Jianxia Zhang

A circulatory system useful both for long-term somatic embryogenesis and genetic transformation in Vitis vinifera L. cv. Thompson Seedless

To establish an efficient regeneration protocol for functional validation and variety resistance improvement, a long-term system that useful for embryogenic culture maintenance and transformation was developed through recurrent cycles of secondary embryogenesis from Vitis vinifera L. cv. Thompson Seedless. Three media and five types of somatic embryo in secondary embryogenesis were evaluated. Somatic embryos (SE) in the torpedo and mid-cotyledonary stages gave the best embryogenic responses with re-induction rates of about 80xa0%. Embryogenic callus, proembryonic masses and SE produced in the system, could be propagated for over 3xa0years and all proved competent for Agrobacterium-mediated transformation. Based on this system, different transgenic selection regimes were compared. Addition of kanamycin at 4xa0weeks after co-cultivation was optimal for embryo recovery. Plant conversion was improved by alternating culture on two media: one containing 0.2xa0mgxa0l−1 BA and the other 0.25xa0mgxa0l−1 kinetin. To further test the efficiency of the system, a ubiquitin ligase gene (VpPUB23) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. Of the 351 transgenic plants obtained, those overexpressing VpPUB23 exhibited decreased resistance to powdery mildew compared with non-transgenic plants.

Establishment of a picloram-induced somatic embryogenesis system in Vitis vinifera cv. chardonnay and genetic transformation of a stilbene synthase gene from wild-growing Vitis species

To facilitate genetic improvement, an efficient system of embryogenic culture induction, maintenance and transformation in Vitis vinifera cv. Chardonnay was developed using picloram. Whole flowers produced the most embryogenic calluses on induction media containing Murashige and Skoog’s (MS) basal salts and 3.0xa0mgxa0L−1 picloram with different concentrations of 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Proembryonic masses (PEM) cultured on maintenance and proliferation media (MPM) containing MS basal salts and picloram had the highest proliferation efficiency within 2xa0months. Additionally, the best PEM formation was found with MPM containing MS basal salts and 2.0xa0mgxa0L−1 picloram. Then this PEM cultured on MPM were transferred to embryo germination medium containing MS basal salts, 0.2xa0mgxa0L−1 kinetin and 0.1xa0mgxa0L−1 2-naphthoxyacetic acid, the optimal medium for normal plantlets regeneration. A single copy of the stilbene synthase gene VpSTSgDNA2 from Chinese wild Vitis pseudoreticulata was transferred into regenerated Chardonnay via Agrobacterium tumefaciens-mediated transformation and confirmed by Southern blot analysis. The positive transgenic grapevine lines exhibited higher levels of stilbene and H2O2 than wild-type vines and could slightly reduced the growth of powdery mildew.

Genetic transformation of a fruit-specific, highly expressed stilbene synthase gene from Chinese wild Vitis quinquangularis

AbstractMain conclusionThe stilbene synthase gene VqSTS6, from Chinese wild typeVitis quinquangularisaccession Danfeng-2, increases the resveratrol content and pathogen resistance of transgenic plants ofV. viniferaThompson Seedless.n This study successfully created transgenic plants of V. vinifera Thompson Seedless which overexpressed VqSTS6, cloned from Chinese wild type V. quinquangularis accession Danfeng-2. Western blot and qRT-PCR showed a variable range in transcript levels among transgenic lines. The resistance to powdery mildew (Uncinula necator) was particularly enhanced in lines most highly expressing VqSTS6. Compared with the non-transformed controls, trans-resveratrol and other stilbene compounds were significantly increased in the transgenic lines. The correlation between high resveratrol content and high pathogen resistance in transgenic grapes is discussed. We hypothesize that the fruit-specific, highly expressed gene VqSTS6 from Chinese wild V. quinquangularis accession Danfeng-2, is directly involved in the resveratrol synthesis pathway in grapes, and plays an important role in the plant’s defense against pathogens. Genetic transformation of VqSTS6xa0explored the potential of the wild Chinese grape species for use in breeding, the results of which would raise both the disease resistance and the fruit quality of V. vinifera grapevines.

Resveratrol derivatives in four tissues of six wild Chinese grapevine species

Resveratrol is well known for its strong antioxidant properties. To accelerate research on resveratrol metabolism and to evaluate the genetic potential for breeding, the stilbenoid contents of four tissues (leaf and berry skin, flesh and seed) were examined in 21 accessions of six wild Chinese grapevine species (Vitis quinquangularis, V. romanetii, V. piasezkii, V. shenxiensis, V. amurensis and V. davidii). Resveratrol derivatives in V. quinquangularis, V. romanetii, V. piasezkii and V. shenxiensis are reported for the first time. Three kinds of stilbenoid (trans-resveratrol, cis-piceid and trans-piceid) were detected in the berry skins, flesh, seeds and leaves but cis-resveratrol was found to be absent. Stilbenoid content was predominantly affected by genetic background and by tissue type. In most accessions, berry skins produced the highest total stilbenoid, followed by leaves, seeds and flesh. As a proportion, cis-piceid was the major stilbenoid component in berry skins, seeds and leaves, whereas trans-piceid was the major component in the flesh.

