Lifei Yang

Enhanced salt tolerance of transgenic vegetable soybeans resulting from overexpression of a novel Δ1-pyrroline-5-carboxylate synthetase gene from Solanum torvum Swartz

Vegetable soybeans [Glycine max (L.) Merrill] are susceptible to salt stress and, thus, soil salinity can severely affect their growth and productivity. To enhance the salt tolerance of vegetable soybeans, a novel Solanum torvum Swartz Δ1-pyrroline-5-carboxylate synthetase gene (StP5CS, GenBank accession number: JN606861) that encodes a critical regulatory enzyme in proline biosynthesis was transformed into the cultivar NY-1001 using an Agrobacterium-mediated transformation method. PCR and Southern blot analyses indicated that two independent T0 fertile transgenic plants were generated. The transgenic plants transmitted the transgenes into their T1 progeny in a 3:1 ratio. The T2 and T3 homozygous transgenic lines (HTLs) were examined for salt tolerance in pot and hydroponic cultures, respectively. The StP5CS overexpression conferred salt tolerance in T2 and T3 HTLs. Under NaCl stress conditions, the leaf scorch scores of T2 and T3 HTLs were significantly lower than those of wild-type (WT) plants. The plant height, leaf area, relative chlorophyll content, and number of fresh pods of T2 and T3 HTLs were significantly higher than those of WT plants. Compared with WT plants, T2 and T3 HTLs had significantly higher levels of proline and significantly lower levels of membrane lipid peroxidation. These results indicate that StP5CS overexpression in HTLs results in enhanced salt tolerance associated with higher levels of proline accumulation under salinity stress and that StP5CS can be utilized to improve salinity tolerance in vegetable crop genetic engineering.

Overexpression of StNHX1 , a Novel Vacuolar Na + /H + antiporter gene from Solanum torvum , enhances salt tolerance in transgenic vegetable soybean

Soil salinity is the primary limiting factor affecting the growth and production of vegetable soybean [Glycine max (L.) Merrill] worldwide. Plants can survive in salinity conditions through the mechanism of sodium ion (Na+) compartmentalization, during which the vacuolar Na+/H+ antiporter (NHX) genes play a critical role. In the present study, a novel vacuolar NHX gene from Solanum torvum Swartz ‘Torvum Vigor’ (StNHX1; GenBank accession number: JN606860.1) was isolated and transferred into the vegetable soybean ‘Xinliaoxian’ through an Agrobacterium-mediated protocol. The PCR and southern blot analyses confirmed the successful integration of the exogenous genes. The GUS assay and semi-quantitative RT-PCR analysis showed that the alien genes were inherited by the progenies and that the StNHX1 gene was overexpressed in the T3 generation. The overexpression of this gene conferred high tolerance to salt stress. Under 100 mM NaCl conditions, the scorch scores and the Na+ and malondialdehyde (MDA) contents of the leaves of the transgenic lines were significantly lower as compared with the wild-type (WT) plants, while the K+/Na+ ratio, the contents of K+, relative chlorophyll and relative water, and some morphological traits of the transgenic plants were significantly higher than those of the WT. These results indicate that the overexpression of StNHX1 enhances the salt tolerance of vegetable soybean and that StNHX1 is one of the promising target genes that can be manipulated to improve the salinity tolerance of crops.

Overexpression of rice phosphate transporter gene OsPT2 enhances nitrogen fixation and ammonium assimilation in transgenic soybean under phosphorus deficiency

Phosphorus (P) deficiency is one of the major factors that limit legume nodulation and nitrogen (N) fixation, and thus legume productivity. In our previous study, we showed that three T2 transgenic soybean lines overexpressing rice phosphate transporter gene OsPT2 showed enhanced tolerance to low P stress. This study aimed to determine whether OsPT2 overexpression would increase N2 fixation and ammonium assimilation in three T3 homozygous transgenic lines (HTLs) under P deficiency in pot culture. Under low inorganic phosphate (Pi) conditions, the P accumulation, total N and total ureide concentrations were significantly higher in the T3 HTLs than in the wild type (WT) plants. Further, the T3 HTLs showed significantly better plant growth performance and nodule development than the WT plants under low-Pi conditions. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression levels of GmENOD40-1, GmENOD40-2 (two early nodulin genes), and GmLba (one leghemoglobin gene) were significantly increased in T3 HTLs under P deficiency at 24 and 32 d after inoculation (DAI). The increased transcript levels of GmGS1β1 and GmGS1β2 (two cytosolic glutamine synthetase genes) in the T3 HTLs were consistent with the increase in glutamine synthetase (GS, EC 6.3.1.2) activity at 32 DAI. Our results indicated that the overexpression of OsPT2 in T3 HTLs enhances N2 fixation and ammonium assimilation activity under low P stress.

Construction and characterization of a full-length cDNA library and identification of genes involved in salinity stress in wild eggplant (Solanum torvum Swartz)

The objectives of this paper were to construct a full-length cDNA library and to isolate genes that confer salt tolerance from the leaves of salinity-tolerant wild eggplant variety, ‘Torvum Vigor’ (Solanum torvum Swartz). A full-length cDNA library from the leaves was successfully constructed by a switching mechanism at 5′-end of RNA transcript (SMART) approach and a long-distance PCR (LD-PCR) technique. The titer of the primary cDNA library was 3.6 × 106 cfu·mL−1 and that of the amplified library was 1.2 × 1010 cfu·mL−1. Gel electrophoresis results showed that most of the cDNA inserts ranged from 0.40 to 2.5 kb, with a recombination rate of 99%. A total of 427 randomly selected positive clones were sequenced. After removing the unsuccessful reads, 364 datasets were obtained and have been submitted to the NCBI Nucleotide Sequence Database under GenBank accession numbers JK265131-JK265494. Among the 364 submitted sequences, 74.45% of them contained full-length coding regions. BLASTX analysis revealed that 62.36% of the ‘Torvum Vigor’ expressed sequence tags (ESTs) possessed homology to known or putative proteins of other organisms. Seven genes that might be responsible for the encoding of known proteins in other organisms were identified to confer salt tolerance. This evidence demonstrated that the cDNA library constructed was a full-length library of high quality. It could be a useful resource for further research in the cloning of stress-related genes, which could be utilized in the genetic improvement of vegetable crops using transgenic technology.

The phytotoxic effects of selenium–mercury interactions on root growth in Brassica rapa (LvLing)

Rapid industrial and agricultural development has dramatically increased the emission of selenium (Se) and mercury (Hg) into the environment. Combined soil pollution by Se and Hg poses a potential threat to crop production. However, no toxic effects of Hg–Se interactions on plants have been reported previously. In this study, we investigated the effects of Hg–Se interactions on biochemical and physiological indices in the roots of Brassica rapa (LvLing). Seedlings were treated hydroponically with solutions of mercury chloride (1 μM), sodium selenite (4 μM), or a combination of the two. Combined Hg+Se treatment significantly inhibited root growth, reduced root biomass, and enhanced reactive oxygen species (ROS) and malondialdehyde accumulation and led to a loss of plasma membrane integrity. The combined treatment increased glutathione peroxidase, glutathione S-transferase, and peroxidase activity, reduced superoxide dismutase activity, and had no effect on catalase activity. In addition, we detected increased glutathione concentrations in root tips and reduced ascorbic acid concentrations in the presence of Hg+Se relative to individual treatments with these elements. Thus, Hg–Se interactions enhanced oxidative injury, cell death, and phytotoxicity in B. rapa roots.