Preparation of Polyclonal Antibody and Expression ...

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Wild-type tomato fruits (Lycopersicon esculentum cv Ailsa Craig) was grown in a .... C. Chang, REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene ...
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Physics Procedia 33 (2012) 111 – 117

2012 International Conference on Medical Physics and Biomedical Engineering

Preparation of Polyclonal Antibody and Expression Analysis of GR in Tomato Yuanhong Xie1, Benzhong Zhu2, Yunbo Luo2, Xiangning Chen1, Hongxing Zhang1 1. Beijing Key Laboratory of agricultural product Safety Detection and Control, Department of Food Science, Beijing University of Agriculture, Beijing, China 102206 2. College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China 100083

Abstract The fruit ripening of Green-ripe (Gr) mutant tomato was inhibited dramatically. To determine the expression patterns of Gr in tomato, we first produced the polyclonal antibody of Gr protein. RT-PCR was used to amplify the Gr gene from green ripe tomato fruit. And the PCR product was subcloned into prokaryotic protein expression vectors pET-30a to generate recombinant plasmid. The Gr protein was induced by IPTG in BL21 (DE3) and purified by Ni-NTA agarose column. The anti-Gr serum was produced by immunizing rabbits, and the titer of the anti-Gr serum was above 5000 by ELISA analysis. Purified by the DEAE-52 ion-column, the high purification level of anti-Gr polyclonal antibody was obtained. Furthermore, RT-CPR was used in the RNA level to demonstrate that the expression of Gr gene was specialized in some cultures of tomato. For example, the expressions of Gr were higher in seed, flower and green ripe fruit than others, and the expression level were reduced by exogenous ethylene treatment in the flower and green ripe fruit. Moreover, Polyclonal antibody of Gr was used to investigate the expression pattern of Gr in protein level by the Western blotting. Our results show that the expression level of Gr in protein level was complied with the expressions in RNA. So, we suggested that the regulation of Gr was transcriptional.

©2012 2011Published Published Elsevier Ltd. Selection and/or responsibility of [name organizer] © byby Elsevier B.V. Selection and/or peerpeer-review review underunder responsibility of ICMPBE International Committee. Open access under CC BY-NC-ND license. Keywords: Green-ripe mutant, Prokaryotes expression, Polyclonal antibody, Ethylene

1.

Introduction

Tomato fruit ripening mutant Green-ripe (Gr) is an autosomal dominant mutant, obtained by natural mutation (Kerr. 1958). The fruit of Gr mutant was obviously immature, and the softening and lycopene accumulation of the Gr fruit was significantly inhibited during the fruit ripening (Jarret et al., 1984). Further studies showed that, during the fruit ripening, there was no obvious difference of the ethylene production between the mutant fruit and wild type tomato ethylene and system ethylene were both detected (Barry et al., 2005). It indicated that the fruit, the system ethylene synthetic of the mutant tomato fruit was not affected, and the immaturity was due to the ethylene insensitivity of fruit. In addition, previous study found that the mutant fruit of Gr was obvious ethylene insensitive, exogenous ethylene treatment did not cause obvious ethylene reaction in Gr fruit, and the expression of ethylene relative genes were no change. Also, in the stem, leaf, organization and organs of the Gr mutant, also show different degree of ethylene insensitivity (Barry et al., 2006). And research suggested that Gr gene locate in the chromosome 1 of the tomato, and the length is 732bp, coding protein for 244 amino acid. The molecular weight of Gr protein is about 28KD, isoelectric point is 6.8. Moreover, research indicated that, Gr mutant is cased by the lack of a 334 bp at the 5’ sequence of Gr gene, which led to insensitivity to ethylene (Barry et al., 2006). The purpose of this study was to clone Correspondent Author: Hongxing Zhang, Beijing Key Laboratory of agricultural product Safety Detection and Control, Department of Food Science, Beijing University of Agriculture, Beijing, China, 102206. E-mail: [email protected]

1875-3892 © 2012 Published by Elsevier B.V. Selection and/or peer review under responsibility of ICMPBE International Committee. Open access under CC BY-NC-ND license. doi:10.1016/j.phpro.2012.05.037

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the tomato Gr gene, express the Gr protein in vitro, and prepare the polyclonal antibody of the Gr. Further, we discusses the expression patterns of Gr in both RNA and protein level. 2.

