Dehydrins, known as group 2 or D-11 family late-embryogenesis-abundant (LEA) proteins, play important tasks in flower growth and stress tolerance. chilly and drought stress. These results shown that were involved in flower reactions to chilly and drought. and whose expressions were induced by salt and osmotic stress were recognized in moss. The transgenic vegetation with overexpressed and genes showed different effects on rosette and root growth in stress conditions (Ruibal et al., 2012). In cowpea, a 35-kDa dehydrin was demonstrated to cosegregate with chilling tolerance during seedling emergence (Ismail et al., 1999). In was recognized, the transgenic banana vegetation performed improved drought and salt tolerance (Shekhawat et al., 2011). More and more dehydrin genes have been recognized in tomato, and so on. The transgenic vegetation performed improved tolerance in drought or chilly stress (Cheng et al., 2002; Park et al., 2006; Brini et al., 2007; Peng et al., 2008; Munoz-Mayor et al., 2012; Jyothi-Prakash et al., 2014; Kosava et al., 2014; Qiu et al., 2014). Sieb. et Zucc. is definitely a deciduous tree that generates fragrant blossoms in late winter BMN673 season to early spring of China. It originated in the southern China round the Yangtze River, and was later on introduced to northern China (Jiang and Chen, 2011). Southern China is definitely warm and damp, with lowest temp around 0C and average rainfall exceeding 1,000 mm per year. However, for most part of northern China, the annual precipitation is around 500 mm, and its lowest temperature can drop as low as -20C (Zhai et al., 2005; Song et al., 2011). Low temperature and water deficiency were considered as the key ecological factors BMN673 that affected the distribution of (Jiang and Chen, 2011). Therefore, many studies have been carried out to enhance the stress tolerance of since 1957 (Chen et al., 2008). The role of dehydrins in stress resistance had Rabbit Polyclonal to OR8K3. never been studied before. Understanding the functions of dehydrins identified from the genomic data of Beijingyudie, which has better freezing tolerance than other varieties. Expression and transgenic analyses showed that four of them were involved in stress tolerance. Materials and Methods Plant Materials and Treatments is also called mei in Chinese. The wild mei found in BMN673 Tibet was used for genome sequencing (Zhang et al., 2012). Beijingyudie is a new cultivar that can withstand temperatures as low as -19C (Chen et al., 1995). Annual branches approximately 60 cm in length with newly mature leaves were collected at 10 am from a 10-year-old Beijingyudie tree on the campus of Beijing Forestry University, Beijing, China. Stress treatments were performed following Liu et al. (2009). Approximately 12 branches were placed in flasks containing 200 mM NaCl or 20% PEG-6000 for imposing salt or osmotic stress, respectively. Branches in water served as controls. For the temperature treatments, branches were maintained in distilled water at 4C or 37C for low and high temperature treatments, respectively. Branches maintained at 20C served as controls. For the ABA treatment, branches were transferred to flasks containing 100 M ABA, and for the SA treatment, branches were treated with 2 mM SA. Untreated branches were used as controls. The leaves were sampled 4 h after treatment. Approximately 3 leaves from different branches were sampled, and immediately frozen in liquid nitrogen and stored in a -80C freezer until use. For each treatment, 3 independent replications were performed. Gene Cloning and Plasmid Construction Total RNA was extracted from leaves using Trizol reagent (Invitrogen, USA) following the manufacturers instructions, and then treated with RNase-free Dnase (Promega). First-strand cDNAs were synthesized from the total RNAs using M-MLV reverse transcriptase (Promega). The coding sequences were cloned using RT-PCR (see Table ?Table11 for primers). The amplicons were inserted into the GATEWAY donor vector pGWC (Chen et al., 2006), and at least three independent clones were sequenced. The expression vectors (Actin (ID: (ID: 104112550) gene was used as an internal control (Wang et al., 2014). The specificity of PCR reaction was verified by melting curve analysis. The primers used in the real-time PCR are shown in.