Jingsheng Xu

ScMT2-1-3, a Metallothionein Gene of Sugarcane, Plays an Important Role in the Regulation of Heavy Metal Tolerance/Accumulation

Plant metallothioneins (MTs), which are cysteine-rich, low-molecular-weight, and metal-binding proteins, play important roles in detoxification, metal ion homeostasis, and metal transport adjustment. In this study, a novel metallothionein gene, designated as ScMT2-1-3 (GenBank Accession number JQ627644), was identified from sugarcane. ScMT2-1-3 was 700 bp long, including a 240 bp open reading frame (ORF) encoding 79 amino acid residues. A His-tagged ScMT2-1-3 protein was successfully expressed in Escherichia coli system which had increased the host cells tolerance to Cd2+, Cu2+, PEG, and NaCl. The expression of ScMT2-1-3 was upregulated under Cu2+ stress but downregulated under Cd2+ stress. Real-time qPCR demonstrated that the expression levels of ScMT2-1-3 in bud and root were over 14 times higher than those in stem and leaf, respectively. Thus, both the E. coli assay and sugarcane plantlets assay suggested that ScMT2-1-3 is significantly involved in the copper detoxification and storage in the cell, but its functional mechanism in cadmium detoxification and storage in sugarcane cells needs more testification though its expressed protein could obviously increase the host E. coli cells tolerance to Cd2+. ScMT2-1-3 constitutes thus a new interesting candidate for elucidating the molecular mechanisms of MTs-implied plant heavy metal tolerance/accumulation and for developing sugarcane phytoremediator varieties.

Establishment of genetic transformation system and obtaining transgenic sugarcane (var. badila) transformed with RS gene

Resveratrol Synthase (RS) is one of the key enzymes in resveratrol biosynthesis, which catalyzes one molecule of coumaroyl CoA and three molecules of malonyl CoA to form one molecule of resveratrol. In this study, the genetic transformation system for badila, a chewing cane variety, was established. The optimized media for regeneration were as follows: induction medium was MS+2,4-D 2mg.L−1; differentiation medium was MS+NAA 0.2 mg.L−1+BA 2 mg.L−1; rooting medium was 1/2 MS+NAA 2 mg.L−1. The addition of 0.5 % activated carbon in the differentiation and rooting medium could decrease the phenol poison and promote rooting. At the same time, the optimal concentration of G418 in subculture and differentiation period was determined as: 30 mgL−1 for differentiation and 25 mgL−1 for rooting. RS gene was transferred into badila via gene gun bombardment through the constructed plant expression vector pBIL-RS. PCR detection and southern blot analysis showed that seven putative transgenic plants had been obtained. Therefore, the genetic transformation system of badila had been elementarily established and proved to be efficient.

Molecular marker application in sugarcane

Molecular markers, such as RAPD, ISSR and ITS, were used to assess genetic diversity of Erianthus and S. spontaneum, to clarify the molecular classification of related Saccharum species and to characterize the genuine hybrid from Saccharum and Erianthus. The results were summarized in this paper.

cDNA-SRAP and its application in differential gene expression analysis: a case study in Erianthus arundinaceum.

Erianthus arundinaceum is a wild relative species of sugarcane. The aim of this research was to demonstrate the feasibility of cDNA-SRAP for differential gene expression and to explore the molecular mechanism of drought resistance in E. arundinaceum. cDNA-SRAP technique, for the first time, was applied in the analysis of differential gene expression in E. arundinaceum under drought stress. In total, eight differentially expressed genes with length of 185–427 bp were successfully isolated (GenBank Accession numbers: EU071770, EU071772, EU071774, EU071776, EU071777, EU071779, EU071780, and EU071781). Based on their homologies with genes in GenBank, these genes were assumed to encode ribonuclease III, vacuolar protein, ethylene insensitive protein, aerobactin biosynthesis protein, photosystem II protein, glucose transporter, leucine-rich repeat protein, and ammonia monooxygenase. Real-time PCR analysis on the expression profiling of gene (EU071774) encoding ethylene-insensitive protein and gene (EU071781) encoding ammonia monooxygenase revealed that the expression of these two genes was upregulated both by PEG and ABA treatments, suggesting that they may involve in the drought resistance of E. arundinaceum. This study constitutes the first report of genes activated in E. arundinaceum by drought stress and opens up the application of cDNA-SRAP in differential gene expression analysis in E. arundinaceum under certain stress conditions.