Materials and methods

2.1 Plant materials Wild-type tomato fruits (Lycopersicon esculentum cv Ailsa Craig) was grown in a heated glasshouse using standard cultural practices with regular additions of N, P, K fertilizer and supplementary lighting when required. Flowers were tagged at anthesis, and fruits were harvested at the following stages: green ripe stage, broken stage, and red ripe stage. 2.2 Cloning of Gr gene and construction of prokaryotic expression vector Total RNA was extracted from the tomato fruit (Lycopersicon esculentum cv Ailsa Craig) by the method (Xie et al., 2006), and contaminating DNA was removed using Dnase (Promega) according to the manufacturer’s instructions. First-strand cDNA was synthesized with the Reverse Transcription System Kit (Promega), using equal amounts of oligo(dT), according to the manufacturer’s instructions. Primers was used to amplify the Gr gene , sense primer 5 CGGAATTCATGGCTAGGGCACAAC 3 containing a EcoRI site, antisense primer 5 CCGCTCGAGTCACTGGATTTGGTG with a Xho site. The condition for amplification was at 94 for 3 min followed by 35 cycles at 94 for 30 s, at 57 for 30 s and at 72 for 30 s, plus a final extension at 72 for 10 min. Three independent PCR products were purified and cloned into pGEM T-easy vector (Promega) and sequenced. The PCR product of Gr and the expression vector pET30a were digested both with EcoRI and Xho , and connected with each other by T4 DNA ligase. The restruction was named as pET-Gr. 2.3 Protein expression, purification and polyclonal antibody preparation The recombinant was transformed into Escherichia coli BL21 (DE3) after pET-Gr was selected by restriction analysis. Gr protein expression and purification were performed as previous methods (Zhu et al., 2007) with small modification. The preparation of polyclonal antibody was preformed according to the method (Zhu et al., 2007) and modified. Four New Zealand adult male rabbits were simultaneously immunized with a 200 g mixture of fusion protein and an equal volume of complete Freund’s adjuvant. Rabbits were re-immunized again after 2 weeks with a 100 g mixture of fusion proteins and the same volume of incomplete Freund’s adjuvant. Seven days after the final immunization, the rabbit’s blood serum was harvested from the carotid artery and the polyclonal antibodies purified by salting (between 0.3 and 0.5 saturation with ammonium sulfate). The purified antibody was supplied in phosphate-buffered saline, sterile-filtered, and containing 0.02% sodium azide. 2.4 Transcript expression of Gr gene by RT-PCR Isolation of total RNA from tomato was performed according to Xie et al. (2006). Semi-quantitative RT-PCR was performed as described by Fu et al. (2005). First-strand cDNA was synthesized using 2.5 g total RNA, 0.5 g oligo d(T)18 primer and M-MLV reverse transcriptase (Promage) to a final volume of 20 L. RT-PCR analysis was used to investigate the expression pattern of Gr according to the method (Fu et al, 2007). PCR was performed in 50 L reactions using 1 L cDNA as a template. The specific primers were designed as 5’-CGGAATTCATGGCTAGGGCACAAC 3 5’-CCGCTCGAGTCACTG GATTTGGTG 3’. And 129 bp fragment of endogenous tomato ubiquitin gene Ubi3 was amplified as a control using the primers 5’-CAGGACAAGGAAGGGATT-3’and 5’-GTAGAGCAC GAGGCAGAG-3’. The condition for amplification was at 94 for 3 min followed by 30 cycles at 94 for 30 s, at 53 for 30 s and at 72 for 30 s, plus a final extension at 72 for 10 min. The PCR products were separated on a 1% agarose gel and compared to the amount of Gr transcript. 2.5 Western blotting analysis 5g of Wild type and ethylene treated tomato samples were ground in liquid nitrogen, and solubilized with the protein extracting buffer (400 mmol/L Tris-HCl pH7.5 200 mmol/L NaCl 800 mmol/L sucrose 20 mmol/L EDTA 10 mmol/L DTT 2 mmol/L PMSF 0.1% Tween-20 . All the samples were centrifuged at 13000g for 10min. The suspension was collected as crude extraction for further analysis. Total protein was quantified by the method (Bradford, 1976). The western blotting was preformed. And the polyclonal antibody of Gr was used as primary antibody, and alkaline phosphatase marked IGg was used as detection antibody. The BCIP/NBT was used to detect the result.

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3.

