Weiwei Ren

Molecular cloning and characterization of 4-hydroxyphenylpyruvate dioxygenase gene from Lactuca sativa

Vitamin E has been found to be associated with an important antioxidant property in mammals and plants. In photosynthetic organisms, the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD; E.C. 1.13.11.27) plays an important role in the vitamin E biosynthetic pathway. The full-length cDNA encoding HPPD was isolated from Lactuca sativa L. by rapid amplification of cDNA ends (RACE). The cDNA, designated as LsHPPD, was 1743 base pairs (bp) long containing an open reading frame (ORF) of 1338 bp encoding a protein of 446 amino acids. Sequence analysis indicated that LsHPPD shared high identity with HPPD from Medicago truncatula L. Real-time fluorescent quantitative PCR (qPCR) analysis revealed that LsHPPD was preferentially expressed in mature leaves compared with other tissues and that the LsHPPD expression was sensitive to high light and drought stress treatments. Transient expression of LsHPPD via agroinfiltration resulted in 12-fold increase in LsHPPD mRNA expression level and 4-fold enhancement in α-tocopherol content compared with the negative control. A decrease in chlorophyll content and inhibition of photosystem II were observed during stress treatments and agroinfiltration.

Overexpression of transcriptional factor ORCA3 increases the accumulation of catharanthine and vindoline in Catharanthus roseus hairy roots

The AP2/ERF-domain transcription factor ORCA3 of Catharanthus roseus has been recognized earlier as a regulator of the expression of some important genes involved in the terpenoid indole alkaloid (TIA) biosynthetic pathway in C. roseus cells. Therefore, this factor was postulated to play an important role for the biosynthesis of secondary metabolites in plants. To investigate the effect of ORCA3 overexpression on the TIA biosynthesis in C. roseus hairy roots, biotransformation of Orca3 gene was conducted with the disarmed Agrobacterium rhizogenes C58C1 harboring pCAMBIA1304+-Orca3, a plasmid that contains an Orca3 gene, a Gus gene, and a hygromycin-resistance (hyg) gene, all driven separately by the cauliflower mosaic virus 35S promoter (35SCaMV). Putative transgenic hairy root lines were induced and subcultured. The T-DNA fragment of pCAMBIA1304+-Orca3 was proved to be integrated into C. roseus hairy root genome by genomic-PCR analysis, real-time quantitative PCR analysis, and GUS staining assay. Metabolite analysis using HPLC established that the average content of catharanthine and vindoline in the transgenic hairy root extracts was increased 2.49-fold and 4.2-fold in comparison to the control hairy root lines, respectively. However, vinblastine content could not been detected by HPLC method in transgenic and control hairy root cultures.

Expression and analysis of thymosin α1 concatemer in Escherichia coli

Tα1 (thymosin α 1) is important in treating immunodeficiency and other diseases. In order to study the feasibility of expressing Tα1 in plants, as the first attempt, we designed and synthesized the Tα1 gene according to the plant codon usage preference and constructed the 4×Tα1 concatemer (four copies of a DNA sequence arranged end‐to‐end in tandem). The latter was inserted into Escherichia coli expression vector pQE30, resulting in a recombinant plasmid that was subsequently transformed into E. coli M15. The 4×Tα1 concatemer protein was successfully expressed in E. coli in a soluble form. The expressed protein was purified and its bioactivity was analysed by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay. Preliminary results showed that the 4×Tα1 concatemer protein could stimulate the mice spleen lymphocyte proliferation. This is the first report on the expression of 4×Tα1 concatemer that was synthesized according to plant codon usage preference in an E. coli expression system. The present study provides the basis for expressing the synthesized active Tα1 gene in plants in the future.

Expression of human coagulation Factor IX in transgenic tomato (Lycopersicon esculentum)

In the present study, a plant binary expression vector PG‐pRD12‐hFIX (where PG is polygalacturonase) harbouring the hFIX (human coagulation Factor IX) gene was constructed and introduced into tomato (Lycopersicon esculentum) via Agrobacterium tumefaciens‐mediated transformation. After kanamycin selection, 32 putative independent transgenic tomato plants were regenerated. PCR and Southern‐blot analyses confirmed the transgenic status of some plants. RT (reverse transcription)–PCR analysis for the expression of the introduced gene (hFIX) demonstrated that the hFIX gene was expressed specifically in fruits of the tomato. Western‐blot analysis confirmed the presence of a 56 kDa band specific to hFIX in the transformed tomatoes. ELISA results showed that the expression of hFIX protein reached a maximum of 15.84 ng/g fresh weight in mature fruit. A blood‐clotting assay demonstrated the clotting activity of the expressed hFIX protein in transgenic tomato fruits. This is the first report on the expression of hFIX in plants, and our research provides potentially valuable knowledge for further development of the plant‐derived therapeutic proteins.

Overexpression of homogentisate phytyltransferase in lettuce results in increased content of vitamin E

Vitamin E is a group of lipid soluble compounds, which are important antioxidants and play a crucial role in mammals and plants. They can protect the membrane from photooxidation, and they are involved in signal transduction and transcription regulation as well. The enzyme homogentisate phytyltransferase (HPT) has been demonstrated to be a key enzyme limiting tocopherol biosynthesis. In this study, HPT gene isolated from Lactuca sativa L. (LsHPT) was transferred into L. sativa (lettuce) via Agrobacterium-mediated method and 15 transgenic plants were obtained. The expression of LsHPT