Production and characterization of recombinant human lactoferrin in transgenic Javanica rice cv Rojolele
1 Gene Research Center Ibaraki University, Japan www.grc15.agr.ibaraki.ac.jp Email firstname.lastname@example.org
2 Faculty of Biology, Gadjah Mada University, Indonesia. www.biology.ugm.ac.id Email email@example.com
3 National Institute Agrobiological Science, Japan www.nias.affrc.go.jp Email firstname.lastname@example.org
We have introduced two different constructs containing either the native signal peptide from human lactoferrin (pIG211) or the signal peptide from rice glutelin (pIG200) fused to mature human lactoferrin into Javanica rice cv Rojolele by an Agrobacterium-mediated transformation system. The expression of the recombinant human lactoferrin (rhLF) under the control of the maize ubiquitin1 promoter was detected in all tissues of transgenic plants. We found the transgenic rice produced considerable amount of rhLF in its seeds. The rice rhLF showed a molecular weight slightly smaller than the native hLF. The rhLF was purified from mature seeds by cation-exchange chromatography and were analysed by the Edman degradation procedure for N-terminal sequencing. The N-terminal sequences of purified rhLF from rice seeds were identical to native LF from human milk for both constructs. Immunofluorescence microscopy revealed that the rhLF was localized in the intracellular endosperm and was abundantly accumulated in the outer portion of the endosperm.
A transgenic Javanica rice cv Rojolele with high expression level of human lactoferrin, an iron rich protein, was generated by Agrobacterium- mediated transformation system.
Recombinant human lactoferrin, Javanica rice, Rojolele, Agrobacterium-mediated transformation
Being one of the most important food crops in the world, rice has received considerable attention in genetic engineering research. The application of biotechnology to rice is potential for increasing rice production and improving their nutritional quality. Rojolele is a famous of Javanica rice variety that is commercially cultivated in Indonesia. Rojolele has become a popular local rice variety in Central Java (Silitonga and Soetjipto, 1994). It is high-quality rice that has long slender grains, intermediate amylose content, an intermediate gelatinization temperature, a high elongation ratio, and is strongly aromatic (Graham, 2002).
Human lactoferrin is an 80 kDa iron-binding glycoprotein that has been proposed to have many biological roles such as facilitation of iron regulation, protection against microbial and virus infection, stimulation of the immune system and cellular growth promotion. Recombinant hLF has been already expressed in tobacco (Nicotiana tabacum L. cv Bright Yellow) cell culture (Mitra and Zhang, 1994), tobacco plants (Salmon et al., 1998) and potato (Solanum tuberosum) plants (Chong and Langridge, 2000). Lactoferrin has also been expressed in rice obtained through particle bombardment (Anzai et al., 2000; Nandi et al., 2002).
Previously, we have established an efficient Agrobacterium-mediated transformation system for Javanica rice cv Rojolele (Rachmawati et al., 103rd Meeting of the Japanese Society of Breeding, 2003). In this study, we describe the transgenic rice expressing rhLF using two different signal peptides under the control of the constitutive promoter ubiquitin1 from maize through Agrobacterium-mediated transformation system. Characterization of transgenic plants includes rhLF expression, Southern blot analysis, N-terminal amino acid sequencing and immunolocalization of rhLF in transgenic rice seeds.
An hLF structural gene and its signal peptide from a plant expression vector pAFT105 (Anzai et al., 2000) were excised using BamHI and SacI. The isolated fragment was used to replace the gus coding region from pAFT14 that had been pre-digested with BamHI and SacI. The resulting plasmid was called pIG211. Whereas for pIG200, the human lactoferrin signal peptide of pIG211 was replaced by the rice glutelin signal peptide. The expression of human lactoferrin transgene was driven by the constitutive ubiquitin1 promoter of Zea mays and under the control of nopaline synthase terminator. Hygromycin phoshotransferase gene was used as a selectable marker gene. The schematic of the binary plasmid pIG200 and pIG211 are shown in Figure 1.
Figure 1. Construction of a binary vector containing mature hLF for rice transformation. (A) pIG200 and (B) pIG211. Gt: Glutelin signal peptide; hLF: human lactoferrin; Ubi: Ubiquitin1 promoter; hpt: hygromycin phosphotransferase.
