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Gene expression of lactoferrin-derived antimicrobial peptides in rice

Taku Funakoshi, Takehiko Hosaka, Eiichi Inoue and Hiroyuki Anzai

Gene Research Center, Ibaraki University, grc15.agr.ibaraki.ac.jp/ Email anzai@mx.ibaraki.ac.jp

Abstract

LactoferricinH (LFcinH) derived from human lactoferrin (hLF) is an antimicrobial peptide against a broad spectrum of microbes. Transgenic rice plants (Oryza sativa L. cv. Nipponbare) overexpressing a LFcinH analogue were generated by an Agrobacterium tumefaciens-mediated transformation system. In this experiment, antimicrobial activity of a heat stable LFcinH analogue was detected in the crude extracts from calli before regeneration and in leaves of individual T0 transformants using several in vitro assays against gram positive and negative bacteria. The 7.5 kDa peptide, corresponding to the processed form of LFcinH analogue, was detected by western blot analysis. The thermostability of LFcinH analogue derived from transgenic rice plants was maintained.

Media summary

Gene expression and functional analysis of mammalian antimicrobial peptides in rice plant.

Key Words

Transgenic rice/ Antimicrobial peptide/ LactoferricinH/ Gene expression/ Agrobacterium tumefaciens

Introduction

Lactoferrin (LF) is an approximately 80kDa iron-binding glycoprotein present in breast milk, saliva, tears, and mucous secretions of mammals (Masson 1966). LF was identified to have multi physiological function, for example iron sequestering (Brodie 1994; Singh 2002), immunomodulation (Lash 1983), and antimicrobial activity (Ellison 1994; Leitch EC and Willcox 1999; Arnold 1981). Lactoferricin (LFcin), bactericidal domain of LF, was isolated by gastric pepsin cleavage of LF (Bellamy 1992).A derivative of the human LF (LFcinH) has ten times stronger antimicrobial activity than the native protein against broad spectrum of microbes in vitro and in vivo.

Mechanisms of interaction between antimicrobial peptides such as magainin, defensin or LFcin and microbes are gradually being revealed. Conventional antibiotics such as penicillin would generate resistant microbe species. Because these antimicrobial peptides widely take effect and suppress against regenerating antibiotic-resistant microbes, these peptides could be anticipated to improve severe clinical situations such as nosocomial infection (Zasloff 2002).

Recently, genetic engineering has been developed as an alternative way to create new crops which can resist plant disease pathogens and produce vaccines or medicines. Many antimicrobial peptides against several plant or animal pathogens have been identified. In some plant species, transgenic plants overexpressing foreign antimicrobial peptides actually acquired resistant to pathogens.

Here we report gene expression of the lactoferricin gene in transgenic Japonica rice plant (Oryza sativa L. cv. Nipponbare) and functional analysis of sequence modified LFcinH analogues . We confirmed that LFcinH analogues have strong antimicrobial activity and thermostability using an agar-well expansion assay and a microtiter plate assay.

Methods

Construction of binary plasmids and transformation of rice plant

The binary plasmids pIG260 and pIG261, containing the 276 bp and 252 bp coding region of the LFcinH analogues fused with rice glutelin and hLF signal peptide sequence and driven by the maize ubiquitin1 promoter (Christensen 1992). The selectable marker was the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter. These were transformed into Agrobacterium tumefaciens strain EHA105. The transformation of rice plant (Oryza sativa L. cv. Nipponbare) was carried out according to optimized methods (Toki 1997). Hygromycin (50mg/L) was used for selection of transformants during in vitro culture. After acclimatization, transformants were grown in soil in a greenhouse.

Protein electrophoresis and western blot analysis

To carry out western blot analysis, total protein was extracted from T0 callus and leaf by grinding in protein extraction buffer (50 mM Tris-HCl, pH 6.0, 1 mM EDTA, 100 mM NaCl, 0.1 mM PMSF, 0.1 % Triton X-100). Protein concentration was determined by using a protein assay rapid kit (Wako). Total protein (10 μg) was subjected to the following assay. Seven μl of protein sample was mixed with 1μl of 10% sodium dodecyl sulfate (SDS), 1 μl of 2-mercaptoethanol and 1 μl of 1 % bromophenol blue solution, and boiled for 5 minutes. Ten μl of sample was analysed using 20% SDS-poylacrylamide gel electrophoresis using Tris-Tricine as the SDS-PAGE electrophoresis buffer. Protein bands were transferred to a Hybond-ECL nitrocellulose membrane (Amersham Biosciences). After blocking with 5% skim milk powder (PBS-Tween 20) for 2 hours at room temperature, membranes were incubated with rabbit polyclonal LFcinH antiserum (1:2,000 dilution by PBS-T) for 1 hour at 37℃ and after blotting washed three times (each 15 minutes) by PBS-T, and incubated with a secondary antibody goat anti-rabbit IgG conjugated with horseradish peroxidase (1:4,000 dilution by PBS-T) for 1 hour at 37℃ and then washed three times (15 minutes each) with PBS-T. The detection of signal was visualized with ECL-Western blotting detection reagents (Amersham Biosciences) (Figure 2).

