Overexpression of chitinase gene with a GC-rich synthetic enhancer in tobacco plant ( Nicotiana tabacum L . )

Perakitan tanaman perkebunan toleran terhadap serangan cendawan patogenik dilakukan dengan mengoverekspresikan gen penyandi kitinase. Untuk itu elemen DNA peningkat ekspresi (enhancer E52) yang berupa oligonukleotida 52 pb dan kaya kandungan basa purin (GC) disisipkan di ujung 5’ konstruk 35S-chi. Penyisipan E52 tersebut dilakukan secara lebih terarah pada situs ganda HindIII-SalI dari MCS pCAMBIA2301. Melalui situs HindIII yang terletak tepat di ujung 3’ E52, konstruk 35S-chi kemudian disambungkan dengan E52 pada pCAMBIA tersebut. Transformasi DNA rekombinan ke dalam sel tembakau dikerjakan melalui perantaraan Agrobacterium tumefaciens LBA4404. Sel tanaman transgenik diseleksi dan diregenrasi dalam media yang mengandung 3% sukrosa, 0,5 mg/L diregenerasikan pada media MS padat benzilaminopurin (BAP) dan 50 mg/L kanamisin. Pada media ini tunas transgenik tembakau mulai terbentuk setelah 5 minggu penanaman. Analisis tingkat aktivitas enzimatis menunjukkan bahwa aktivitas kitinase pada tembakau transgenik 40 hingga 80 kali lebih tinggi daripada non-transgenik. Pengujian hibridisasi protein menggunakan antibodi anti-kitinase, dot blot dan western blot, membuktikan bahwa enhancer tersebut dapat meningkatkan ekspresi transgen chi pada tanaman tembakau. Summary


Summary
Development of estate crops tolerant to pathogenic fungi is conducted by overexpressing chi gene.For this purpose, a synthetic enhancer consisting of 52 base pairs and GC-rich was inserted at immediate 5' end of a 35S-chi cassette.Insertion of the E52 was directed at HindIII-SalI restriction sites of the pCAMBIA2301 MCS.
With HindIII restriction site located just after the 3' end of the E52 sequence, the 35S-chi construct was then ligated with the E52 of the pCAMBIA.Transformation of the resulting recombinant DNA into tobacco cells was mediated by Agrobacterium tumefaciens LBA4404.The transgenic cells were selected and regenerated on a solid MS medium supplemented with 3% sucrose, 0.5 mg/L benzylamino purine (BAP) and 50 mg/L kanamycin.Tobacco shoots were initiated after 5 weeks inoculation on the selection media.Enzymatic analysis demonstrated that chitinase activity of transgenic tobacco was 40 to 80 folds higher than that of the control plant.Analysis of enzymatic activity using hybridization with anti-chitinase antibody indicated that the level of chitinase activity in the transgenic tobacco carrying the enhancer is higher than that without enhancer.These data suggest that the enhancer improved the expression of chi transgene in tobacco.
Key words: Gene expression, recombinant DNA, transgenic tobacco 

Introduction
An attempt to develop estate crops tolerant to fungal deseases has led to utilization of chi gene.This gene encodes chitinase, an enzyme capable of degrading chitin which is a cell wall constituent of most pathogenic fungi.Genetic engineering of plant species using chi gene for resistance to pathogenic fungi was reported on rice (Lin et al., 1995), tobacco and canola (Anonim, 2000).With similar approach, estate crops tolerant to fungal diseases such as Ganoderma sp. in oil palm, and Hemilia vastatrix of Arabica coffee should be regenerated with chitinase transgene.
The resistance of plant to pathogenic fungus is determined by the effectiveness of the enzyme in degrading the fungal chitin.In addition to the specificity, the enzymatic activity is also affected by the amount of the protein expressed in the plant.The more chitinase protein present in the plant, the more resistant the plant to the pathogenic fungi.Therefore plants possessing the chitinase transgene are not always tolerant to the pathogenic fungus due to low expression of the transgene or low activity of the enzyme (Lin et al., 1995;Siswanto, 1999).
DNA fragments having a high content of purin bases (GC) and specific motives, at some degree capable of enhancing gene expression in eukaryotic cells through interaction with transacting element (Kay et al.,1987;Balcells & Coupland, 1994;Wilde et al., 1994).This type of interaction is required for the cells to form initiation complex between proteins of transcription factors and the DNA promoter.With this complex, RNA polymerase binds to the DNA and starts the transcription process at the start site (Santoso, 1995).Involvement of regulatory DNA to enhance such gene expression was also reported currently on flowering stage of Arabidopsis (Deholos & Sieburth, 2000).
This research is aimed to construct recombinant DNA carrying chi under a synthetic enhancer and examine the expression of the transgene in tobacco plant.The enhancer is a 52bp oligonucleotide and GC rich.The expression was assayed at RNA level with Northern dot blot analysis using DIG-labeled DNA probe and at protein level using immunoassay.

