and artificial-controlled cold stress condition were investigated by using real-time and semiRT-PCR method, respectively. The results provide a fairly comprehensive molecular biology background to the research on hazelnut development, particularly with overwintering of floral bud. In addition, we choosed CBF and DHN to do further functional identification. Overall, these sequences will provide valuable resources for the hazelnut community. Additionally, these results also suggested that transcriptome analysis based on Illumina paired-end sequencing is a powerful tool for gene discovery for non-model species. This work will improve our understanding on overwintering of hazelnut. And results will lay the foundation for carrying out genetic improvement of cold-tolerant hazelnut cultivar. Main results are as follows:1. This study investigated the transcriptome profiles of hazelnut using the Solexa/Illumina RNA-seq technologies, high-throughput sequencing, assembly, and annotation of expressed sequences from hazelnut were obtained. The substantial amount of transcripts obtained provides a strong basis for future genomic research on hazelnut organisms. More than 13 million sequencing reads were generated using Illumina paired-end sequencing technology. De novo assembly yielded 43,285 Unigenes with an average length of 637 bp. These assembled unigenes were annotated with various methods, Out of these annotated Unigenes, 3,890 and 65,536 unigenes were assigned to gene ontology and clusters of orthologous group, respectively. Based on sequence similarity search with known proteins, a total of over 50 genes were identified to cold regulated. Among those genes some involved in osmo-regulation Substances biosynthesis, regulation of transcription, Nitrogen metabolism, antioxidant and stress responses or plant defence, were found, including 14-3-3, HSP22, GPAT, ERD7, WCOR413-PM, ADF, KT, bzip78, ICE, CIP, NAC, PHD, ENO, HD, DOF. SPDS, NIR, BADH, BAM, SATO. 20 genes of interest which belong to different categories were selected to clone and their expression was analyzed in floral buds during natural NA-CA-MW-DA and in leaves under controlled cold stress by quantitative real-time RT-PCR and semi RT-PCR, respectively. Furthermore, most genes accumulated to higher levels in the cold hardy genotype'Corylus heterophylla Fisch'than in'Corylus avellana L.'2. Gene expression analysis by semi-quantitative RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in semi-quantitative RT-PCR for abiotic stress studies in hazelnut. Partial sequences of four Corylus heterophylla Fisch housekeeping genes such as ChACTIN, ChMDH, ChUBQ and ChEF1 were obtained by screening the transcriptome library. Primers for quantitative RT-PCR were designed based on these partial sequences. The housekeeping genes were evaluated for their expression stability in samples from plants subjected to cold stress and different organs. The analysis found that ChActin were the most stable.3. The CBF/DREB1 transcription factors control an important pathway for increased freezing tolerance in plants. We report here the isolation of CBF1 and CBF2, from freezing-tolerant hazelnut (Corylus heterophylla Fischer) and freezing-sensitive hazelnut(Corylus avellana L.). CBF2 expression pattern differ substantially between'Corylus heterophylla Fischer'and'Corylus avellana L.'Hydrophobic residues of C-terminal region may result in transcription activation. The tissue organ-specific expression pattern of the CBF1 and CBF2 gene showed that its transcripts were abundant in male flower and floral bud, respectively. Nuclear localization of CBF2 proteins was confirmed by transient introduction of green ?uorescent protein fusion constructs for a subset of protein genes into onion cells.4. Two cDNA encoding dehydrin-like gene homologues was isolated from hazelnut(Corylus heterophylla Fisch.)by RACE-PCR and designated ChDHN1 (GenBank Accession No. HM228388) and ChDHN2 (GenBank Accession No. HM228389). Sequence analysis of ChDHN1 showed it contained a single open reading frame of 339 bp. The predicted ChDHN1 protein has 112 amino acids; Sequence analysis showed that cDNA of ChDHN2 was 639 bp long and contained a single open reading frame. The predicted ChDHN2 protein has 167 amino acids, qRT-PCR analysis showed that the expression of ChDHN2 was induced by low temperature and peaked at 24 h after exposed to low temperatures of 4℃. The transcripts of ChDHN2 appeared in many hazelnut tissues including male flower, bark, ?ower bud and seeds, but mostly accumulated in seeds. Genomic DNA sequence of DHN was obtained through TAIL-PCR, the results found that a 97-bp and 95-bp intron was present in the genomic sequence of DHN1 and DHN2, respectively..Computer analysis using the PLANTCARE database predicted the presence of several putative cis-regulatory sequences, in addition to these MYC, ABRE and MYB recognition sites, CRT cis-elements were predicted in the DHN1 promoter. In order to ideniify the function of ChDHN2, in vitro functional analysis were performed using an E. coli heterologous expression system. Recombinant pET-ChDHN2 vector was constructed, SDS-PAGE analysis showed that the recombinant plasmid pET-30a(+)-DHN2 could be expressed a relative molecule mass of approximately 30 KDa, Western blot results showed that the recombinant fusion protein could be identified by anti-His antibody. Expression of pET-ChDHN2 in E. coli enhanced its tolerance to cold stress. Under low temperature, ChDHN2-overexpressing tobacco showed lower MDA content, electrolyte leakage and superoxide radical (O₂^.-). These results indicated heterologous expression of ChDHN2 in tobacco could enhance the ability to endure low temperature.