Lin Yuanzhen

Cloning and sequence analysis of a glucose-6-phosphate dehydrogenase gene PsG6PDH from freezing-tolerant Populus suaveolens

A 1 207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freezing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydrogenase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana tabacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.

One rapid and efficient method for isolation of total RNA from shoots regenerated in vitro of populus suaveolens

Total RNA was isolated from shoots regenerated in vitro of Populus suaveolens by the modified method of CTAB, and two clear bands of rRNA (28S and 18S) were observed in agarose electrophoresis. In addition, the values of OD260/OD280 and OD260/OD230 of extracted RNA were 2.12 and 2.23 respectively. The results show that RNA is little decomposed and the purity of RNA is high. Moreover, RNA isolated by the modified method of CTAB reagent had been successfully used for reverse transcription of P. suaveolens cDNAs and ideal special band was observed.

Assessment of rhizospheric microorganisms of transgenic Populus tomentosa with cowpea trypsin inhibitor (CpTI) gene

To have a preliminary insight into biosafety of genetically transformed hybrid triploid poplars (Populus tomentosa × P. bolleana) × P. tomentosa with the cowpea trypsin inhibitor (CpTI) gene, two layers of rhizospheric soil (from 0 to 20 cm deep and from 20 to 40 cm deep, respectively) were collected for microorganism culture, counting assay and PCR analysis to assess the potential impact of transgenic poplars on non-target microorganism population and transgene dispersal. When the same soil layer of suspension stock solution was diluted at both 1:1 000 and 1:10 000 rates, there were no significant differences in bacterium colony numbers between the inoculation plates of both transgenic and non-transgenic poplars. The uniform results were revealed for both soil layer suspension solutions of identical poplars at both dilution rates except for non-transgenic poplars at 1:10000 dilution rates from the same type of soil. No significant variation in morphology of both Gram-positive and Gram-negative bacteria was observed under the microscope. The potential transgene dispersal from root exudates or fallen leaves to non-target microbes was repudiated by PCR analysis, in which no CpTI gene specific DNA band was amplified for 15 sites of transgenic rhizospheric soil samples. It can be concluded that transgenic poplar with the CpTI gene has no severe impact on rhizospheric microorganisms and is tentatively safe to surrounding soil micro-ecosystem.

Plant antifreeze proteins and their expression regulatory mechanism

Low temperature is one of the major limiting environmental factors which constitutes the growth, development, productivity and distribution of plants. Over the past several years, the proteins and genes associated with freezing resistance of plants have been widely studied. The recent progress of domestic and foreign research on plant antifreeze proteins and the identification and characterization of plant antifreeze protein genes, especially on expression regulatory mechanism of plant antifreeze proteins are reviewed in this paper. Finally, some unsolved problems and the trend of research in physiological functions and gene expression regulatory mechanism of plant antifreeze proteins are discussed.

Construction and characterization of cDNA library from water-stressed plantlets regenerated in vitro of Populus hopeiensis

In order to isolate and clone water-stress-responsive genes, total RNA was extracted from water-stressed plantlets regenerated in vitro of Populus hopeiensis using a QIAGEN RNeasy Plant Mini Kit. CDNA, synthesized by LD-PCR with the SMART cDNA Library Construction Kit, was in vitro packaged into a phage λTriplEx2 vector. The resulting primary library and amplified library have a titer of 1.68 × 106 and 1.69× 109 pfu·mL−1 respectively. The combination ratio reached 98.8% and the average size of inserts was about 800 bp. In addition, the percentage of inserted fragments (>400 bp) was approximately 90%. The results indicate that a cDNA library has been successfully constructed.