Materials and Methods
Cloning of plasmid DNA was performed with Escherichia coli JM109 of Promega (Madison, WI).Gene construct of 35S-chi in pBS-G11 was obtained from Dr. Swapan Datta at IRRI, The Philippines.Plasmid vector of pCAMBIA2301 was kindly provided by Dr. R.A. Jefferson of CAMBIA, Australia.Restriction endonucleases, alkaline phosphatase and ligase were from Promega.Kits for plasmid preparation of Quantum MiniPrep and Prep-A-Gene were from BioRad (Hercules, CA), Acetosyringone and other reagents were from Sigma Chem.Co. (St Louis, MO) or Gibco BRL (Gaitherburg, MD).

Construction of recombinant plasmid
Recombinant DNA carrying 35S-chi construct under the 52-bp enhancer was made through two steps.First, insertion of the enhancer (E52) in between HindIII and SalI sites at the MCS of pCAMBIA2301.Confirmation for the right construct was conducted using the restriction enzymes flanked the E52 fragment followed by electrophoresis with high concentration of agarose gel (Santoso & Haqqi, 1998).Functionality of the E52 was previously tested by examining the expression of gus in transgenic tobacco (Chaidamsari & Santoso, 1999).The second step was insertion of 35S-chi that was done by ligating the 35S-chi fragment excised from the pBS-G11 using HindIII, with HindIII-cleavaged PCAMBIA 2301/E52 recombinant.The 35S-chi fragment was purified from the 1.5-kb preparative gel slice using Prep-A-Gene kit.To improve efficiency of ligation, the HindIII-digested pCAMBIA was dephosphorylized using calf intestine alkaline phosphatase (CIAP).After ligation at 4 o C for overnight, the recombinant DNA was then transferred into JM109 competent cells, which were prepared following the method of Schnable (1991).Afterward, the transformation products

Overexpression of chitinase gene under a GC-rich synthetic…
were plated on selection media containing kanamycin 50 mg/L.
To screen plasmid-carrying bacterial clones, plasmid DNA was prepared from selected colonies using Speedy method (Schnable, 1991) then electrophorezed using 0.7% agarose gel.To confirm the identity for true recombinants harbored in the selected bacterial clones, plasmid prepared with Quantum MiniPrep kit was examined for its map of restriction sites.Restriction enzymes of SphI or PstI were used to confirm the presence of E52, HindIII for the presence of the 35S-chi insert and BamHI to determine the orientation of the DNA fragments.
The presence of chitinase gene in the DNA fragment was reconfirmed with Southern blotting using non radioisotop-labeled probe of DIG.The probe was synthesized with linearized 35-chi DNA template.The transfer of DNA from the agarose gel to the nylon membrane was perfomed based on capillary force as described in Sambrook et al. (1989).Procedures for hybridization, detection of hybridized DNA with antibody and staining were accomplished as recommended in the user manual.Specvific DNA bands detected on the membrane were recorded.

Plant genetic transformation and regeneration
Genetic transformation of tobacco plant of local variety was mediated by Agrobacterium LBA4404 with a slightly modified method from Sain et al. (1994).Acetosyringone at final concentration of 100 mg/L was added at the steps of inoculation and co-cultivation.After cocultivation, the leaf explants were rinsed with MS liquid media containing 500 mg/L cefotaxime (Murashige & Skoog, 1962) to remove Agrobacterium stuck on the leaf surface.The clean transformed explants were then inoculated on MS solid media containing 500 mg/L cetofaxime, 100 mg/L kanamycin, 0.5 mg/L BAP and 3% sucrose.Planlets with 1-2 pair of leaves were cut at the bottom of the stem and transferred onto the MS solid media with no hormone to induce roots.The planlets were subcultured every 8-10 weeks.The culture for plantlet regeneration was incubated at 25 o C under a 12-h photoperiod provided by a bank of 40-W cool white fluorescent tubes.

Chitinase assay
Procedure to quantitatively determine the activity of chitinase was previously described by Siswanto (1999).Enzymatic extract containing 0.3-0.9mg protein was added with 1 mM substrate of p-nitrophenyl -D-glucosamine and phosphate buffer 50 mM pH 6.5 up to total volume of 250 L.The reaction mixture was incubated at 37C for 0 and 15 minutes.After stopping the reaction with 125 L TCA 20% and centrifugation 12.000 g for 10 minutes, 0.3 mL of the supernatant was added with 0.7 mL NaOH 0.5 M, and absorbance was measured at  405 nm for 30 minutes.Chitinase activity was calculated with a standard curve based on the concentration of pure p-nitrophenyl phosphate (pNP).

