Báo cáo khoa học: Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis A key factor participating in natural rubber biosynthesis

Molecular Cloning, Expression and Characterization of cDNA Encoding cis-Prenyltransferases from Hevea brasiliensis

Tác giả: Kasem Asawatreratanakul, Yuan-Wei Zhang, Dhirayos Wititsuwannakul, Rapepun Wititsuwannakul, Seiji Takahashi, Atiya Rattanapittayaporn, Tanetoshi Koyama

Lĩnh vực: Sinh hóa, Công nghệ sinh học, Sinh học thực vật

Nội dung tài liệu: Nghiên cứu này tập trung vào việc phân lập và đặc tính hóa các bản sao cDNA từ cây cao su (Hevea brasiliensis) mã hóa các yếu tố chức năng tham gia vào quá trình sinh tổng hợp cao su tự nhiên. Các nhà nghiên cứu đã xác định hai bản sao cDNA, được đặt tên là HRT1 và HRT2, có khả năng mã hóa các enzyme cis-prenyltransferase trong mủ cây cao su. Phân tích trình tự cho thấy cả hai bản sao cDNA đều chứa các vùng bảo tồn cao đặc trưng cho các enzyme kéo dài chuỗi cis-prenyl. Nghiên cứu đã chứng minh rằng protein HRT2 có hoạt tính enzyme cis-prenyltransferase, xúc tác cho sự hình thành các sản phẩm polyprenyl chuỗi dài. Kết quả cũng cho thấy rằng enzyme này có thể cần các yếu tố hoạt hóa trong hạt đáy (WBP) từ mủ cây cao su để sản xuất cao su có khối lượng phân tử cao. Nghiên cứu này là một bước quan trọng trong việc hiểu rõ hơn về cơ chế phân tử của quá trình sinh tổng hợp cao su.

Mục lục chi tiết:

• Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis
• A key factor participating in natural rubber biosynthesis
• Natural rubber from Hevea brasiliensis is a high molecular mass polymer of isoprene units with cis-configuration.
• The enzyme responsible for the cis-1,4-polymerization of isoprene units has been identified as a particle-bound rubber transferase, but no gene encoding this enzyme has been cloned from rubber-producing plants.
• By using sequence information from the conserved regions of cis-prenyl chain elongating enzymes that were cloned recently, we have isolated and characterized cDNAs from H. brasiliensis for a functional factor participating in natural rubber biosynthesis.
• Sequence analysis revealed that all of the five highly conserved regions among cis-prenyl chain elongating enzymes were found in the protein sequences of the Hevea cis-prenyltransferase.
• Northern blot analysis indicated that the transcript(s) of the Hevea cis-prenyltransferase were expressed predominantly in the latex as compared with other Hevea tissues examined.
• In vitro rubber transferase assays using the recombinant gene product overexpressed in Escherichia coli revealed that the enzyme catalyzed the formation of long chain polyprenyl products with approximate sizes of 2 × 10³−1 × 10⁴ Da.
• Moreover, in the presence of washed bottom fraction particles from latex, the rubber transferase activity producing rubber product of high molecular size was increased.
• These results suggest that the Hevea cis-prenyltransferase might require certain activation factors in the washed bottom fraction particles for the production of high molecular mass rubber.
• Keywords: prenyltransferase; rubber transferase; Hevea brasiliensis; isoprenoid.
• Although over 2000 species of higher plants are recognized for producing latex with polyisoprenes [1], only the rubber tree (Hevea brasiliensis) has been established as a key commercial rubber source due to its good yield of rubber and the excellent physical properties of the rubber products.
• Hevea rubber is a high molecular mass polymer of isoprene units in cis-configuration.
• Rubber molecules are produced and aggregated or packaged as rubber particles in latex vessels of the rubber tree [2].
• Although natural rubber is synthesized and made almost entirely of isoprene units derived from isopentenyl diphosphate (IPP), an allylic diphosphate is also required as the priming cosubstrate to initiate the subsequent extensive prenyl chain elongation process for the formation of rubber macromolecules [3-5].