Results

3.1 Construction of prokaryotic expression vector By RT-PCR, the Gr was cloned and sequenced. A 732 bp product was obtained (Fig 1). And by sequencing, we obtained the complete coding region of the Gr. This sequence was completely complied with the original sequence in the NCBI databases (No.DQ372895). Moreover, the Gr was inserted into pET-30a expression vector after digested with EcoRI and Xho . The positive clone of the Gr inserted into pET-30a vector was named as pET-Gr, and identified by digestion (Fig 2). And the recombinant plasmids pET-Gr was transformed into E. coli BL21 cells. 3.2 Protein expression, purification and polyclonal antibody preparation In order to obtain recombinant protein on a large scale in the supernate, expression conditions were optimized using different IPTG concentrations (0.1, 0.5 and 1.0 mM), and no significant difference was observed, all the selected conditions could obviously induced the expression of recombinant protein (Fig 3). The soluble fraction was purified with Ni-NTA agarose column according to the manufacturer’s instructions (Novagen). We obtained the purified Gr recombinant proteins as shown in Figure 4. To prepare the polyclonal antibody of the Gr protein, New Zealand adult male rabbits were immunized. After blood, the titer of the anti-Gr serum was above 5000(Table 1).The polyclonal antibody was firstly precipitated by ammonium sulfate, and then DEAE-52 ion exchange method was used to further purify the polyclonal antibody. As shown in Fig 5, 4-7 tubes were collected and the antibody was dialyzed with deionized water. By western-blotting, the purified Gr polyclonal antibody had immunization specificity and could use for further analysis (date not show). 3.3 The expression patterns of Gr under ethylene treatment To further examine the expression of Gr under ethylene treatment, both RT-PCR and Western-blotting were used. As shown in Fig 6, in different tissues and organs of wild type plant, the expression of Gr gene was not the same. In the tomato seeds, the Gr expression was significantly higher than that in stems, leaves and stems. And in the tomato fruits, the Gr expression in green ripe fruit was significantly higher than that in broken stage and red ripe fruit. When treated with exogenous ethylene, the transcript expressions of Gr had no obviously changed in seed, stem, leave and green ripe fruit of wild type tomato. However, the transcript expressions of Gr in flower and broken stage fruit slightly decreased compared with that in untreated tomato (Fig 6). Further, we also investigated the Gr expression in the protein level using the Gr polyclonal antibody. Western-blotting results demonstrated that the Gr protein was low in stem, leaf and red ripe fruit of the wild type tomato, and could detect slightly. In contrast, Gr protein expression in the seeds and green ripe fruit is much higher than other samples (Fig 7). Overall, the Gr expression in protein level complied with that in RNA level in wild type tomato. When treated with exogenous ethylene, the expression of Gr protein obviously decreased in flower, green ripe fruit and broken stage fruit than control (Fig 7). All these results suggested that exogenous ethylene decreased the expression of Gr in flower, green ripe fruit and broken stage fruit both in RNA and protein level. 4.

Discussion

Tomato is a model plant used to investigate the ripening mechanism of fruit. And the tomato fruit ripening mutants are also important materials. By using these mutants, many ripening related genes were cloned and characterized (Lanahan et al., 1994; Barry et al., 2005). Among these mutants, the Green-ripe (Gr) was also investigated. Previous study indicated that the Gr gene took part in the ethylene pathway of tomato fruit (Barry, et al., 2006). According to homology analysis, Gr gene has a high similarity with the RTE in Arabidopsis, and the homology reached 85%. And studies show that RTE involved in ethylene signal transduction in Arabidopsis. Studied suggested that the RTE negatively controlled the ethylene signal transduction, and over-expression of RTE cased obviously ethylene insensitivity in Arabidopsis (Resnick et al., 2006). Moreover, RTE acted at the upstream of ethylene receptors in the ethylene signal pathway (Barry, et al., 2006). So, the mechanism of fruit ripening controlled by Gr was especially important. Similar as other mutant (Chao et al., 1997; Imanishi et al., 2001; Solano et al., 1998; Tieman et al., 2001) , there was a significantly ethylene insensitivity in flower and fruit of the Gr mutant. However, the ethylene induced triple response of the Gr was similar with wild type plant, which indicated that the ethylene insensitivity of the Gr was tissue specific (Barry, et al., 2005). Barry found that the Northern blotting method could not detect the transcript expression of Gr in wild type tomato, but could only detect the transcript expression in mutant fruit. In the Gr mutant fruit, the Gr expression was highest in green ripe fruit, and exogenous ethylene did not significantly affect the expression of Gr (Barry, et al., 2005). Among these researches, the expression of Gr in protein level was not studied. In this research, we used RT-PCR to investigate the transcript expression of Gr in wild type tomato. Our results suggested that the

