สืบค้นงานวิจัย
Cloning and expression of 3-hydroxy-3-methylglutaryl-Coa Reductase 1 (Hmge 1) from Hevea brasiliensis
P.K. Ambily - ไม่ระบุหน่วยงาน
ชื่อเรื่อง (EN): Cloning and expression of 3-hydroxy-3-methylglutaryl-Coa Reductase 1 (Hmge 1) from Hevea brasiliensis
ผู้แต่ง / หัวหน้าโครงการ (EN): P.K. Ambily
บทคัดย่อ (EN): Natural rubber (cis-1, 4-polyisoprene) an important raw material for many industrial uses is synthesized in the lacticiferous tissue of Hevea tree. The mevalonate pathway, which initiates the synthesis of mevalonate by 3-hydroxy- 3 –methyl – CoA reductase (HMGR) provides prectruors for diverse spectrum of isoprenoid compounds. This is a key regulatory enzyme in the rubber biosynthetic pathway. Earlier workers reported a significant positive correlation between rubber biosynthesis and HMG-CoA reductase activity in Hevea. Different members of hmgr gene were cloned and characterized from the Hevea clone RRIM 600. Earlier reports suggested that hmgrl is expressed more in lacticifers than in leaves and is specifically involved in rubber biosynthesis. The objective of the present study was the in vitro synthesis of the HMGR1 protein. The protein would be used to get the antiserum with an objective to use it as a biochemical marker of yield potential in Hevea. This would further enable screening of newly emerging Hevea clones and wild germplasm accessions to evaluate their yield potential. Latex from trees of Hevea clone RRll 105 was collected and mRNA was isolated using Dynabeads (Invitrogen, USA). CDNA was synthesized using Superscript IIIRT First strand synthesis kit (Invitrogen, USA) and PCR amplification with gene specific primers was performed under standard conditions. The PCR amplified cDNA fragment (1.8 kb fragment) was cloned in to pGEM-T vector and transformed in to E. coli cells (Gen Hunter cells). The selection of transformants was done according to the manufacturer’s instructions. Presence of the insert was confirmed by colony PCR. Restriction digestion and sequencing (Macrogen, Korea0. pRSET-A expression vector (Invitrogen) was used for in vitro protein synthesis in BL21 (DE3) pLysS cells. The clone of pRSET-A/hmgr-1 was inoculated into SOB containing ampicillin (50 µg)/ml) and chloramphenicol (35 µg /ml). The overnight culture was further inoculated in to SOB for protein expression by inducing with 1mM IPTG. The protein from the culture was subjected to SDS polyacrylamide gel electrophoresis (PAGE) analysis and visualized using Coomassie blue stain. A PCR based approach was employed to isolate hmgrl gene from Hevea PCR resulted in the amplification of single 1.8 kb fragment corresponding to the length of hmgrl. The PCR amplified cDNA fragment was cloned in to pGEm-T vector. The colony PCR and restriction digestion analyses confirmed the presence of cloned DNA inserts in pGEM-T vector. Sequencing results showed a 100% homology of our clone to earlier submitted sequence (Acc. No. X54659). Restriction digestion analysis and sequencing confirmed the DNA inserts in pRSET-A expression vector. Orientation of the insert in the expression vector was confirmed by the sequencing data. A protein with a size of 64.6 kDa (including His Tag protein) corresponding to HMGR1 was observed in the IPTG induced culture samples of the recombinant clone. The purification of the protein is in progress. The purified protein would further be employed to develop specific antiserum that could be utilized for quantification of HMG-CoA reductase in different Hevea clones and wild germplasm accessions through immune techniques. The initial steps which involved PCR amplification of specific gene, cloning into the expression vector and in vitro protein synthesis could be achieved successfully.
บทคัดย่อ: ไม่พบข้อมูลจากหน่วยงานต้นทาง
ภาษา (EN): en
เผยแพร่โดย (EN): การยางแห่งประเทศไทย
คำสำคัญ (EN): protein
เจ้าของลิขสิทธิ์ (EN): การยางแห่งประเทศไทย
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การอ้างอิง

P.K. Ambily. "Cloning and expression of 3-hydroxy-3-methylglutaryl-Coa Reductase 1 (Hmge 1) from Hevea brasiliensis". ไม่ระบุหน่วยงาน. ม.ป.ป.

P.K. Ambily. "Cloning and expression of 3-hydroxy-3-methylglutaryl-Coa Reductase 1 (Hmge 1) from Hevea brasiliensis". ไม่ระบุหน่วยงาน. ม.ป.ป.. Print.



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Cloning and expression of 3-hydroxy-3-methylglutaryl-Coa Reductase 1 (Hmge 1) from Hevea brasiliensis
P.K. Ambily
การยางแห่งประเทศไทย
ไม่ระบุวันที่เผยแพร่
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