Scutellum-derived calli from mature seeds of Javanica rice cv Rojolele were transformed with Agrobacterium tumefaciens strain EHA101 that carried either pIG200 or pIG211. Transgenic plants were grown to maturity and seed set in a phytotron.
The transgenic calli, roots and leaves tissues were ground in liquid nitrogen and the soluble proteins were extracted in an extraction buffer [50 mM Tris-HCl (pH 6.5), 1 mM EDTA, 100 mM NaCl, 0.1% Triton X-100]. Individual T1 seeds were cut into halves. The endospermic half was subjected to the rhLF expression analysis and the corresponding positive embryonic half was germinated to generate T1 seedlings. The expression of the rhLF was determined by enzyme-linked immunosorbance assay (ELISA) and Western blot analysis. Extracted proteins from transgenic plants were separated on 10% SDS-PAGE and Western analysis using rabbit anti-hLF antiserum was carried out by the method of Anzai et al., (1996).
Genomic DNA was isolated from mature leaves following the method of Byeong-Ha Lee, University of Arizona (bioProtocol™). For Southern blot analysis, 3 μg of genomic DNA was digested with BamHI. The Gene Images AlkPhos Direct labelling and detection system (Amersham Biosciences) and chemiluminescent detection with CDP-star was used to detect transgenes.
The recombinant human lactoferrin produced in transgenic plants was purified from mature seeds by cation-exchange chromatography with a linear salt gradient from 0 - 1 M NaCl using RESOURCE™S column (Amersham Biosciences). The purified rhLF was separated by 10% SDS-PAGE and electroblotted to a PVDF membrane. The blot was stained with Coomassie Brilliant Blue R-250 (BIORAD) for 1 h and immediately destained in 40% methanol until the rhLF band was clearly visible. The band containing rhLF was excised for N-terminal sequencing by the Edman degradation procedure using a PPSQ-21 protein sequencer (Shimadzu, Kyoto, Japan).
In order to determine the distribution of the rhLF in transgenic rice seed, we examined its localization by in-situ western hybridization and immunofluorescence microscopy. In-situ western hybridization was carried out as the procedures described by Qu et al., (2003). For immunofluorescence studies, immature rice seeds were harvested at 14 days after pollination (DAP). Paraffin-embedded sections at 8-10 μm thickness were reacted with rabbit anti-hLF antiserum primary antibody and secondary anti-rabbit IgG antibody conjugated with FITC. Immunofluorescence detection was performed using a laser scanning confocal microscopy.
Recombinant hLF content was mg/g seed and % of Total Soluble Protein (TSP).
Agrobacterium-mediated transformation of Javanica rice cv. Rojolele with a human lactoferrin cDNA under the control of the maize ubiquitin1 promoter leads to production of recombinant hLF in transgenic plants. The growth of transgenic plants was found to be similar to that of non-transgenic rice plants. A high expression level of rhLF was detected in seed. The rhLF was also expressed in other tissues such as callus, root and leaf but the expression was low (Figure 2A and 2B). The rice rhLF showed a molecular weight slightly smaller than the native hLF (Figure 2C and 2D). We suspect that the size difference between the recombinant hLF from rice and the native hLF from milk might be due to modification of the sugar chain.
Figure 2. SDS-PAGE (A) and Western blot analysis (B) of rhLF in T1 generation. M. Marker (1) native hLF (2) callus (3) root (4) leaf (5) mature seed and (6) 4-days after seeding. Purified rhLF from transgenic rice seeds were analyzed by SDS-PAGE (C) and Western blot analysis (D). M: Marker (1) native hLF (2) IG200R-10 and (3) IG211R-22.
In the ELISA, there was a large variation in rhLF expression level among independent transgenic lines. The expression of the rhLF with the rice glutelin signal peptide reached 2 mg/g seed, while the expression of the rhLF with hLF signal peptide was 1.6 mg/g seed. The expression level of rhLF was up to 15% of total soluble protein (TSP). Based on the expression analysis, selected T1 lines with high expression levels of rhLF were advanced to the T1 generation. Southern analysis was carried out to determine the transgene copy number in the transgenic rice plants. The copy number of transgene varied from one to three copies and was stably integrated in the transgenic rice genome (data not shown).