VaERD15, a Transcription Factor Gene Associated with Cold-Tolerance in Chinese Wild Vitis amurensis

Early responsive to dehydration (ERD) genes can be rapidly induced to counteract abiotic stresses, such as drought, low temperatures or high salinities. Here, we report on an ERD gene (VaERD15) related to cold tolerance from Chinese wild Vitis amurensis accession ‘Heilongjiang seedling’. The full-length VaERD15 cDNA is 685 bp, including a 66 bp 5′-untranslated region (UTR), a 196 bp 3′-UTR region and a 423 bp open reading frame encoding 140 amino acids. The VaERD15 protein shares a high amino acid sequence similarity with ERD15 of Arabidopsis thaliana. In our study, VaERD15 was shown to have a nucleic localization function and a transcriptional activation function. Semi-quantitative PCR and Western blot analyses showed that VaERD15 was constitutively expressed in young leaves, stems and roots of V. amurensis accession ‘Heilongjiang seedling’ plants, and expression levels increased after low-temperature treatment. We also generated a transgenic Arabidopsis Col-0 line that over-expressed VaERD15 and carried out a cold-treatment assay. Real-time quantitative PCR (qRT-PCR) and Western blot analyses showed that as the duration of cold treatment increased, the expression of both gene and protein levels increased continuously in the transgenic plants, while almost no expression was detected in the wild type Arabidopsis. Moreover, the plants that over-expressed VaERD15 showed higher cold tolerance and accumulation of proline, soluble sugars, proteins, malondialdehyde and three antioxidases (superoxide dismutase, peroxidase, and catalase). Lower levels of relative ion leakage also occurred under cold stress. Taken together, our results indicate that the transcription factor VaERD15 was induced by cold stress and was able to enhance cold tolerance.

The Novel Gene VpPR4-1 from Vitis pseudoreticulata Increases Powdery Mildew Resistance in Transgenic Vitis vinifera L.

Pathogenesis-related proteins (PRs) can lead to increased resistance of the whole plant to pathogen attack. Here, we isolate and characterize a PR-4 protein (VpPR4-1) from a wild Chinese grape Vitis pseudoreticulata which shows greatly elevated transcription following powdery mildew infection. Its expression profiles under a number of abiotic stresses were also investigated. Powdery mildew, salicylic acid, and jasmonic acid methyl ester significantly increased the VpPR4-1 induction while NaCl and heat treatments just slightly induced VpPR4-1 expression. Abscisic acid and cold treatment slightly affected the expression level of VpPR4-1. The VpPR4-1 gene was overexpressed in 30 regenerated V. vinifera cv. Red Globe via Agrobacterium tumefaciens-mediated transformation and verified by the Western blot. The 26 transgenic grapevines exhibited higher expression levels of PR-4 protein content than wild-type vines and six of them were inoculated with powdery mildew which showed that the growth of powdery mildew was repressed. The powdery mildew-resistance of Red Globe transformed with VpPR4-1 was enhanced inoculated with powdery mildew. Moreover, other powdery mildew resistant genes were associated with feedback regulation since VpPR4-1 is in abundance. This study demonstrates that PR-4 protein in grapes plays a vital role in defense against powdery mildew invasion.

Downregulation of serum DKK-1 predicts poor prognosis in patients with papillary thyroid cancer.

The Wnt inhibitor dickkopf-1 (DKK-1) has been shown to be closely correlated with tumor initiation and progression in various types of cancers. However, the serum level of DKK-1 in patients with papillary thyroid cancer (PTC) and its potential clinical significance is poorly understood. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the level of serum DKK-1 in patients with PTC (N = 132) and healthy controls (N = 40). The association between serum DKK-1 level and clinicopathological parameters of PTC was examined and independent prognostic markers for PTC were identified. The mean serum DKK-1 level was significantly lower in patients with PTC than healthy controls (44.64 ± 15.13 and 85.51 ± 9.94 ng/mL, respectively; P < 0.01). Following treatment, the mean serum DKK-1 level in PTC patients significantly increased (67.03 ± 17.09 ng/mL; P < 0.01). Serum DKK-1 level was associated with various PTC clinical features including tumor size (P = 0.003), lymph node metastasis (P = 0.001), and tumor-node-metastasis stage (P = 0.004). Survival analysis revealed that PTC patients who had lower serum DKK-1 levels suffered both poorer overall survival (P = 0.036) and relapse-free survival (P = 0.015). Moreover, serum DKK-1 levels were an independent risk factor for predicting the prognosis of PTC (P = 0.031). In conclusion, low DKK-1 serum levels are associated with poor prognosis in PTC patients and DKK-1 could potentially be used as a biomarker leading to earlier diagnosis of PTC.

