Ziqiu Guan

A novel WRKY transcriptional factor from Thlaspi caerulescens negatively regulates the osmotic stress tolerance of transgenic tobacco

A novel member of the WRKY gene family, designated TcWRKY53, was isolated from a cadmium (Cd)-treated Thlaspi caerulescens cDNA library by differential screening. WRKY proteins specifically bind to W-boxes, which are found in the promoters of many genes involved in defense and response to environmental stress. TcWRKY53 contains a 975-bp open reading frame encoding a putative protein of 324 amino acids. Homology searches showed that TcWRKY53 resembles similar WRKY domain-containing proteins from rice, parsley and tobacco, especially AtWRKY53 from Arabidopsis thaliana. Semi-quantitative RT-PCR showed that the expression of TcWRKY53 was strongly induced by various environmental stresses, including an excess of NaCl, drought, cold and the signal molecule salicylic acid (SA). The expression of TcWRKY53 in response to NaCl, drought and cold suggested a possible role of TcWRKY53 in abiotic stress response. However, physiological tests indicated that the expression of TcWRKY53 in tobaccos decreases tolerance to sorbitol during seedling root development. This was consistent with PEG6000 treatment of tobacco seedlings, and together these results indicate a negative modulation of TcWRKY53 in response to osmotic stress. Furthermore, two ethylene responsive factor (ERF) family genes, NtERF5 and NtEREBP-1, were negatively induced in TcWRKY53-overexpressing transgenic plants. In contrast, a LEA family gene, NtLEA5, showed no change, suggesting that TcWRKY53 might regulate the plant osmotic stress response by interacting with an ERF-type transcription factor rather than by regulating function genes directly.

The Thlaspi caerulescens NRAMP homologue TcNRAMP3 is capable of divalent cation transport

The NRAMP gene family encodes integral membrane protein and mediates the transport of Fe, however, its function in transport of toxic metal ions is not very clear in plants. TcNRAMP3 was isolated from Thlaspi caerulescens, and encoded a metal transporter member of the NRAMP family. TcNRAMP3 was predominantly expressed in roots of T. caerulescens by semi-quantitative RT-PCR. The expression of TcNRAMP3 was induced by iron starvation and by the heavy metals Cd and Ni in roots. TcNRAMP3 was able to rescue growth of an iron uptake fet3fet4 mutant yeast strain, suggesting a possible role in iron transport. Expression of TcNRAMP3 in yeast increased Cd sensitivity and Cd content, while it enhanced the Ni resistance and reduced Ni accumulation, indicating that TcNRAMP3 could accumulate Cd and exclude Ni in yeast. Furthermore, overexpression of TcNRAMP3 in tobacco resulted in slight Cd sensitivity of root growth and did not influence Ni resistance. These results suggested that TcNRAMP3 played a role in metal cation homeostasis in plant.

Enhancement of Cd tolerance in transgenic tobacco plants overexpressing a Cd-induced catalase cDNA.

Catalase (CAT), an important enzyme of antioxidant system, was investigated the role in preventing the plant from Cd-induced oxidative stress caused by reactive oxygen species. A CAT gene from Brassica juncea was cloned and up-regulated in response to Cd/Zn. The CAT cDNA (BjCAT3) under the control of CaMV35S promoter was introduced into tobacco via Agrobacterium-mediated transformation. Northern blot analysis verified the BjCAT3 was expressed at high level in different transgenic lines. In morphological observation, we found that seedlings from transgenic tobacco plants grew better and showed longer root length in the presence of Cd versus wild-type (WT) seedlings. Under 100 microM Cd stress, WT plants became chlorotic and almost dead while transgenic tobacco plants still remained green and phenotypically normal. The CAT activity of transgenic T(1) generations was approximately two-fold higher than that of WT plants. In WT, endogenous CAT activity is rapidly reduced as a result of 200 microM CdCl2 exposure. Compared with WT plants, lower level of Cd-induced H2O2 accumulation and cell death were detected in roots of transgenic plants with high level of CAT activity. All our findings strongly support that overexpressing BjCAT3 in tobacco could enhance the tolerance under Cd stress.

Efficient shoot regeneration andAgrobacterium-mediated transformation ofBrassica juncea

Procedures for callus induction, plantlet regeneration, andAgrobacterium-mediated transformation ofBrassica juncea were optimized by studying several factors, including explant types, and various plant growth regulators and adjuvants, such as silver nitrate, sucrose and agar. The highest shoot regeneration frequency was obtained from hypocotyl and cotyledonary petiole explants on MS medium containing 3 mg/L benzylaminopurine (BA) and 2 mg/L α-naphthaleneacetic acid (NAA). Transformation was affected by a number of factors, including explant type, selection agents, preculture duration, pre-selection conditions, and coculture temperature. Transformation efficiencies for hypocotyl and cotyledonary petiole explants were at 65% and 69%, respectively.

In vitro multiplication of heavy metals hyperaccumulator Thlaspi caerulescens

A micropropagation protocol through multiple shoot formation was developed for Thlaspi caerulescens L., one of the most important heavy metals hyperaccumulator plants. In vitro seed-derived young seedlings were used for the initiation of multiple shoots on Murashige and Skoog (MS) medium with combinations of benzylaminopurine (BA; 0.5–1.0 mg dm−3), naphthaleneacetic acid (NAA; 0–0.2 mg dm−3), gibberellic acid (GA3; 0–1.0 mg dm−3) and riboflavin (0–3.0 mg dm−3). The maximum number of shoots was developed on medium containing 1.0 mg dm−3 BA and 0.2 mg dm−3 NAA. GA3 (0.5 mg dm−3) in combination with BA significantly increased shoot length. In view of shoot numbers, shoot length and further rooting rate, the best combination was 1.0 mg dm−3 BA + 0.5 mg dm−3 GA3 + 1.0 mg dm−3 riboflavin. Well-developed shoots (35–50 mm) were successfully rooted at approximately 95 % on MS medium containing 20 g dm−3 sucrose, 8 g dm−3 agar and 1.0 mg dm−3 indolebutyric acid. Almost all in vitro plantlets survived when transferred to pots.

PCR-enriched cDNA pool method for cloning of gene homologues

We have developed a PCR-enriched cDNA pooling method for enrichment of the complete 5′ ends of the target homology cDNA fragments with just 1 conserved region needed. By using reverse transcription and a few rounds of PCR amplification, a full-length cDNA population flanked by T7 and M13 primers was generated. Multiple complete 5′ ends of cDNA members of a gene family can subsequently be enriched via PCR with M13 and degenerate primer mix priming at the 5′ end and the conserved region, and they migrate as a single dense band when separated on an agarose gel. The enriched homologous cDNA fragments could be separated for subsequent cloning and sequencing. The main advantages of our method are its speediness, simplicity, and cost-effectiveness. The method has been successfully applied to the cloning of members of the cation-efflux family inBrassica juncea L. and the natural resistance-associated macrophage protein family inThlaspi caerulescens, which demonstrates that this novel approach permits rapid isolation of novel interspecific gene orthologues. It could also be easily adapted to highly specific cloning of gene homologues identified in target genomes.