Preparation of antibody
Preparative antigen containing 0.6 mg protein/mL was emulsified with complete Freud's adjuvant.The immunogene was then injected intramuscularly to rabbit (Young New Zealand Albino).After 14 days the rabbit was bled for 1 mL serum.The procedure was adopted from Dunbar & Schwoebel (1990).The harvested antiserum was stored in deep freezer until ELISA test.
For ELISA, antigen was 10-1000X diluted with carbonate buffer pH 9.6 then pipeted into micro-well plates and incubated at 4C for overnight.After 3x washing with PBS-Tween 0.05 % and drying for 5 minute, the wells was added with 50 L Ab, incubated for 1.5 hours at room temperature with slow shaking, then washed.Afterward 50 L antirabbits IgG conjugate peroxidase was added into the wells, incubated for 1.5 hours with slow shaking, then washed.After added with 100 L 2,2'-azino-bis(3-ethylbenzthia-zoline-6-sulfonic acid), the mixtures were shaken at room Santoso et al.
temperature for color development.The absorbancy was measured at  405 and 492 nm.

Immunology analyses of proteins
Procedure for dot blot analysis was modified from Timmons & Dunbar (1990).Diluted Ag was spotted onto nitrocellulose membrane.After drying, the membrane was immersed into TEN-TC buffer and incubated with slow shaking for 30 minute then with diluted Ab for 2 hours.After 3x washed with PBS Tween, the membrane was incubated with anti-rabbits IgG peroxidase conjugate for 1.5 hours.After washing, the membrane was stained by incubation with 3,3'-diaminobenzidine tetrahydrocholride (DAB) until color developed, then washed with aquadest.For Western blot analysis, the experimental protocol was basically similar, except the protein bands were transferred from SDS-PAGE gel onto nitrocellulose membrane.

Northern dot blot
A total RNA was isolated from tobacco leaves using miniscale method slightly modified from Wadsworth et al. (1988).Solution of total RNA was spotted slowly onto nylon membrane drop by drop.After air-drying, the RNA on membrane was fixed by baking at 80 o C for 1-2 hours.Hybridization using DIG-labeled probe was conducted as described in instruction manual.The DIG-labeled probe was prepared using DNA template of chi gene excised from pBS-G11.The detail of the protocol was also described in the manual.

