научная статья по теме MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF TRANSGENIC CHERRY TOMATO MUTANT WITH HBSAG GENE Биология

Текст научной статьи на тему «MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF TRANSGENIC CHERRY TOMATO MUTANT WITH HBSAG GENE»

ГЕНЕТИКА, 2011, том 47, № 8, с. 1044-1051

ГЕНЕТИКА РАСТЕНИЙ

УДК 575.17:582.951.4

MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF TRANSGENIC CHERRY TOMATO MUTANT WITH HBsAg GENE

© 2011 Z. J. Guan1, 2, B. Guo1, Y. L. Huo3, H. Y. Hao3, and Y. H. Wei1

1Key Laboratory of Resource Biology and Biotechnology in Western China, Northwest University, Xi'an, 710069 P.R. China e-mail: zhengjunguan@126.com; weiyahui@nwu.edu.cn;guanzj722822@126.com 2Department of Life Sciences, Yuncheng University, Yuncheng, Shanxi, 044000 P.R. China 3Centre of Biological and Chemical Exiperiment, Yuncheng University, Yuncheng, Shanxi,044000 P.R. China

Received July, 23, 2010

HBsAg gene was previously introduced into cherry tomato (Lycopersicum esculentum var. cerasiforme) by Agrobacterium-mediated transformation. To investigate the side effect of HBsAg gene in cherry tomato, we analyzed morphological and physiological characteristics of the transgenic mutant N244. The process was performed under field conditions. The results suggested that the mutant N244 exhibited morphological, cy-tological and physiological variation. First of all, compared with the wild plants NK, N244 had fleshy and dark green leaves, the fewer notches of leaf edge, more adventitious roots and barren seeds. Moreover, the chromosome of N244 were found to be triploid (n = 36) by flow cytometric analysis. Furthermore, N244 has obvious physiological alterations, as compared to NK. It was speculated that transformation of the genes probably led to ploidy variation, and further caused phenotype and physiological changes of plants. Our study will reveal side effects of the mutants, and promote cultivation of transgenic plants in the field.

Hepatitis B virus (HBV) is the pathogen which is able to cause serious deceases. The number of chronic HBV carriers all over the world is estimated to be 350 million. And the annual HBV morbidity rate is more than 1 million. Prophylactic measures against HBV infection may be performed by immunization. Therefore, the development of "edible" vaccines based on transgenic plants is one of the most promising directions in novel types of vaccines. Up to now, HBsAg gene has been expressed in the tobacco [1], potato [2], banana [3], tomato [4] and other fruits and vegetables [5].

The above-mentioned literatures indicate that HBsAg gene used for tomato transformation is well known. However, the biochemical and physiological effects of the gene on transgenic tomato plants have not been recorded. There are no literatures in physiology of trans-genic plants carrying HBsAg gene and there is no data on their sterility in publications of another investigators. Although several metabolic researches of transgenic plants have shown the effects of transformation [6], investigation into the physiological and morphological side effects would probably have a positive impact on the public perception of transgenic plants [7].

In view of the prospects above, our experiments had previously constructed a plant expression vector carrying hepatitis B surface antigen (HBsAg) gene, and transformed cherry tomato (Lycopersicum esculentum var. cerasiforme) mediated by Agrobacterium tumefaciens. ELISA and Western blotting indicated that HBsAg had expressed in transformed plants [8]. A transformed mutant N244 was obtained by Hyg screening, PCR and Southern blotting detection.

The purpose of this work was to investigate and analyze morphological structure and physiological characteristics of transgenic cherry tomato mutant N244. By PCR detection, HBsAg gene had been integrated into its genome, and the mutant plants were well-grown. The paper could provide a detailed evaluation of a transgenic cherry tomato mutant under field environment, with a view to deeply study the growth characteristics of exogenous gene expression products, to demonstrate pheno-typic variation of transgenic plants, to reveal side effects of the transgenic mutants, as well as to promote cultivation of transgenic plants in the field.

MATERIALS AND METHODS

Plant material. Seeds of wild cherry tomato named NK (Lycopersicon esculentum var. cerasiforme) were kindly provided by Deng JJ (Shannxi, Xi'an, Vegetable Research Institute). After immersed into water for two hours, the seeds were sterilized with 70% alcohol for one minite and were washed with sterile distilled water several times. Then they were sterilized with 10% NaClO for five minites and rinsed several times. At last the seeds were placed to germinate on 1/2 Murashige—Skoog medium without any hormone. Cotyledonary segments from 10-day-old seedlings were excised and used for Agrobacterium-mediated transformation. Seedlings were subcultured on 1/2 MS medium and used as the wild (non-transformed) NK.

A transgenic mutant with HBsAg gene named N244 was obtained after co-cultivating explants with Agrobacterium tumefaciens strain LBA4404 harbouring

the vector pCAMBIA1301/HB. The T-DNA of pCAMBIA1301/HB contains the HBsAg gene (ap-prox. 0.7 kb). Transformation procedure used to obtain the mutant N244 was described in Hao et al. [8]. For its seeds were sterile, the mutant plant N244 was subcultured by aseptic seedlings for follow-up research, which were operated under the same culture condition as NK.

