Introduction
Cultivated strawberry (
Fragaria ananassa Duch.) is a highly-valued fruit crop species. Strawberry fruits are appreciated for their unique flavor and nutritious quality, but the fleshy fruits becoming less firm with progressive ripening are liable to post-harvest deterioration that impairs their preservation, resulting in great losses. Therefore, efforts have long been made to understand the regulatory mechanisms of fleshy fruit ripening. Dihydroflavnol 4-reductase and pectate lyase gene, two transcripts previously shown to be associated with anthocyanin synthesis and fruit firmness, respectively, were identified in a subtractive library from ripening fruit (
Medina-Escobar et al., 1998). Jimenez-Bermudez et al. (2002) transformed strawberry with antisense pectate lyase constructs driven by the CaMV 35S promoter. Antisense plants with demonstrated repression of pectate lyase gene expression exhibited the decreased fruit set, yet there were no differences in general fruit qualities. A gene encoding cystathionine
γ-synthesis, an enzyme required for methionine synthesis, was also characterized in developing strawberry fruits. The gene was identified via its abundant transcript in
F.
vesca fruit (
Marty et al., 2000). This report indicated that the transcript and protein levels were high in ripening fruits and that levels were influenced by auxin application. The unusual correlation of accumulation of this protein led the authors to posit that it may be important in the ripening process. A cell with a gene encoding an endo-1,4-
β-glucanase was also cloned from ripening fruit (
Harpster et al., 1998).The corresponding transcript was present only in ripening fruit, with increasing steady-state levels as the tissue progressed towards reddening and ripening. The expression of two other members of the endo-1,4-
β-glucanase gene family (
FaEG1 and
FaEG3) was studied, indicating that the transcripts were both present during ripening (
Trainotti et al., 1999). Both genes were used to produce antisense plants, yet neither had an effect on fruit softening (
Wooley et al., 2001).
Despite the conspicuous influence of achenes and auxin on strawberry fruit ripening and coincident gene expression, little is known about the role of ethylene in the strawberry ripening process. Although strawberry is a non-climacteric fruit, ethylene is quite abundant in green strawberry fruit (
Knee et al., 1977). Two cDNAs coding for enzymes of the ethylene biosynthetic pathway (i.e.
FaACO1 and
FaACO2), and three cDNAs coding different ethylene receptors have been isolated recently. The receptor
FaEtr2, with a degenerate histidine-kinase domain belonging to type-Ⅱ, (
Cancel et al., 2002) is mostly expressed in ripening strawberries. Perhaps even the little ethylene produced by ripening strawberries might be sufficient to trigger ripening-related physiological responses (
Trainotti et al., 2005). So a specific PCR product of
FaEtr2 was obtained and an antisense expression vector was constructed for further research on gene function to improve the varieties of strawberry through transgenic technology.
Materials and methods
Materials
TIAN gel Midi Purification Kit and pGEM-T cloning kit were products from TIANGEN BIOTECH (BEIJNG) CO., LTD., TaqDNA polymerase and T4 DNA ligase were purchased from SLE. Biology Tech. (Baoding) Co., Ltd., various restriction enzymes were purchased from TakaRa Biotechnology (Dalian) Co., Ltd., and E. coli DH5α and pBI121 plant expression vector were stored in our laboratory. Primer synthesis and sequencing were done by Beijing Sunbiotech Co., Ltd.. Agrobacterium strain LBA4404 was provided by Dr. Wang Lei from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.
Methods
Cloning of FaEtr2 gene
Specific primers (sense 5'-TGCTTACCCAAGAGATTCGC-3', antisense 5'-ATCATTCCTCCCCTGACTTCC-3') were designed according to the reported cDNA sequence of Chandler FaEtr2, and a partial sequence of FaEtr2 was obtained. The PCR product was purified and ligated with pGEM-T vector, then transformed into E. coli DH5α competent cells. White colonies were screened in the medium with X-gal/IPTG. The plasmid was digested with EcoR I to select the correct recombinant plasmid. Bacterium liquid with recombinant plasmid was sent to Beijing Sunbiotech Co., Ltd. and sequenced. The sequencing results were blasted in NCBI and analyzed with Omiga software.
Amplification of reverse fragments
One pair of primers was designed according to the restriction enzyme sites of the amplified fragments and the pBI121 vector. The primers were 5'-TGGATCCAGTGGTGATTCTGGTGAG-3' for sense (with BamH I restriction site) and 5'-CCACTATCCTTGTCTAGATTATCCA-3' for antisense (with Xba I restriction site). Using a sequenced plasmid as template, the PCR product of the reverse fragment was obtained.
Construction of plant expression vector
The expression vector of pBI121 and the PCR product of the reverse fragments were double-digested with Xba I and BamH I and linked (T4 DNA ligase 16°C for 16 h) directionally. The linked product was transformed into E.coli DH5 α competent cells. Positive clones were screened on solid LB medium containing kanamycin (100 μg•middlt;mL-1). A positive plasmid was identified by restriction enzyme digestion and the recombinant plasmid was recorded as pBI121-Anti- Etr2.
