Cloning And Expression Analysis Of Two Plant Anti-stresses Genes: GbTPS And BnPrx

Plants meet various environmental stresses during their growth and development and establish some corresponding mechanisms against these stresses. Some mechanisms are developed against specific stress such as the role Na⁺/H⁺ exchanger (NHX) plays in salt tolerance. However, some are adaptive to various stresses such as the trehalose-6-phosphate synthase and peroxidase in this study. Cloning the related genes is instrumental to know the exact mechanism. On the other hand, introducing these genes into other plants helps to study their specific functions. Furthermore, it may be applicable in improving quality of corps. In our study, we cloned the trehalose-6-phosphate synthase is cloned from a so-called "living fossil" gymnosperm: Ginkgo biloba and a peroxidase is cloned from an important corp: Brassica napus. Both species display good resistance against various environmental stresses.1) Trehalose-6-phosphate synthase from GinkgoIn many organisms, trehalose acts as protective metabolite against harsh environmental stresses, such as freezing, drought, nutrient starvation, heat and salt. Herein a cDNA, trehalose-6-phosphate synthase, (designated as GbTPS, GenBank Accession Number AY884150) encoding a trehalose-6-phosphate synthase homologue was isolated and characterized from the living fossil plant, Ginkgo biloba, which is highly tolerant to drought and cold. GbTPS encoded an 868-amino-acid polypeptide with a predicted isoelectric point of 5.83 and molecular mass of 97.9 kD. Amino acid sequence alignment revealed that GbTPS shared high identity with class II trehalose-6-phosphate synthase homologues (67% identical to AtTPS7), but had only 17% and 23% of identity with OstA from Escherichia coli and ScTPS1 from S. cerevisiae, respectively. Its genome DNA sequence contains two introns. And its promoter region is isolated by genome-walking. DNA gel blot analysis indicated that GbTPS belonged to a small multi-gene family. The expression analysis by RT-PCR showed that GbTPS expressed in a tissue-specific manner in G. biloba and might involve in leaf development. GbTPS was also found to be induced by a variety of stresses including cold, salt, drought and mannitol. GbTPS could not restore the yeast mutation tps1 and tps2. It might have some unknown functions. To test their function, its promoter and ORF was constructed into vectors and introduced into plants by agrobacterium strain EHA102) Peroxidase from oilseed rapeA new gene, designated as BnPrx (GenBank accession number DQ078754), was isolated from oilseed rape (Brassica napus) by SMART Rapid Amplification ofcDNA Ends (RACE), The full-length cDNA is 1307 bp long and contains a 1062 bp open reading frame (ORF), which encodes a 354 amino acid peroxidase precursor, with a 31 aa N-terminal signal peptide and a 15 aa C-terminal propeptide.o The putative protein has a molecular weight of 38.86 kDa and a calculated pI of 5.85. BnPrx shares high identity with HRPC (89%). BnPrx possesses all active residues and two Ca²⁺ sites present in Horseradish peroxidase isoenzymes C (HRPC) as well as six N-glycosylation sites. The predicted three-dimensional structure of BnPrx is very similar to that of HRPC. Assisted by genome-walking technology, the genome DNA of BnPrx was also cloned, consisting of 3 introns and 4 exons. Thirty-two TATA boxes, 18 CAAT boxes and many cis-elements, such as WUN and MeJR, were found in its promoter region. We constructed a series of deletion mutation of its promoter with pCAMBIA1304 vector and transformed tobacco with EHA105 strains to test its function. Southern blot analysis indicated that BnPrx belonged to a small gene family. Northern. Northern blot analysis revealed that BnPrx was constitutively expressed in all tested tissues, including roots, stems and leaves, with the high expression in leaves and stems. The expression of BnPrx could be induced by methyl jasmonate (MeJA), salicylic acid (SA), cold and H₂O₂. The cloning and characterizing of BnPrx might not only help us understand the physiological function and molecular evolution of the large peroxidase gene family more comprehensively, but also provide an alternative way of seeking a more effective and economical substitute for HPRC. In addition the analysis of the promoter region of BnPrx might provide us a promoter with high-efficiency, used in the genetics engineering.