N-terminal sequences of purified recombinant hLF from rice seeds were determined as GRRRRSVQW, which is identical to native LF from human milk for both constructs. It indicated that hLF molecules are correctly processed independently of whether the native hLF signal peptide or the glutelin signal peptide is used in the recombinant construct. This result suggests that processing of the amino acid sequence in plant cells was identical to that in mammalian cells.
In situ Western hybridization revealed the presence of recombinant hLF in the transgenic rice seeds as indicated by the blue color staining both in the aleurone layer and the endosperm (Figure 3a-b). Meanwhile, non-transgenic rice seed did not show any detectable color reaction. The result demonstrated the clear difference between the non-transgenic (control) and transgenic rice seeds.
For immunofluorescence microscopy, immature seeds of non-transgenic and transgenic rice seed were tested with anti-hLF antibody. The result showed that no immunofluorescence signal was detected in non-transgenic seed (Figure 3c), while strong immunofluorescence signals of recombinant hLF were detected in the intracellular region of the endosperm (Figure 3f). We used Propidium iodide (PI) as a counterstain by binding to DNA showing a red fluorescence (Figure 3d, g). Merging the two separately recorded images (Figure 3e, h) visualized green fluorescence for rhLF and a yellow colour signal for nucleus. In addition, the immunofluorescence signal of the rhLF was expressed more strongly in the outer portion of the endosperm.
Figure 3. In-situ western hybridization of rhLF from a median-longitudinal (a) and transversal section (b) in transgenic (left) and non-transgenic rice seed (right). Immunofluorescence localization of rhLF in the endosperm of non-transgenic (c-e) and transgenic Javanica rice cv Rojolele (f-h). Endosperm was incubated with antibody against hLF. Images were obtained with appropriate wavelength. c, f: FITC (488 nm); d, g : PI (543 nm); e: merged Images of c and d; h : merged images of f and g. Bar = 25 μm.
We reported here the expression and characteristic of rhLF in the transgenic Javanica rice cv Rojolele obtained through Agrobacterium-mediated transformation. Transgenic rice produced considerable amount of the recombinant hLF in its seed. The efficiency of glutelin signal peptide and hLF signal peptide in the rhLF expression is almost same. Although the exact mechanism is unknown, the expression of rhLF was localized into the intracellular of endosperm.
Our research was supported by a grant of a Research and Development Program New Bio-industry Initiatives of the Bio-oriented Technology Research Advancement Institution (BRAIN).
Anzai H, Ishii Y, Shichinohe M, Katsumata K, Nojiri C, Morikawa H, and Tanaka M (1996). Transformation of Phalaenopsis by particle bombardment. Plant Cell Mol Biol Lett 13, 265-272.
Anzai H, Takaiwa F, and Katsumata K (2000). Production of human lactoferrin in transgenic plants. Proceedings of the 4th Interntional Conference on Lactoferrin, Sapporo, Japan.
Chong DK and Langridge WH (2000). Expression of full-length bioactive antimicrobial human lactoferrin in potato plants. Transgenic Res. 9, 71-78.
Graham R (2002). A proposal for IRRI to establish a grain quality and nutrition research center. IRRI Discussion Paper Series No.44. Los Banos (Philippines): International Rice Research Institute. 15p.
Mitra A and Zhang Z (1994). Expression of human lactoferrin cDNA in tobacco cell produces antibacterial protein(s). Plant Physiol.106, 977-981.
Nandi S, Suzuki YA, Huang J, Yalda D, Pham P, Wu L, Bartley G, Huang N, and Lonnerdal B (2002). Expression of human lactoferrin in transgenic rice grains for the application in infant formula. Plant Sci. 163, 713-722.
Qu LQ, Tada Y, and Takaiwa F. (2003). In situ western hybridisation: a new, highly sensitive technique to detect foreign and endogenous protein distribution in rice seeds. Plant Cell Rep. 22, 282-285.
Salmon V, Legrand D, Slomianny MC, ElYazidi I, Spik G, Gruber V, Bournat P, Olagnier B, Mison D, Theisen M, and Merot B. (1998). Production of human lactoferrin in transgenic tobacco plants. Protein Exp. Purif. 13, 127-135.
Silitonga TS and Soetjipto K. (1994). Rice germplasm exploration, collection, conservation and evaluation in Indonesia. Report of an Action Plan Meeting: Safeguarding and preservation of the biodiversity of the rice genepool. Los Banos (Philippines): International Rice Research Institute. pp 32-35.