Molecular Cloning And Characterization Of Dehydrin Genes From Ginkgo Bilobal.,Brassica Napus And Capsella Bursa-Pastoris

Dehydrins, which are also known as late-embryogenesis-abundant protein group II (LEA), have been most extensively studied in relation to drought and cold stresses. Dehydrins are a family of plant proteins that are induced by stimuli that have a dehydrative component such as drought, low temperature, salinity and ABA. Dehydrins are characterized by a highly conserved 15-mer lysine rich sequence, called K-segment. The K-segment can form an amphiphathic a-helix structure. Transgenic research showed that over-expression dehydrin genes in plants can improve tolerance abilities to environmental stresses.In this paper, dehydrin genes including Gbdhn, BnDHN ERD10 and CbDHN were successfully isolated from Ginkgo biloba L., Brassica napus and Capsella bursa-pastoris by using rapid amplification of cDNA ends (RACE)-PCR method respectively, and their functions were also studied.A full-length cDNA encoding a dehydrin was cloned from the living fossil plant Ginkgo biloba by rapid amplification of cDNA ends (RACE) for the first time. The cDNA, designated as GbDHN, was 820 bp long containing an open reading frame of 489 bp. The deduced GbDHN protein had 163 amino acid residues, which formed a 17 kDa polypeptide with a predicted isoelectric point (pI) of 5.75. GbDHN had an S-segment and a K-segment, indicative of dehydrins. Homology analysis indicated that the S-segment and K-segment of GbDHN shared identity with those of other reported dehydrins, indicating that GbDHN belonged to dehydrin superfamily. Genomic sequence of GbDHN was also cloned using genomic walker technology. By comparing genomic DNA with the cDNA, it was found that there was a 257-bp intron in this gene. Promoter analysis indicated that it contained two ABREs in the 5'-flanking region. Southern blot analysis revealed that GbDHN belonged to a single copy gene family. RT-PCR analysis revealed that GbDHN constitutively expressed in stems and roots. The increased expression of GbDHN was detected when G. biloba seedlings were treated with exogenous abscisic acid (ABA), salt stress and drought stress. These results indicate that the GbDHN has the potential to play a role in response to ABA and environmental stresses that can cause plant dehydration.A new dehydrin gene is cloned and characterized from Brassica napus successfully (designated as Bndhn ERD10). The full-length cDNA of Bndhn ERD10 is1114 bp long and contains an open reading frame of 816 bp encoding a protein of 271 amino acid residues. The deduced amino acid sequence of Bndhn ERD10 contained 271 residues with a predicted isoelectric point (pI) of 5.09 and calculated molecular weight of about 31 kDa. The deduced Bndhn ERD10 protein contains an S-segment and two conserved repeats of the characterized lysine-rich-K-segment (KIKEKLPG). The promoter of Bndhn ERD10 was further obtained by genomic walking technology, and analysis of the promoter indicated that the regulation of Bndhn ERD10 was ABA-dependent. Semi-quantitative RT-PCR of different tissues in unstressed plants indicated that the transcript of Bndhn ERD10 gene was more abundant in leaf tissue than in stem and root tissues. The expression profiles of Bndhn ERD10 in B.napus seedlings under various stress conditions including cold, salt and ABA were also investigated. Upon cold, salt and ABA stresses, increased expression accumulations of the Bndhn ERD10 were detected in young leaves.A new dehydrin gene was also cloned from Capsella bursa-pastoris by rapid amplification of cDNA ends (RACE). The cDNA, designated as CbDHN, was 983 bp long containing an open reading frame of 720 bp. The deduced CbDHN protein had 239 amino acid residues, which formed a 27 kDa polypeptide with a predicted isoelectric point (pI) of 5.2. CbDHN had an S-segment and three K-segments, indicative of dehydrins, Homology analysis indicated that the S-segment and K-segment of CbDHN shared identity with those of other reported dehydrins, indicating that CbDHN belonged to dehydrin superfamily. RT-PCR analysis revealed that CbDHN constitutively expressed in leaves, stems and roots. The increased expression of CbDHN was detected when Capsella bursa-pastoris seedlings were treated with exogenous abscisic acid (ABA), salicylic acid (SA), salt stress, cold stress and drought stress.Four kinds of univalent plant expression vector were constructed based onpCAMBIA1304, with constitutive expression CaMV35S promoter activating the genes. Vector 1: pCaMV35S: BnDHN, vector 2: pCaMV35S::GbDHN, vector 3: pCIP::CbCOR15b, CIP: cold-induced promoter, and vector 4: pGBP::GUS. The recombinant vector was introduced into Agrobacterium tumefaciens with freeze-thaw method. The leaf discs of tobacco plants infected by A. tumefaciens were selected with hygromycin. PCR testing or gus analysis of the regenerated plants are in proceeding.The cloning of GbDHN, Bndhn ERD10 and CbDHN provides a basis for further investigating their functions in seed maturation and stress resistance. This study is of potential importance not only for the obtaining of intellectual properties of more stress tolerance genes but also for the improvement of crops for stress resistances by genetic engineering.