• Synthesis of the allylic prenyl diphosphates are catalyzed by IPP isomerase and trans-prenyltransferase enzymes, the enzymatic activities of which were found in both the bottom fraction and the supernatant cytosol (C-serum) of centrifuged fresh Hevea latex [6-8].
• The enzyme responsible for cis-1,4-polymerization of isoprene units from IPP onto the allylic primer has been identified as a particle-bound rubber transferase (EC 2.5.1.20) [3,9,10].
• The particle-bound rubber transferase activities were demonstrated in various rubber producing plants; guayule [4,5], Ficus elastica [11] and Ficus carica [12].
• It has been shown that IPP is incorporated into rubber at the surface of the rubber particles in latex, by reaction with a terminal allylic diphosphate group of the rubber molecules [13-15].
• This indicates that rubber transferase is bound to the rubber particles and is still present even after the particles are washed repeatedly.
• However, the precise mechanism for the biosynthesis of rubber molecules has not yet been established.
• Moreover, the exact site of the formation of new rubber molecules still remains unknown.
• It has been suggested that the bottom fraction membrane could possibly serve as the site for initiation of new rubber formation [16,17].
• In the biosynthesis of polyisoprenoid compounds, the prenyl chain elongation, catalyzed by prenyltransferases, proceeds consecutively and terminates precisely at certain chain lengths according to the specificities of individual enzymes [18-20].
• These enzymes have been classified into two major groups, referred to as trans- or (E)-prenyl diphosphate synthases (trans-prenyltransferases) and as cis- or (Z)-prenyl diphosphate synthases (cis-prenyltransferases), depending on the stereochemistry of the condensation reaction of IPP with the corresponding allylic prenyl diphosphate initiator.
• During the past 16 years, many different genes encoding trans-prenyltransferases have been cloned and characterized [19,20].
• On the other hand, very limited information was available on cis-prenyltransferases until the recent cloning and characterization of the genes encoding cis-prenyltransferases from Micrococcus luteus B-P 26, Escherichia coli, Haemophilus influenzae, Streptococcus pneumonica, Saccharomyces cerevisiae and Arabidopsis thaliana [21-25].
• Natural rubber has been thought to be made almost entirely of cis-isoprene units derived from IPP, and the enzyme responsible for polymerization is believed to have characteristics similar to the cis-prenyl diphosphate synthases.
• However, the genes encoding Hevea cis-prenyltransferases or rubber transferase (HRT) from H. brasiliensis have not yet been reported.
• It was suggested that rubber biosynthesis in H. brasiliensis is mediated by the association of a soluble trans-prenyltransferase with a rubber elongation factor, a 14.6 kDa protein, tightly bound to the rubber particles in the laticifers [26].
• However, Cornish [10] has demonstrated that the soluble trans-prenyltransferase functions as farnesyl diphosphate synthase, and almost certainly plays no direct role in the cis-1,4-polyisoprene elongation.
• A detailed understanding of rubber biosynthesis processes at the molecular level is important for genetic manipulation of the isoprenoid biosynthesis pathway enzymes.
• In this study, by using the sequence information from the conserved regions of the cis-prenyl chain elongating enzymes [20-22], we isolated and characterized two Hevea cis-prenyltransferases cDNAs designated as HRT1 and HRT2 from the H. brasiliensis latex.
• Sequence analysis showed that all five regions that are conserved among cis-prenyl chain elongating enzymes were present in each of the deduced amino acid sequences encoded by the two cDNAs.
• In vitro assay of the rubber transferase activity of the recombinant HRT proteins was carried out in the presence of washed bottom fraction particles (WBP) of fresh Hevea latex.
• These results suggest that the HRT2 protein catalyzes the synthesis of new rubber molecules with the active involvement of a number of factors in WBP of fresh Hevea latex.
• Materials and methods
• Plant materials and RNA isolation
• Latex and various tissue samples were obtained from ten-year old rubber plants (H. brasiliensis clone RRIM 600) being grown at the Rubber Research Center of Songkla, Thailand.