Results and Discussion
Ligations of the DNA fragments of E52, 35S-chi gene, and linearized vector provide several possible recombinants.To select the right recombinant, their restriction site map was examined.For this purpose, plasmid DNAs was prepared from three bacterial clones using QuantumPrep kit, then digested with three restriction enzymes SphI, BamHI and HindIII.Electrophoresis of the digested plasmids showed varying DNA banding pattern (Figure 1).Digestion with SphI of clone 1, clone 2 and clone 3 resulted in dominant DNA bands of 6.7, 6.7 & 5.0, and 8.2 & 7.8 kb respectively.Digestion of the clones with BamHI provided DNA bands of >11.6, 11.6, and 11.6 & 1.2 kb.Whereas bands from digestion with HindIII respectively were 11.6 & 1.4, 11.6, and 11.6 and 1.4 b.The 1.4 kb-DNA of clone 3 with HindIII is more intense than that of the others.The DNA fragments sizing of 8.2 kb of the SphI digestion, 1.2 kb of the BamHI and 1.4 kb of the HindIII were likely those carrying the chitinase gene which is 1.1 kb (Lin et al., 1995).
Analysis of the DNA banding patterns suggested that the tested three recombinant plasmid clones possess different restriction maps or characteristics (Figure 2).Clones 1 and 3 could be recombinant of pCAMBIA-chi.Whereas clone 2 is pCAMBIA-E52 without 35S-chi construct.Based on the result from BamHI digestion, it is assumed that the orientation of 35S-chi in clone 3 is in opposite orientation of clone 1.In the clone 3, 35S promoter of 35S-chi located across the E52, whereas in clone 1 the E52 resides just next to the 35S promoter.
Recombinant plasmids confirmed to harbor E52 and 35S-chi were then transferred into explant slices of tobacco via Agrobacterium LBA4404.
Selection regeneration on the kanamycin-containing media supplemented with 0.5 mg/L BAP resulted in plantlets able to growth on the selection media (Table 1).Shoot initiation from the transformed explants on the selection media was seem slower than from untransformed control explants on the media without kanamycin.Green spots representing shoot initiation were observable after about 6 weeks.Furthermore the number of regenerated transgenic planlets from transformed explants was less than that from the control regeneration.Whereas untransformed explants which had been cultured for 6 weeks on the selection media regenerated no shoot.Note: *) N/A = not applied because the explant died Table 2. Activities of chitinase in the leaves of transgenic tobacco, assayed using two types of chromogenic substrates The plant cells growing on the selection media containing lethal dose of kanamycin, must theoretically be the cells bearing and expressing the transgenes in between the T-DNA borders, which includes NPTII gene encoding the kanamycin resistance.Expression of the NPTII gene indicated by the ability of the transformed cells to grow and regenerate on the selection media.To examine the expression of the disease resistance chi gene, enzymatic assays were done on the activity of chitinase.The axtivity of chitinase in transgenic planlets were 40-80 fold higher than that of the nontransgenic planlets (Table 2).With double 35S cCMV promoter, chi transgene from Trichoderma viride was expressed in plant Vitis vinifera at varying levels 10-100 fold higher than chitinase activity in the untransformed control plant (Kikkert et al., 1998).This concludes that the transgene of the rice chi was integrated into the chromosomal DNA Overexpression of chitinase gene under a GC-rich synthetic … and expressed properly on the transgenic tobacco planlet.
The increase of chitinase activities in the transgenic tobacco was likely the consequence of increase amount of chitinase protein expressed from the chi transgene.To prove this pressumption, the amount of the protein was measured with protein dot blot using anti-lane chitinase antibody.The result of the blotting analysis demonstrated that the intensities of the spots from transgenic tobacco transformed with E52-35S-chi was the highest, followed by 35Schi and no colored spot from non-transgenic plantlet.The data suggested that expression of chi transgene construct with E52 was the highest, meaning that the DNA sequence E52 enhanced the expression of the chi constructed under its influence.The expression was also confirmed with Western blot analysis ( the right panel of Figure 3).More color on the representing expression improved by E52 enhancer.This enhancement function of E52 DNA sequence was also reported in our previous publication for reporter gene of gus (Chaidamsari & Santoso, 1999).The recombinant DNA used in the previous study harboring the enhancer at position about a half kb away from the 5' end of the 35S promoter driving the expression of the reporter gene.These experimental data collected so far indicate that E52 enhances gene expression in a fashion of a bit position independent.This is in agreement with general rule for DNA enhancer of gene expression.In monocotyledon, a regulatory sequence shown to modulating high gene expression was characterized as a GC-rich regulatory sequence (Zhu, 1995).
. Plants including tobacco possess chitinase activity with metabolic function obviously similar to those of most species.At molecular level, protein hybridization approach has degree of less specificity.To reconfirm the overproduction of chitinase activities as well as proteins were merely as the result of chitinase transgene expression, a simplified Northern blot was conducted to examine the expression of the gene at RNA level.The result of RNA dot blot analysis is presented in Figure 4.The presence of color dots on some spots representing transgenic tobacco indicates that the transgene chi was expressed in the transgenic plantletsPlants including tobacco possess chitinase activity with metabolic function obviously similar to those of most species.At molecular level, protein hybridization approach has degree of less specificity.To reconfirm the overproduction of chitinase activities as well as proteins were merely as the result of chitinase transgene expression, a simplified Northern blot was conducted to examine the expression of the gene at RNA level.The result of RNA dot blot analysis is presented in Figure 4.The presence of color dots on some spots representing transgenic tobacco indicates that the transgene chi was expressed in the transgenic plantletsLess intensity of color is expected in non-radioisotope-label probe.Since the spotted samples were solutions of about one μg total RNA prepared miniscally from tobacco plantlets, it is reasonable that the spot color is not as strong as if 32 P labeled probes were used (Santoso & Thornburg, 1998).Beside, total RNA is dominated by population of rRNA, whereas the mRNA is usually less than 1% of the total RNA.Furthermore chitinase mRNA maybe only one over thousands of mRNA population in the total RNA solution.Nevertheless, the chitinase transcript was still detectable confirming that the chitinase transgene was really overexpressed in the transgenic tobacco harboring E53-35S-chi construct.
Regulation of gene expression can occur at several different points.In eukaryotic cells, it is mostly at transcription process and sometime at translation step (Anonim, 2001).
Examinations at enzymatic activity, protein and transcript levels suggest that the regulation of the transgene chi under 35S promoter and E52 enhancer does not seemingly rule out the common mechanism.Like those involved in the metabolism of pyrimidine nucleotide (Santoso & Thornburg, 1998), expression regulation of such fungal resistance transgene also occurs at transcription level.A current report on Arabidopsis also indicated the presence of regulation at transcription process particularly at initiation of transcription for expression of genes in the process of cell differention (DellaPenna, 2001).

Conclusion
1. Transformation of 35S-chi constructs into tobacco explants via Agrobacterium LBA4404 carrying pCAMBIA/35S-chi with or without E52 resulted in plantlets with increased chitinase activities.2. DNA cis-acting element consists of 52 bp and GC rich was able to enhance the expression of chi transgene in tobacco plants.

Table 1 .
Selection of 35S-chi carrying cells and the regeneration for the tobacco transgenic plantlets on the selection media. E