Lijie Cui

Expression of thymosin α1 concatemer in transgenic tomato (Solanum lycopersicum) fruits

Tα1 (thymosin α1), an immune booster, plays an important role in the maturation, differentiation and function of T‐cells. It can also activate the production of cytokines in dendritic cells. Tα1 is one of two thymosin proteins that have potential future clinical applications. In order to express Tα1 protein in plants, we designed and synthesized the Tα1 gene according to the plant codon usage bias and created a novel 4×Tα1 concatemer (four copies of the Tα1 gene arranged end‐to‐end in tandem, designated 4×Tα1). Subsequently, a plant binary expression vector, PG‐pRD12‐4×Tα1, was constructed and introduced into tomato via Agrobacterium tumefaciens‐mediated transformation. Through selection, 54 regenerated tomato plants resistant to kanamycin were obtained, and four transgenic tomato plants were further confirmed by PCR and Southern blotting. RT–PCR (reverse transcription–PCR) analysis showed that the 4×Tα1 gene was transcribed specifically in tomato [Solanum lycopersicum (formerly Lycopersicon esculentum)] fruits. ELISA analysis showed that the content of the 4×Tα1 protein reached a maximum of 6.098 μg/g fresh weight in mature tomato fruit. Western‐blot analysis further confirmed the expression of 4×Tα1 protein in transgenic tomato fruits. The MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay showed the 4×Tα1 protein derived from transgenic tomatoes exhibited bioactivity that can stimulate the proliferation of mice splenic lymphocytes in vitro, and the specific activity of Tα1 protein from the artificial system was higher than that from the synthetic Escherichia coli system. This study is the first to report successful expression of bioactive Tα1 in plants, and also it will provide the basis for further development of the plant system to produce Tα1.

Molecular detection of Torque teno sus virus from tissues samples of sick pigs in China.

In the present study, Torque teno sus virus (TTSuV) was detected from different tissues, stool and serum samples of 25 sick pigs. The total prevalence of TTSuV1 and TTSuV2 were 64% (16/25) and 28% (7/25), 24% (6/25) were co-infected with both TTSuV1 and TTSuV2. The prevalence of TTSuV infection in spleen is a slightly higher, with positive rates of 52% (13/25) for TTSuV1 and 24% (6/25) for TTSuV2. Phylogenetic analysis of TTSuV1 showed that 21 isolates were distributed into two clusters (genotype TTSuV1a and TTSuV1b), with genotype TTSuV1b was the dominant genotype. Phylogenetic analysis of TTSuV2 showed that the nine isolates shared 80.9-99.2% nucleotide homology with each other, and were distributed in different genotypes (TTSuV2a-TTSuV2f). TTSuV2d was the most prevalent genotype in this study, which contained five Spanish strains and nine Chinese strains, and shared 94.2-96.8% homology.

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.

Molecular detection of Saffold Virus in children in Shanghai, China.

Saffold Virus (SAFV) was initially characterized in stool sample rom an 8-month-old girl presenting with fever of unknown origin n the United States.1 Recently, such infections had been recorded in ermany, Brazil, China and USA.2–5 SAFVs were also detected in resiratory secretion of children with respiratory symptoms in Canada nd USA.6 In Pakistan and Afghan, SAFVs were identified from chilren with nonpolio acute flaccid paralysis.7 Although SAFVs are ifficult to propagate on cell lines, the SAFV-1 and SAFV-2 have been ultured in human fetal diploid kidney(HFDK) and rhesus monkey idney cells (LLC-MK2) respectively.1,6 Additionally, SAFV-3 had een isolated on HELF and tMK cells and produced a rapid cytoathic effect on HeLa cells.8 Hitherto, the pathogenic role of SAFV emains unclear. From November 2008 to March 2010, stool specimens were colected from 577 hospitalized children with diarrhea and 60 healthy ontrols in Shanghai, China. Diarrhea was defined as >3 loose stools n the previous 24 h and none of them had any apparent clinical

Sequence analysis of an isolate of minute virus of canines in China reveals the closed association with bocavirus

In the present study, we have cloned and sequenced the nearly-full-length genome of minute virus of canines (MVC), SH26, in China. The genome of MVC, 5,132 nucleotides (nts) in length, contains three open reading frames (ORFs), which are 2,325-bp of NS1, 561-bp of NP1 and 2,112-bp of VP1/VP2 encoding three proteins of 774, 186 and 703 residues, respectively. Predicted amino acids sequence of NS1 of MVC has 44% identity with human bocavirus (HBoV) and human boacvirus 2 (HBoV2), NP1 has 48 and 45% identity with HBoV and HBoV2, VP1/VP2 has 45 and 46% identity with HBoV and HBoV2, respectively. Phylogenetic analysis showed that the present Chinese MVC strain was also closely clustered with the previous American and Japanese MVC isolates, and MVCs formed a different branch together with bovine parvovirus and HBoVs from other parvoviruses classified into Parvovirinae.

Recombinant hHscFv-RC-RNase protein derived from transgenic tobacco acts as a bifunctional molecular complex against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common clinical primary malignant tumor; however, efficient drugs for the treatment of HCC are still lacking at the present time. To develop a new approach for liver cancer therapy, we designed a chimeric gene (his‐HR) encoding a single‐chain variable fragment of human HAb25 (hHscFv) fused to a cytotoxic ribonuclease from Rana catesbeiana (RC‐RNase) and expressed the corresponding fusion protein in transgenic tobacco (Nicotiana tabacum). Eleven positive transgenic plant lines were identified from 204 regenerated tobacco plants by PCR and Southern blot analysis, and the immunocompetence of the recombinant his‐HR protein was confirmed by Western blotting. The expression levels of his‐HR protein ranged from 0.75 to 1.99 µg/g in the fresh tobacco leaves. To characterize the bifunction of the expressed his‐HR protein in tobacco, binding specificity and cell toxicity to several cell lines were examined by the indirect immunocytochemical streptavidin–biotin complex method and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide assay. Data indicated that the his‐HR protein had stronger specific binding affinity to HepG2 (human liver HCC cell line) than to the other tumor cell lines and normal liver cell line, and the capacity to kill the HCC cell lines SMMC7721 and HepG2 with an half maximal inhibiting concentration of 2.0 and 2.4 nM, respectively. The results suggest that recombinant bifunctional his‐HR protein derived from transgenic plants may provide a novel strategy to treat HCC in the future.

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