• Latex collection was performed as described by Kush et al. [27].
• The latex total RNA was extracted by using RNAgents Total RNA Isolation System (Promega).
• Total RNAs of rubber leaf and other tissues were obtained using QuickPrep Total RNA Extraction kit (Amersham Biosciences) and RNA Isolation kit (Qiagen), respectively.
• Poly(A)+ RNA was isolated from the total RNA with Oligodex-dT30 mRNA Purification kit (TaKaRa, Ohtsu, Japan).
• RT-PCR amplification of Hevea cis-prenyltransferase cDNA fragment
• RT-PCR was carried out using Ready-To-GoTM RT-PCR Beads (Amersham Biosciences).
• First strand cDNA synthesis was performed by reverse transcription with 100 ng of poly(A)+ RNA isolated from latex using poly-d(T)12–18 primer.
• Two oligonucleotide degenerate primers were designed to amplify the Hevea cis-prenyltransferase cDNA fragment according to the highly conserved regions among cis-prenyl chain elongating enzymes; sense primer, P1 (AFIMDGN, region I) 5′-GCTTTTATTATGGAYG GGHAA-3′ and antisense primer, P2 (IRTSGE, region V) 5′-CTCACCAGAWGTWCKWAT-3′, where H is A, C or T; K is G or T; W is A or T and Y is C or T.
• PCR was performed in a final volume of 50 µL containing 50 pmol of amplification primer pair for 45 cycles of 30 s at 95 °C, 30 s at 45 °C and 1 min at 72 °C with a 5 min preheat and a 10 min final extension at 72 °C.
• The resulting band of PCR products were extracted from agarose gel and subcloned into pT7Blue T vector (Novagen) for sequencing.
• One of the resulting clones, which showed homology to cis-prenyltransferases, was termed LT600.
• 3′- and 5′-RACE reaction and cloning of Hevea cis-prenyltransferase cDNA
• Cloning of the full-length cDNA of Hevea cis-prenyltransferase, used poly(A)+ RNA from latex as the template for performing both 3′- and 5′-RACE reactions based on the cDNA sequence of LT600.
• The procedures applied for 3′- and 5′-RACE reaction were according to the manufacturers instructions (3′-Full RACE Core Set, TaKaRa; 5′-RACE kit, Roche).
• The primer, F1, used for 3′-RACE was 5′-AGGGCTACTGCCAACAATTCC-3′ and the primers, R1 and R2, used for 5′-RACE reaction were 5′-GCTTCCAGTTGCATTTGCCTCCTCC-3′ and 5′-GCTAAAGGCATAGATAGTCGC-3′ respectively.
• According to the sequence information obtained by the 5′- or 3′-RACE reaction, the cDNA was amplified by RT-PCR with the latex poly(A)+ RNA as the template and then sequenced.
• Finally, two cDNAs were obtained and designated HRT1 and HRT2, respectively.
• DNA sequencing analysis
• Sequencing reactions were performed using Thermo Sequenase Cycle Sequencing kit (Amersham Pharmacia Biotech) with fluorescent labeled primers.
• Nucleotide sequences were determined by the dideoxy chain termination method [28] with a DNA sequencer (LI-COR, model 4200, LI-COR Inc., Lincoln, NE, USA).
• Computer analysis and comparison of DNA sequences were carried out using GENETYX genetic information processing software (Genetyx Corp., Tokyo, Japan).
• For Northern hybridization, total RNAs (15 µg) from various tissues were subjected to electrophoresis on 0.8% agarose gel containing 1% formaldehyde, and blotted onto a positively charged nylon membrane (Roche).
• The membranes were hybridized with 32P-labeled HRT cDNAs for 1h at 68 °C in ExpressHyb solution (Clontech).
• High stringency washes were performed twice at 50 °C in 2 × NaCl/Cit, 0.05% SDS and twice in 0.1 × NaCl/Cit, 0.1% SDS.