Transformation of plant expression vector into agrobacterium
Plant expression vector was transformed into agrobacterium LBA4404 competent cells by the freeze-thaw method. Positive colonies were screened on solid YEB medium containing Kan (100 μg•middlt;mL-1) and rif (50 μg•middlt;mL-1). Using pBI121-Anti-Etr2 plasmid (positive control), non-transformed Agrobacterium liquid (negative control) and transformed Agrobacterium liquid as the template, transformation was verified by PCR amplification with specific primers.
Results and analysis
Cloning of FaEtr2 gene
It was inferred that the PCR product should be about 1.0 kb, according to the sites of primers on the FaEtr2 gene. Analysis of PCR products by 1% agarose gel electrophoresis showed that the fragment was about the same size as that expected (Fig. 1).
The purified fragment was linked with pGEM-T vector and transformed into the competent E. coli DH5α. The recombinant fragment was obtained by blue-white spots screening. EcoR I was chosen to digest the recombinant plasmid according to the multi-cloning sites at both ends of the vector restriction sites. If the recombinant plasmid contained an inserted fragment, it should be digested into a small fragment of about 1.0 kb and a 3.0 kb fragment of the T-vector. Digestion results as seen on agarose gel electrophoresis detection were consistent with expectations, which indicated that a DNA fragment of about 1.0 kb was inserted into the pGEM-T vector (Fig. 2).
Positive clones were sent to Beijing Sunbiotech Co., Ltd. and sequenced. The results showed that the sequencing PCR amplification product was 1049 bp (Fig. 3), containing the primer sequences and no intron sequences. Sequence analysis showed that the All Star-Etr2 nucleotide sequence had 100% identity with Chandler-Etr2 (AJ297513) in the GenBank.
Amplification of reverse fragment
We used the diluted sequencing plasmid as a template to amplify the reverse fragment. The amplified products should be 516 bp according to the site of the primers on the FaEtr2 gene. The result seen on electrophoresis was consistent with expectations (Fig. 4).
Construction and identification of antisense expression vector
Double-digestion and purification of plant expression vector and PCR product
The PCR product and pBI121 plant expression vector were digested with Xba I and BamH I. The sizes of the fragments did not change significantly, with only a few bases cut (Figs. 5, 6).
Identification of antisense expression vector
If the antisense expression vector was digested with Xba I and EcoR I, the small fragment should be about 2.6 kb. If it was digested with Xba I and BamH I the small fragments of the electrophoresis band should be about 500 bp. After digestion with Hind Ⅲ and Xba I or Hind Ⅲ and BamH I the small fragments should be about 0.9 kb and 1.3 kb, respectively. Electrophoresis results (Fig. 7) were in line with expectations.
Identification of expression vector in agrobacterium by bacterium liquid PCR
The pBI121-anti-Etr2 expression vector plasmid was transformed into Agrobacterium fumefeciens LBA4404 competent cells. Positive colonies were screened on solid YEB medium containing Kan (100 μg•middlt;mL-1) and rif (50 μg•middlt;mL-1). A single colony was chosen and shaken in the YEB liquid medium containing the same antibiotic. Using the pBI121-anti-Etr2 plasmid (positive control), non-transformed Agrobacterium liquid (negative control) and transformed Agrobacterium liquid as the template, transformation was verified by PCR amplification with specific primers. If the pBI121-anti-Etr2 expression vector was transformed into Agrobacterium, the PCR product should be about 500 bp. The PCR product electrophoresis (Fig. 8) was in line with expectations. That is to say, the expression vector was transformed into Agrobacterium fumefeciens LBA4404.
Discussion
Since the 1980s, antisense RNA technology has become a powerful tool for regulation and control of plant gene expression. Antisense RNA technology can delay or suppress fruit ripening through the suppression of the fruit mature gene expression (
Luo et al., 1995;
Waterhouse et al., 1998). Strawberry is a non-climacteric fruit, but it can also produce a small amount of ethylene. Only when ethylene binds with the receptor can it be effective in the signal transduction process (
Klee, 2002).
In contrast to the great deal of information regarding the regulation of ripening in climacteric fruits (
Giovanoni, 2001;
Giovanoni, 2004), much less is known about non-climacteric ones. Since non-climacteric fruits are also able to synthesize ethylene, and in some cases it has been seen that ethylene can hasten post-harvest deterioration, the possible involvement of this hormone in the ripening of non-climacteric fruits has been studied in different laboratories (
Ferrarese et al., 1995;
Harpster et al., 1997;
Harpster et al., 1998;
El-Kereamy et al., 2003;
Katz et al., 2004;
Tesniere et al., 2004). However, no results have been obtained that can demonstrate a clear relation between ethylene and the ripening of these fruits.
In our study, an antisense expression vector of
FaEtr2 was constructed for further research on gene function to improve the varieties of strawberry through transgenic technology. Transgenic experiments with antisense plasmids are in progress. However, attempting to construct the antisense expression vector is difficult due to the fact that the two fragments to be connected vary widely. Connection is not successfully done in accordance with the recommended mole concentration ratio (
Sambrook et al., 2001). In this study mole concentration ratio was optimized, so this issue was resolved fundamentally.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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