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Functional Characterization of a Novel Hevea â-1, 3-Glucanase Gene Promoter and Its Regulatory Role in Hevea and Tobacco Tissues
Supriya R. - ไม่ระบุหน่วยงาน
ชื่อเรื่อง (EN): Functional Characterization of a Novel Hevea â-1, 3-Glucanase Gene Promoter and Its Regulatory Role in Hevea and Tobacco Tissues
ผู้แต่ง / หัวหน้าโครงการ (EN): Supriya R.
บทคัดย่อ (EN): Plant â-1,3-glucanases (EC 3.2.1.39) coming under the family of PR-2 proteins, comprises of large and highly complex gene families involved in pathogen defense as well as a wide range of normal developmental processes. The diverse physiological functions might force these enzymes to occur as multiple structural isoforms that differ in their size, iso-electric point, primary structure, cellular localization and pattern of regulation. In Hevea, â-1,3-glucanase play a major role in combating the abnormal leaf fall disease (ALF) caused by Phytophthora spp. Earlier studies at RRII showed a differential Expression pattern of â-1,3-glucanase in tolerant and susceptible clones of Hevea towards abnormal leaf fall disease. To learn the mechanism behind this differential regulation. understanding of the regulatory elements residing in the â-1,3-glucanase gene promoter is essential. In the present study, a 913 bp promoter region of a novel form of â-1,3-glucanase gene has been isolated through inverse PCR. Inverse PCR comprises an initial restriction enzyme digestion of the total genomic DNA, circularization of the digested products via ligation linearization of the circularized products and finally PCR amplification. The enzyme, Ssp 1 was chosen for digestion of the total genomic DNA and Bgl II for linearization of the circularized product. The inverse PCR primers were designed based on a reported sequence of â-1,3-glucanase from H. brasiliensis (Acc. No. AY325498) primers; 5´ glucanase: TAG GAA ATT CCT ACC CTC CTT CTG;3´ glucanase: CCT GTT ATA CCA AGG CTT GCTG). Four bands were amplified in the inverse PCR ranging in size from 0.4-1.2 kb. The amplified band which showed 1.2 kb fragment size in the 1.2% agarose gel was preferred for further characterization. The fragment has been cloned in TOPOTA® cloning vector and sequenced. Upon sequencing using the M13 forward and the reverse primer, the result revealed that the sequence comprised of 913 bp promoter region of a â-1,3-glucanase upstream to the 'ATG' initiation codon. Within the 913 bp promoter, the 5' UTR region comprises 39 nucleotides Zidentified on alignment with the cDNA report by Chye et al., 1996) extending upto the 'ATG' initiation codon. The BLASTn analysis of the obtained promoter sequence with other reports showed that the 5' UTR region is showing a maximum identity of 97% towards â-1,3-glucanase mRNA reported from RRIM 600 (NCBI Acc No:U22147). It is also interesting to observe that a portion of the promoter region is showing a 100% similarity towards the Zebrafish (Danio reiro) DNA sequence from clone CH211-236H18 (NCBl Acc. No. CR385064). The 913 bp promoter sequence is found to contain the essential cis-elements that are usually present in a biotic / abiotic stress related gene promoters. It contains the TATA, CAAT, GATA, WRKY, W box elements along with other complex regulatory regions. The promoter region is rich in 'AT' base pairs similar to any other promoters. The Profiles of nucleosome potential dentified through the RECON programme showed the probability of nucleosome formation along the promoter region amplified. With the 913 bp promoter a very strong nucleosome forming potential was found in the region (5'-3') between the nucleotides 85 – 220, 399 – 486 and also from 626 – 834 nucleotide (the value ranges between 0.5 – 0.9). Two CpG islands were also observed (using the FMBOSS CpG plot online softeare), in the region 449-521 with about 32.88% CG and in the region 609-695 with 36.78% CG. Attempt was also made to understand whether the promoter form identified is functional so that the gene form identified will also be functional. Therefore, promoter: reporter gene fusion binary vector was constructed and the reporter gene expression was studies in the heterlogous system, tobacco, through Agrobacterium mediated genetic transformation. Attempts were also done to underdtand the reporter gene expression in Hevea callus through transient assay. Â-glucuronidase gene was selected as the reporter gene here. Total 3 binary vectors were developed such as one basic construct with 913 bps and two deletions of the 913 bp promoter with 550 and 200 bps. The vector used was pCAMBIA 1381 Z TDNA vector, a plant expression vector to check promoter efficiency. The GUS activity through transient assay showed a fair level of the reporter gene expression in the Hevea callus where as in the tobacco leaf discs no GUS expression was observed. The transformed ex-plants with the Agrobacterium harbouring the promoter-less vector (negative control) did not show any GUS activity in both Hevea callus and tobacco leaves. The construct with 913 bp promoter showed increased level of GUS activity within the Hevea callus than two deletion constructs. The induction experiments with 0.1% salicyilic acid also did not show any detectable levels of GUS activity in the regenerated transgenic tobacco plants. The Hevea â-1,3-glucanase gene promoter characterized here can be exploited for the pathogen induced expression of transgenes in the future crop improvement programmers in Heave and in other crops.
บทคัดย่อ: ไม่พบข้อมูลจากหน่วยงานต้นทาง
ภาษา (EN): en
เผยแพร่โดย (EN): การยางแห่งประเทศไทย
คำสำคัญ (EN): 3-Glucanase Gene Promoter
เจ้าของลิขสิทธิ์ (EN): การยางแห่งประเทศไทย
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Supriya R.. "Functional Characterization of a Novel Hevea â-1, 3-Glucanase Gene Promoter and Its Regulatory Role in Hevea and Tobacco Tissues". ไม่ระบุหน่วยงาน. ม.ป.ป.

Supriya R.. "Functional Characterization of a Novel Hevea â-1, 3-Glucanase Gene Promoter and Its Regulatory Role in Hevea and Tobacco Tissues". ไม่ระบุหน่วยงาน. ม.ป.ป.. Print.



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Functional Characterization of a Novel Hevea â-1, 3-Glucanase Gene Promoter and Its Regulatory Role in Hevea and Tobacco Tissues
Supriya R.
การยางแห่งประเทศไทย
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