• The hybridized membranes were exposed for 12 h on a Fuji imaging plate and then analyzed with a Fuji BAS 1000 Mac Bioimage analyzer.
• RT-PCR for the analysis of HRT expression was performed by using total RNAS (2 µg) from various Hevea tissues, amplified with HRT1 or HRT2 specific primers.
• The PCR reaction was carried out with 25 cycles of programmed temperature control of 30 s at 95 °C, 30 s at 50 °C and 1 min at 72 °C with a 5 min preheat at 95 °C and a 10 min final extension at 72 °C using primers, S1 (5′-GCAAATGCAACTGGA AGCGG-3′) and A1 (5′-ACAGCCTGCTAGCAAAGA GG-3′) for amplification of HRT1, and primers S2 (5′-GAAGAATCCTCTAAGGATAA-3′) and A2 (5′-TA CAAGGATTAATCCCTTGC-3′) for amplification of HRT2.
• The PCR products were analyzed by agarose gel electrophoresis with ethidium bromide staining.
• Construction of expression vector systems in E. coli, and purification of Hevea cis-prenyltransferase
• Expression vector systems for the HRTs were constructed using pET32b(+) vector (Novagen), which is designed to express the gene product as a thioredoxin- and His-tagged fusion protein, suitable for production of soluble protein in E. coli cytoplasm and rapid purification.
• The restriction enzyme recognition site for Ncol or BamHI was introduced by PCR at either the 5′-end or 3′-end of the coding regions of the two HRT cDNAs.
• The resulting fragments were sequenced, digested with NcoI and BamHI, and ligated into the NcoI-BamHI vector of pET32b(+), yielding the expression plasmids pETHRT1 and pETHRT2.
• Each of the expression plasmids was used for transformation of E. coli BL21(DE3), and 1 mL of an overnight culture of the transformant in Luria-Bertani medium containing 50 µg·mL¯¹ ampicillin was inoculated into 200 mL of M9YG medium [29] containing 50 µg-mL¯¹ ampicillin.
• The cells were grown at 37 °C to an A600 value of 0.4.
• Isopropyl thio-ẞ-D-galactoside (IPTG) was added to a final concentration of 0.5 mm, and then a further incubation at 30 °C for 4 h was carried out.
• Overproduction of the proteins was confirmed by SDS/PAGE according to the standard method of Laemmli [30].
• The cells were harvested by centrifugation (5000 g for 10 min) and then disrupted by sonication.
• The cell homogenates were fractionated into soluble and insoluble proteins by centrifugation at 8000 g for 10 min.
• The expressed proteins were purified essentially according to the protocol of Xpress Protein Purification System (Invitrogen), using a Ni²+ nitrilotriacetic acid-agarose column.
• The soluble proteins were applied to a Ni²+ resin column, and the His-tagged fusion protein was eluted with a gradient of 50-500 мм іmidazole in 20 mm phosphate buffer, pH 6.0.
• The insoluble proteins were solubilized with 6 м guanidine hydrochloride lysis buffer, pH 7.8 and subjected to a Ni²+ resin column.
• The column was washed and the tagged protein was eluted with 8 м urea in 20 mm phosphate buffer, pH 4.0.
• The purified protein was renaturated by removal of urea via stepwise dialysis.
• The portion of purified fusion protein was treated with enterokinase to remove the N-terminal fused thioredoxin.
• The digested protein was used for the rubber transferase activity assay and for product analysis.
• Protein concentration was measured by the Bradford method [31].
• In vitro rubber transferase activity assay
• The washed bottom fraction particles (WBP) were prepared by ultracentrifugation (49 000 g, 45 min, 4 °C) of fresh Hevea latex followed by repeated washing of the fresh bottom fraction with 50 мм Tris/HCl buffer, pH 7.4 containing 0.9% NaCl (w/v) according to the method of Wititsuwannakul et al. [17].
• The rubber transferase activity assay was performed by the modified method of Tangpakdee et al. [16].
• The reaction mixture contained, in a final volume of 0.2 mL, 50 мм Tris