Construction of a cDNA library of the Chinese wild Vitis amurensis under cold stress and analysis of potential hardiness-related expressed sequence tags.

A cDNA library of Chinese wild Vitis amurensis, which is the most cold-resistant species in the genus Vitis, was constructed using young leaves of seedlings of the clone Heilongjiang potted and subjected to cold stress. The leaves were harvested at various times after cold stress for total RNA extraction, which was used to generate expressed sequence tags (ESTs). The titer of the original library was 2.67 x 10(6) pfu/mL and the corresponding combination frequency was 98.5%. The test values of the amplified library were 1.53 x 10(9) pfu/mL and 98.2%, respectively. After randomly choosing, cloning and sequencing, 227 ESTs with high quality were obtained and submitted to GenBank database. Using BLASTX, 79.7% of the ESTs shared homology with known functional DNA sequences and 20.3% shared significant homology with unknown proteins. Some potential hardiness-related ESTs were obtained, which were involved in signal transduction, stress inducement, defense reactions, transcription factors, etc. Some functional genes identified from the cDNA library have potential for plant defense. These sequences will be subjected to further researches on hardiness genes and their molecular mechanisms.

Overexpression of VpEIFP1, a novel F-box/Kelch-repeat protein from wild Chinese Vitis pseudoreticulata, confers higher tolerance to powdery mildew by inducing thioredoxin z proteolysis

An F-box protein (VpEIFP1) induced by Erysiphe necator was isolated from Vitis pseudoreticulata, a wild Chinese grapevine species naturally resistant to powdery mildew (PM). It contains an F-box domain and two Kelch-repeat motifs. Expression profiles indicate the VpEIFP1 is strongly induced at both transcriptional and translational levels by PM infection. A subcellular localisation assay showed that VpEIFP1 is predominantly located in the nucleus and cytoplasm. Overexpression of VpEIFP1 accelerated the accumulation of hydrogen peroxide (H2O2) and up-regulated the expressions of ICS2, NPR1 and PR1 involved in defence responses, resulting in suppression of PM germination and growth. As an F-box protein, VpEIFP1 interacts with thioredoxin z (VpTrxz) in the yeast-two-hybrid (Y2H) assay and in the bimolecular fluorescence complementation (BiFC) assay. Decreased amounts of VpTrxz protein in transgenic grapevine leaves overexpressing VpEIFP1 were restored by proteasome inhibitor MG132, implying that VpEIFP1 mediated VpTrxz for degradation through the SCFVpEIFP1 (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. The RNA interference line of VpTrxz showed increased H2O2 accumulation following PM inoculation. We propose VpEIFP1 positively modulates the grapevine defence response to PM by inducing the degradation of VpTrxz via the ubiquitin/26S proteasome system.

RING-H2-type E3 gene VpRH2 from Vitis pseudoreticulata improves resistance to powdery mildew by interacting with VpGRP2A

Grapevine is one of the worlds most important fruit crops. European cultivated grape species have the best fruit quality but show almost no resistance to powdery mildew (PM). PM caused by Uncinula necator is a harmful disease that has a significant impact on the economic value of the grape crop. In this study, we examined a RING-H2-type ubiquitin ligase gene VpRH2 that is associated with significant PM-resistance of Chinese wild-growing grape Vitis pseudoreticulata accession Baihe-35-1. The expression of VpRH2 was clearly induced by U. necator inoculation compared with its homologous gene VvRH2 in a PM-susceptible grapevine V. vinifera cv. Thompson Seedless. Using a yeast two-hybrid assay we confirmed that VpRH2 interacted with VpGRP2A, a glycine-rich RNA-binding protein. The degradation of VpGRP2A was inhibited by treatment with the proteasome inhibitor MG132 while VpRH2 did not promote the degradation of VpGRP2A. Instead, the transcripts of VpRH2 were increased by over-expressing VpGRP2A while VpRH2 suppressed the expression of VpGRP2A. Furthermore, VpGRP2A was down-regulated in both Baihe-35-1 and Thompson Seedless after U. necator inoculation. Specifically, we generated VpRH2 overexpression transgenic lines in Thompson Seedless and found that the transgenic plants showed enhanced resistance to powdery mildew compared with the wild-type. In summary, our results indicate that VpRH2 interacts with VpGRP2A and plays a positive role in resistance to powdery mildew.