Molecular cloning and expression analysis of a zeta-class glutathione S-transferase gene in sugarcane

Glutathione S-transferases (GSTs) play an important role in stress tolerance in plants. This is the first report of cloning and characterization of a zeta-class GST gene in sugarcane (GenBank Accession number: GQ246461). Sequence analysis showed that the cDNA sequence of Sc-GST gene was 829 bp, contained a 621 bp open reading frame (ORF), the 5’ untranslated region (UTR) of 65 bp and 3’UTR of 143 bp, plus the typical AATAA region and poly (A) tail. It encoded the 206 amino acid residues with a molecular mass of 23.1 KD and isoelectric point of 6.10. Protein domain prediction and multiple sequence alignment demonstrated that the conserved domain in Sc-GST at N-terminus was SSCXXRXRIA, while that at C-terminus was quebec platelet disorder (QPD), both of which were specific for zeta-type GST in eukaryotes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and enzyme activity assay indicated that the prokaryotic expression product was a fusion protein with a molecular weight of about 30 KD, which also possessed GST enzyme activity. It was revealed in real-time quantitative polymerase chain reaction (qPCR) analysis that the Sc-GST gene had induced expression under H 2 O 2 and Ustilago scitaminea stresses, while it was inhibited and then induced by salicylic acid (SA) stress, suggesting that it is a type of stress-tolerant gene playing a certain role in sugarcane resistance response. Key words: Saccharum officinarum, glutathione S-transferase, homology, prokaryotic expression, real-time quantitative PCR.

Molecular cloning and characterization of a cytoplasmic cyclophilin gene in sugarcane

Cyclophilins are ubiquitous proteins with an enzymatic activity of peptidyl-prolyl cis-trans isomerase (PPIase), which play important roles in a variety of stress responsiveness. In this study, we reported the cloning and characterization of a full-length cytoplasmic cyclophilin gene in sugarcane. Sequence analysis showed the cDNA of this gene (GenBank accession number:GQ246462), termed as Sc-CyP, was 904 bp long, including a 519 bp complete ORF, the 5’ UTR of 74 bp and 3’UTR of 311 bp, plus a typical AATAA motif and poly (A) tail. It encoded the 172 amino acid polypeptide with a molecular weight of 18.4 KD and the isoelectric point of 8.68. The Sc-CyP encoding protein had the conserved site Trp128 (W128) ubiquitious of all cyclophilins in eukaryotes and the KSGKPLH48-54 region specific to cytoplasmic cyclophilins in plants. SDS-PAGE analysis and PPIase assay revealed that the expression product, with PPIase activity, was a fusion protein with a molecular weight about 25 and 18.4 kD of Sc- CyP plus 7 kD of His • Tag peptides. In real-time qPCR analysis, the Sc-CyP gene showed induced expression under PEG, NaCl, SA and H2O2 stresses, indicating it a stress-related gene for drought and salt stress, signal transduction and disease resistance response in sugarcane. Key words : Sugarcane (Saccharum officinarum), cyclophilin, PPIase, real-time quantitative PCR.

Construction of expression vector of CryIA(c) gene and its transformation in sugarcane

A plant expression vector, pGCryIA(c), was constructed by linking CryIA(c) gene which has UBI as promoter and NOS as terminator into pGreenII 0229. The size of pGCryIA(c) is 8,602 bp with bar (phosphinothricin acetyl transferase) gene as screening marker gene by using herbicide phosphinothricin (PPT) or Basta selection. The embryonic callus of GT94-119 was transformed with the plasmid of pUBCG0229 by bombardment. 203 regenerated plants were obtained after screening with PPT or Basta. Two transformation events were proved sucessful by PCR, PCR products sequencing and Dot-Southern blotting.