Plants Under Arid Environmental Stress Molecular Adaptation Mechanism And Its Application

Drought is one of the major abiotic stress factor affecting the normal metabolism, growth and development of plants, which have a serious influence on the urban-greening construction and agricultural development in china. Dehydrins and CBF (CRT binding factors) are groups of proteins produced in the process of the drought-environmental stress. They play a big role in anti-drought respectively. But by far it is lack of data applying in the plant genetic engineering of anti-drought. And it still need further research. In this research, a novel dehydrin gene called cor29 was cloned from Capsella bursa-pastoris. The full-lenth cDNA of cor29 and Cbcbf25 gene were transformed into tobacco by the Agrobacterium tumefaciens-mediated transformation, so as to provide references for the plant genetic engineering research of anti-drought. The research results are as follows:1. Molecular cloning and characterization analysis of the full-length cDNA of dehydrin gene cor29 from Capsella bursa-pastorisCombined by the homo logy-based cloning method and RACE technology, a novel dehydrin gene designated as cor29 (GenBank, AY513787) was cloned from Capsella bursa-pastoris. The full-length cDNA of cor29 was 1110 bp long with a 783 bp open reading frame (ORF) encoding a putative protein of 261 amino acids. The deduced polypeptide (designated as COR29) had a predicted molecular weight of 29.36 kD and an estimated pI of 4.93.Sequence analyses showed that COR29 shared several typical features with dehydrins family, including a high proportion of hydrophilic amino acids which makes it a highly hydrophilic polypeptide, three copies of lys-rich domains (K-segments) and a Ser-rich domain (S-segment) prior to the first lys-rich domain that was common to many dehydrins. Search of the sequence database in EXPASY/UniProt through the BLAST algorithm implied that the COR29 sequence had high homology with that of dehydrins.Analyses of structure and function indicated COR29 protein would play a important role by its abundant hydrophilic amino residues and K-segment which can form the amphipathic α-helix in preventing cell from dehydration injure resulted by drought-environmental stress.Semi-quantitative RT-PCR revealed that cor29 gene could be induced by drought, low-temperature and exogenous abscisic acid (ABA). The further analysis suggestedthat there exist at least two independent signal transduction pathways of cor29 gene expression: one pathway was ABA-dependent pathway but does not require protein biosynthesis(IV), and the other one was ABA-independent pathway.2. Plant expression vector construction of cor29 and Cbcbf25 full-length cDNAThe plant expression vector pCAMBIA2301 was adopted as the backbone. The gus box of plant expression vector pBI121 was cut down with EcoRI/HindIII double digestion and recovered to be inserted into the multiple cloning sites (MCS) of pCAMBIA2301 to form an intermediate vector p2301-gus. In this vector, the usable restriction sites after the CaMV 35S promoter of the inserted gus box are XbaI, BamHI and XmaI/SmaI, while only one SacI site can be used before the Nos terminator. PCR primers with adequate restriction site sequences were designed and the full-length cDNA fragments of cor29 and Cbcbf25 genes were amplified from reverse-transcribed total cDNAs. After verified with sequencing, pGEM-T Easy Vectors containing the target gene fragments were double digested completely and the following gene fragments were recovered: Cbcbf25 full-length cDNAs with XmaI/SacI cohesive ends in their 5' and 3' ends respectively, and the cor29 full-length cDNAs with BamHI/SacI cohesive ends in their 5' and 3' ends respectively. Meanwhile the p2301 -gus was double digested with BamHI/SacI and XmaI/SacI respectively to cut away the gus gene fragments and the corresponding linearized vector fagments were also recovered. The plant expression vectors p2301 -cor29 and p2301 -Cbcbf25 containing the cor29 and Cbcbf25 full-length cDNA respectively were finally obtained by lig(?)ting the corresponding vector fragments and the target gene fragments with compatible cohesive ends. The 2 target genes were drived by the CaMV 35S promoter and terminated for transcription by the Nos terminator respectively. Both of the constructed plant expression vectors contain an nptII marker gene box and a gus reporter gene box. They were first transformed into Escherichia coli strain DH5 α , and then into Agrobacterium tumefaciens strains EHA105.In addition, the expression vector p2301-Cbcbf25 was constructed, in which the target gene Cbcbf25 was drived by the Ubiquitin-promoter which was cloned from the maize and the hpt gene was used as marker gene. And it could be used to transform the monocotyledon.3. The research of cor29 and Cbcbf25 full-length cDNA into tobacco using Agrobacterium tumefaciens-mediated transformationAgrobacterium tumefaciens EHA105 harboring the plant expression vector p2301-cor29 and p2301-Cbcbf25 was used to co-culture with tobacco leaf discs for the transformation respectively. After successive induction of the indefinete buds and roots, 35 regenerated tobacco plants transplanted by p230\-cor29 and 23 regenerated tobacco plants transplanted by p2301 -Cbcbf25 were finally obtained respectively.The result of the identification of these seedlings with 0.5% Kan for leaf inoculation indicated that, in the plants transplanted by p2301-cor29, there were 6 resistant, 6 partially resistant and 23 non-resistant ones; and in the plants transplanted by p2301 -Cbcbf25, there were 5 resistant, 3 partially resistant and 15 non-resistant. PCR identification results of the target gene (cor29 or Cbcbf25) and nptII gene show the kan resistant seedlings contained the target gene respectively. Based on the result showed above, we can confirmed that the dehydrin gene cor29 and CBF gene Cbcbf25 from Capsella bursa-pastoris has been inserted into the genomic DNA of tobacco.