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transcript expression of Gr was higher in seeds, flower and green ripe tomato fruit than other tissues (Fig 6). Different from the Gr mutant, exogenous ethylene decreased the Gr expression in flower and green ripe fruit of wild type tomato (Fig 6). To further investigate the Gr expression in protein level, we first expressed the Gr protein in prokaryotic cells (Fig 3), and purified the recombinant protein (Fig 4). By immunized the New Zealand adult male rabbits, the polyclonal antibody of the Gr protein were prepared and purified (Fig 5). Furthermore, western-blotting was preformed to examine the expression of Gr protein. Our results suggested that the Gr protein was higher in seeds, flower, green ripe fruit and broken fruit than other tissues. And exogenous ethylene decreased the Gr protein in flower, green ripe fruit and broken fruit (Fig 7). All these results indicated that the regulation of Gr was transcriptional. 5.

Acknowledgment

The item of Beijing University of Agriculture young science fund provided financial assistance. Reference [1]

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6, no.4, pp 521-530, 1994 [10] J.S. Resnick, C.K. Wen, J.A. Shockey, C. Chang, REVERSION-TO-ETHYLENE SENSITIVITY1, a conserved gene that regulates ethylene receptor function in Arabidopsis, Proc. Natl. Acad. Sci. USA, vol 103, no. 20, pp 7917-7922, 2006 [11] R. Solano, A. Stepanova, Q. Chao, J.R. Ecker, Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSEFACTOR1, Genes Dev, vol 12, no.23, pp 3703–3714, 1998 [12] D.M. Tieman, M.G. Taylor, J.A. Ciardi, H.J. Klee, Members of the tomato LeEIL (EIN3-like) gene family are functionally redundant and regulate ethylene responses throughout plant development, The Plant Journal, vol 26, no.1, pp 47-58, 2001 [13] Y. H. Xie, B. Z. Zhu, X. L. Yang, H.X. Zhang, D.Q. Fu, H.L. Zhu, Y. Shao, Y.C. Li, H.Y. Gao, Y.B. Luo, Delay of postharvest ripening and senescence of tomato fruit through virus-induced LeACS2 gene silencing, Postharvest Biology and Technology, vol 42, no.1, pp 8-15, 2006 [14] H. L. Zhu, B. Z. Zhu, Y. L. Zhang, Y. Shao, X.G. Wang, Y.H. Xie, A.J. Chen, Y.C. Li, H.Q. Tian, Y.B. Luo, Expression of a truncated ripening inhibitor (RIN) protein from tomato and production of an anti-RIN antibody, Biotechnology Letters, vol 29, no.9, pp 1425–1430, 2007 [15] FIGURE:

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M 2000 1000 750 500 250

Gr

100

Figure 1. RT-PCR amplication of Gr DL2000 marker 1 PCR production of Gr M

1

M

PET30a

2000 1000

Gr

750 500 250 100 Figure 2. Identification of recombinant pET-Gr plasmid M DL2000 Marker 1 EcoRI / Xho digestion product

M 98 KD

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32 KD

Gr i

14.4KD

Figure 3. Induced expression of Gr protein in E. coli Marker; 1 control; 2 Induced by 0.1mmol/L IPTG; 3 M

Induced by 0.5mmol/L IPTG

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Induced by 1mmol/L IPTG

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M

1

98 KD

32 KD

Gr protein

14.4 KD

Figure 4. Purification of Gr protein M Marker; 1 Purification of Gr protein Table 1. The titer of the anti-Gr serum Diluted times of Serum OD492 Negative control OD492

1/1000000 0.04

1/100000 0.3

1/5000 1.1

1/1000 1.8

1/100 >2

0

0

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Figure 5. Purification of Gr polyclonal antibody by DAEA-52

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Gr in wild type tomato Ethylene treatment

Ubi Figure 6. RT-PCR analysis of Gr gene in tomato 1: Seed 2: Stem 3: Leaf 4: Flower 5: Green ripe fruit 6: Broken fruit 7: Red ripe fruit.

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Gr in wild type tomato Ethylene treatment Figure 7. Western blotting analysis of the Gr protein in tomato 1: Seed 2: Stem 3: Leaf 4: Flower 5: Green ripe fruit 6: Broken fruit 7: Red ripe fruit.

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