Agar-well expansion assay

A suspension of Bacillus subtilis strain ATCC6633 (50 μl, 8.01015cell/ml) was added into 50 ml high salt tryptone (HST) agar medium (1 % tryptone, 4 % NaCl, 0.5 % agar, pH 7.0 per 1L) and 25 ml poured into sterilized plates. Holes were cut into the congealed HST agar medium using 5 mm cork borer. 100 μl of antimicrobial samples was prepared from calli or leaves of rice plants. Heat treatment of antimicrobial samples were demonstrated into the water bath at 100℃ for 50 minutes. These samples were dropped into the wells created in the agar. Plates were then incubated at 37℃ for 12 hours (Figure 3).

Microtiter plate assay

Single colony of B. subtilis strain ATCC6633 was inoculated into HST liquid medium and cultured at 37℃ overnight. Two hundreds μl of this suspension was added to 10 ml new HST liquid medium and cultured at 37℃ until 5.0109cell/ml determined by dilution plating. A hundred μl of this optimized suspension was dropped into one well of 96-well microtiter plate and 100 μl of antimicrobial samples were added to each well. In this experiment, crude extracts from untransformed calli and leaves were used as negative control samples. These plates were cultured at 37℃ with constant shaking (200 rpm) and the absorbance monitored at OD600. Colony-forming units per milliliter were determined by doing dilution series and spread plating from each plate each hour (Figure 4). This detection system is a modified version of the microtiter plate assay as described by O’Toole (1999).

Results

Construction of binary plasmids and transformation of rice plants

The binary plasmids pIG260 and pIG261 was constructed and used to generate transgenic rice plants via Agrobaterium infection. LFcinH analogues were cloned downstream of the constitutive maize ubiquitin1 promoter and each signal peptide was sequenced (Figure 1a). Polymerase chain reaction analysis of genomic DNA confirmed the presence of full length transgene in all hygromycin resistant plants (Data not shown).

Figure 1. (a) T-DNA region of the binary plasmids pIG260 and pIG261 used for transformation of rice plants. LB and RB, left and right border sequences of the T-DNA region; P35S, cauliflower mosaic virus 35S promoter; hpt, hygromycin phosphotransferase gene; T3’6b, transcriptional terminator of 6b gene; Pubi, the maize ubiquitin1 promoter; Sglutelin, rice glutelin signal peptide; ShLF, human lactoferrin signal peptide; TNOS, transcriptional terminator of nopaline synthase gene. (b) Amino acid sequences of signal peptide-LFcinH analogues. Underlined, signal peptide sequences; asterisks, predicted signal peptide cleavage site.

Western blot analysis

As expected 7.5 kDa LFcinH analogues were detected in T0 calli and mature leaves by using a rabbit polyclonal antiserum against LFcinH (Figure 2).

Figure 2. Detection of LFcinH analogues in crude extracts derived from IG260 and IG261 T0 mature leaves by western blot analysis (Data of calli were not shown). Arrow indicates 7.5kDa corresponding to the processed form of LFcinH analogues. N, untransformed; underlined, transformants.

Detection of antimicrobial activity and thermostability of LFcinH analogues

We could detect antimicrobial activity of LFcinH analogues from crude extracts of T0 calli and leaves against B. subtilis strain ATCC6633 by agar well expansion assay (Figure 3a, b) and microtiter plate assay (Figure 4). Protein samples from calli and leaves were respectively used at a rate of 0.1 mg per well of agar plate and 0.04 mg per well of microtiter plate. Figure 3b suggested that LFcinH analogues had thermostability which suggest that LFcinH analogue would be stable after cooking processes. In addition, antimicrobial activities against Escherichia coli strain K12 were confirmed (Data not shown).

(a)

(b)

Figure 3. Agar well expansion assay of crude extracts from IG260 and IG261 T0 calli (Data of leaves not shown). (a) N, untransformed; IG260, transformed; 1, crude extract; 2, crude extract after boiling for 50 minutes; bars, 5mm; indicator strain, B. subtilis strain ATCC6633. (b) Diameter of growth inhibition circle of B. subtilis strain ATCC6633 by using crude extracts of IG260 and IG261 T0 calli (asterisks, P<0.001, t-test). hole diameter, 5mm; bars= SE.

Figure 4. Microtiter plate assay of crude extracts from IG260 and IG261 T0 leaves (P < 0.001 at every hour except 0 hr, t-test) (Data of calli were not shown). Indicator strain, B. subtilis strain ATCC6633; bars= SE.

Conclusions

We were able to confirm that LFcinH analogues were expressed in callus and seedling of rice plants had strong antimicrobial activity against B. subtilis or E. coli and had thermostability after boil treatment for 50 minutes. These results suggested that transgenic rice of IG260 and IG261 might be disease-resistant and with the added benefit that the crop would act as a neutraceutical for improving daily human health.

References

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