สืบค้นงานวิจัย
Cloning and expression of HbACO2 of Hevea brasiliensis
M. B. Mohamed Sathik - ไม่ระบุหน่วยงาน
ชื่อเรื่อง (EN): Cloning and expression of HbACO2 of Hevea brasiliensis
ผู้แต่ง / หัวหน้าโครงการ (EN): M. B. Mohamed Sathik
บทคัดย่อ (EN): Ethylene has been known to stimulate latex production in Hevea and is being widely used for increasing the crop productivity. Ethephon (chloro-2-ethyl phosphonic acid) which is an ethylene releaser is commonly used as an yield stimulant. Ehtylene stimulation in Hevea has been found to enhance the expression of genes involved in ethylene biosynthesis such as ACC synthase and ACC oxidase (ACO) revealed that they belong to a multigene family and are regulated by a complex network signals as a response to both internal and external stimuli. Existence of three types of ACO (HbACO1, HbACO2 and HbACO3) have been reported with open reading frames encoding polypeptides of 312, 318 and 318 amino acids, (1115, 1174 and 1074 bp long) respectively. It was also reported that HbACO1 may be responsible for basal levels of ethylene production while HbACO2 and HbACO3 are up-regulated in response to external factors such as wounding. Further investigations are needed to identify which of these ACOs are involved directly in increasing the ethylene levels in response to ethephon stimulation. Hence, this study was initiated with an objective to understand the role of ACO2 in ethylene biosynthesis in Hevea. For this purpose, the ACO2 has been cloned into an expression vector for getting the protein with an aim to develop antiserum which could be utilized for the screening ACO levels through ELISA technique. Bark samples from Hevea brasiliensis (clone RRII 105) was collected from which mRNA was isolate using Dyanbeads (Invitrogen, USA) and cDNA was synthesized using Superscript IIIRT First strand synthesis kit (Invitrogen, USA). ACO2 specific primers flanked with suitable restriction sites (to enable cloning into specific restriction sites in the expression vector) were synthesized and employed to PCR amplify the coding region of ACO2 from Hevea. The PCR amplified cDNA fragment was cloned in to pGEM-T vector and transformed in to E. coli cells (GenHunter, USA). The selection of transformants was done by blue white screening after which the clones were further confirmed by colony PCR and by restriction digestion using BamH I and Pst I restriction enzymes. The coding region of ACO2 cloned in pGEMT vector was digested with BamH I and Pst I restriction enzymes and ligated into the appropriate sites of pRSET-A vector. This ligate mix was successfully transformed into E. coli (BL 21 (DE3) PLysS) cells and the transformants were selected based on colony PCR and restriction digestion analysis. The above results indicate the successful cloning of HbACO2 gene in pRSET-A expression vector. The selected clone was inoculated into Super Optimal Broth (SOB) containing ampicillin (50 µg/ml) and chlouamphenicol (35 µg /ml). The overnight culture was further inoculated in to SOB for protein expression bey inducing with 1mMIPTG. The protein was subjected to SDS polyacrylamide gel electrophoresis (PAGE) analysis and was visualized using Coomassie blue stain. A band of recombinant protein (with size of approximately 36 KDa) was observed in the 1PTG induced culture samples of the recombinant clone. The protein after purification would further be employed to develop specific antiserum that could be utilized for quantification of HB ACO2 in different Hevea clones and wild germplasm accessions through ELISA.
บทคัดย่อ: ไม่พบข้อมูลจากหน่วยงานต้นทาง
ภาษา (EN): en
เผยแพร่โดย (EN): การยางแห่งประเทศไทย
คำสำคัญ (EN): ethylene
เจ้าของลิขสิทธิ์ (EN): การยางแห่งประเทศไทย
หากไม่พบเอกสารฉบับเต็ม (Full Text) โปรดติดต่อหน่วยงานเจ้าของข้อมูล

การอ้างอิง

M. B. Mohamed Sathik. "Cloning and expression of HbACO2 of Hevea brasiliensis". ไม่ระบุหน่วยงาน. ม.ป.ป.

M. B. Mohamed Sathik. "Cloning and expression of HbACO2 of Hevea brasiliensis". ไม่ระบุหน่วยงาน. ม.ป.ป.. Print.



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Cloning and expression of HbACO2 of Hevea brasiliensis
M. B. Mohamed Sathik
การยางแห่งประเทศไทย
ไม่ระบุวันที่เผยแพร่
Cloning and expression of 3-hydroxy-3-methylglutaryl-Coa Reductase 1 (Hmge 1) from Hevea brasiliensis Expression of NAC Transcription Factor under Drought Stress in Hevea brasiliensis IDENTIFICATION OF DROUGHT TOLERANT GENES BY QUANTITATIVE EXPRESSION ANALYSIS IN HEVEA BRASILIENSIS CLONAL VARIABILITY IN THE INCLINATION OF LATICIFERS AND PHLOIC RAYS IN HEVEA BRASILIENSIS IMPROVEMENT AND APPLICATION OF THE TECHNIQUE OF MINI-SEEDLING BUDDING OF HEVEA BRASILIENSIS THE YIELD AND QUALITY OF RUBBER (HEVEA BRASILIENSIS) AS AFFECTED BY IRRIGATION AND FERTILIZER APPLICATIONS Yield Modeling and Analysing the Critical Factors of Productivity of Natural rubber (Hevea brasiliensis) ANALYSING THE GENETIC RESPONSE TO ENVIRONMENTAL STRESS IN YOUNG RUBBER PLANTS (HEVEA BRASILIENSIS) FOR PREDICTING ADULT PHENOTYPIC EXPRESSION Seasonal Variation and Evolution of Some Latex Physiological Parameters of Hevea brasiliensis over Consecutive Tapping Years A Mini Juvenile Bud Stick and Mini Seedling Budding of Hevea brasiliensis
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