Guoan Shen

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 Cloning and Sequence Analysis of a Novel Chalcone Synthase cDNA from Ginkgo biloba

A chalcone synthase (CHS) gene was cloned from Ginkgo biloba for the first time and it was also the first cloned gene involved in flavonoids metabolic pathway in G. biloba. The full-length cDNA of G. biloba CHS (designated as Gbchs) was 1608 bp with poly(A) tailing and it contained a 1173 bp open reading frame (ORF) encoding a 391 amino acid protein. Gbchs was found to have extensive homology with those of other plant chs genes via multiple alignments. The active sites of the CoA binding, coumaroyl pocket and cyclization pocket in CHS protein of Medicago sativa were also found in GbCHS. Molecular modeling of GbCHS indicated that the three-dimensional structure of GbCHS strongly resembled that of M. sativa (MsCHS2), implying GbCHS may have similar functions with MsCHS2. Phylogenetic tree analysis revealed that GbCHS had closer relationship with CHSs from gymnosperm plants than from other plants. Gbchs is a useful tool to study the regulation of flavonoids metabolism in G. biloba.

cDNA cloning and expression analysis of the rice (Oryza sativa L.) RNase L inhibitor.

A subtractive cDNA library was constructed using rice (Oryza sativa L.) callus cDNA as driver and differentiating callus cDNA as tester. A novel cDNA fragment encoding RNase L inhibitor (RLI) was isolated by screening the subtractive library, which had a higher expression level in differentiating callus than in callus. The full-length cDNA of rice-RLI was obtained by the method of rapid amplification of cDNA ends, which contained a 1812-bp open reading frame encoding a 604 amino acid polypeptide. Homologous analysis showed that rice-RLI contained the conserved motifs (two repeated P-loops, two ATP-binding boxes and an iron–sulfur binding motif). The fluorescence quantitative PCR analysis showed that it was a constitutive expressed gene but up-regulated in abiotic stress (low temperature and NaCl treatment) and down-regulated under the treatments of NAA and IAA.

Molecular cloning and characterization of a novel lectin gene from Zephyranthes candida

A new lectin gene was cloned from Zephyranthes candida by using RACE-PCR. The full-length cDNA of Zephyranthes candida agglutinin (ZCA) was 647 bp and contained a 477 bp open reading frame encoding a 159 amino acid protein. Zephyranthes candida lectin gene was found to encode a precursor lectin with signal peptide and had extensive homology with those of other plant lectins. Molecular modeling of ZCA indicated that the three-dimensional structure of ZCA strongly resembles that of the snowdrop lectin, implying ZCA may have the similar insecticidal functions with GNA.

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.

Cloning and characterization of a novel Hsp100/Clp gene (osClpD) from Oryza sativa

A novel osClpD gene, encoding a highly conservative ClpD subfamily member, was first isolated and characterized from Oryza sativa. The full-length cDNA of osClpD gene was 3140 bp and contained a 2884 bp open reading frame encoding a 938 amino acid protein. The phylogenetic tree and blast search showed that OSClpD belonged to the ClpD subfamily of the Hsp100/Clp family, and contained all protein motifs characteristic for the ClpD subfamily of Hsp100/Clp proteins. The real-time quantitative PCR analysis proved that it was inducible by water deficit and temperature stress in vegetative tissues.