Culture conditions. The subcultured clone seedlings of both non-transformed plant NK and transformed mutant N244 were cultured in the same 1/2MS medium (25 ± 1°C, 15-h light photoperiod, 2500 lx). After four weeks, aseptic seedlings were transferred to the greenhouse for hardening (25 ± 2°C, 14-h light photo-period, 2000 lx). Eventually all the plants were transplanted to the field environment after two weeks of hardening.

PCR analysis. Genomic DNA from the clone plant leaves of N244 and NK in the field were extracted according to Dellaporta et al. [9]. PCR conditions were those described by the Taq polymerase manufacturer (Life Technologies, Gaithersburg, MD). Reaction mixtures were incubated for 5 minutes at 94°C followed by 35 cycles at: 30 s at 94°C, 40 s at 59°C and 1 minute at 72°C. Primer sequences designed to amplify the HBsAg gene was 5'AACGGGATCCCGCAC-CATGGAGA ACACAACATCA3' and the reverse primer 5'CCCGGAATTCCGGCTTAAATGTATACCCA AAGAC3'.

Morphological analysis. Based on the field conditions, phenotypic traits of the transgenic mutant N244 were examined in comparison to the wild NK by using a sample of 20 subcultured clone plants per group. The characteristics included stem and leaf morphology, fruit morphology and seed setting.

Observation of stomata of leaves. The leaves of both N244 and NK were selected, with the dust wiped off, coated with the nail polish and covered with the lucid rubberized fabric after the nail polish air-dried naturally on the surface of leaves. The lucid rubberized fabric were later ripped off, observed and photographed under an Olympus AH2 microscope.

Chromosome analysis. Root tip chromosomes from both wild plants and mutants were counted by colchi-cines pretreatment and Feulgen stain. Root tips were pretreated with colchicines (0.04%) for 2 h and then fixed in absolute ethanol/glacial acetic acid (3 : 1 v/v) for 2 h at 4—8°C. After fixing, the samples were hydrol-ysed in 1M HC1 for 12 min at 60°C and stained in Feul-gen. Ten-metaphase cells were analysed per sample. Moreover, the chromosome numbers of each plant material were determined according to Bor Y. et al. [10].

Flow cytometric analysis. Flow cytometry was used to analyze the ploidy level of N244 and NK. Leaf samples (1 cm2) were chopped with a razor blade and suspended in an ice-cold neutral Otto I buffer (100 mM citric acid; 0.5% (V/V) Tween 20, pH 2-3). The samples were filtered through a 75 ^m nylon mesh and

stained with nucleic acid staining fluid (5% (W/V) propidium iodide; 5% (W/V) RNase). The amount of nuclear DNA was measured in a FACStar PLUS flow cytometer (Becton Dickinson, USA) according to Shiba T and Mii M. Data were subjected to analysis of Cell Quest software (Becton Dickinson, USA).

Determination of physiological indexes. Healthy, fully-expanded clone plant leaves from the mutant N244 and the wild NK were detached and washed briefly in distilled water. Determination of leaf tissue water status was evaluated by calculating the relative water content in the leaves following the method of[11]. Hydrogen peroxide levels were determined spectrophoto-metrically at 415 nm according to Brennan T. and Frenkel C. Catalase (CAT) activity was measured by following the decomposition of H2O2 at 240 nm according to the method of Aebi [12]. Chlorophyll content, soluble protein content, superoxide dismutase (SOD) activity, and membrane permeability were estimated following the method of Li H.S. [13]. Malond-ialdehyde (MDA) content was determined by the thiobarbituric acid method, peroxidase (POD) activity was analyzed by the guaiacol reduction method and IAA oxidase (IAAO) were measured in percentage of IAA destroyed (100% IAA is in the control without en-zymic fraction) calculated from the absorbance of Salkowski's reagent at 530nm, all of which as previously described by Zhang [14].

Statistical analysis. All the experiments were carried out in triplicate, and 20 clone seedlings per group were examined each time. The values shown in the figures are mean values ± SD.

RESULTS

Identification of transgenic cherry tomato mutant

Transgenic cherry tomato plants expressing HBsAg gene were produced by introduction of Agrobacterium tumefaciens strain LBA4404 harbouring the constructed vector pCAMBIA-1301/HB. Expression of HBsAg in the mutant N244 was confirmed by PCR amplification. The total DNA of transformed cherry tomato mutant N244 presented a clear band of about 700 bp on agarose gel electrophoresis map compared with the wild NK (Fig. 1). The result indicated that HBsAg was constitutively expressed in the transformed mutant N244. Further, the subcultured clone seedlings of the transgenic plant N244 showed obvious differences in both morpholo

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