Hong-Mei Zhang

Enhancement of salt tolerance in alfalfa transformed with the gene encoding for betaine aldehyde dehydrogenase

Gene BADH was transformed into alfalfa (Medicago sativa L.) through Agrobacterium-mediated transformation method, and salt tolerance of the transgenic progenies was assessed. Among 247 plants obtained after transformation, 43 were positive by polymerase chain reaction (PCR) detection using the primers specific for BADH gene. Reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that gene BADH was expressed in 42 plants at transcriptional level. Southern blotting analysis also showed that the target was integrated into Medicago genome. The transgenic plants grew vigorous in salt stress condition, whereas the wild type plants was retarded and did not survive after cradle. The relative electrical conductivity and malondialdehyde (MDA) contents in the T1 transgenic plants were lower, but peroxidase (POD) and superoxide dismutase (SOD) activities were higher than those of the wild type plants. These results demonstrated that the expression of foreign BADH gene enhanced the salt tolerance in transgenic alfalfa.

The dynamic changing of Ca2+ cellular localization in maize leaflets under drought stress

Maize cultivar zhengdan958 was selected as materials. The sub-cellular distribution of soluble calcium at different phases was shown by the potassium-pyroantinonate-precipitation method and transmission electron microscopy. The results showed that the deposits of calcium antimonate as the indicator for Ca(2+) localization were mainly concentrated within the vacuoles and intercellular spaces without PEG treatment. Firstly, when the leaf was treated with PEG, the Ca(2+) level increased remarkably in the cytoplasm, but considerably decreased in vacuoles and intercellular gaps. Meanwhile, the level of Ca(2+) also increased in chloroplast and nucleus. When the treatment continued, the level of Ca(2+) in chloroplasts and nucleus continued to increase and some cells and chloroplasts finally disintegrated, showing that there is a relationship between the distribution of Ca(2+) and the super-microstructure of cells. Ca(2+) plays a role in the plant drought resistance. The changes of cytosolic Ca(2+) localization in cells treated by ABA, EGTA, Verapamil and TFP were investigated too. The increase of cytosolic calcium induced by ABA was mainly caused by calcium influx. Calmodulin participated in ABA signal transduction, which was indicated by the variation of cytosolic Ca(2+)/CaM concentration change induced by ABA. The above results provided a direct evidence for calcium ion as an important signal at the experimental cellular level.

The vacuolar Na+–H+ antiport gene TaNHX2 confers salt tolerance on transgenic alfalfa (Medicago sativa)

TaNHX2, a vacuolar Na+-H+ antiport gene from wheat (Triticum aestivum L.), was transformed into alfalfa (Medicago sativa L.) via Agrobacterium-mediated transformation to evaluate the role of vacuolar energy providers in plant salt stress responses. PCR and Southern blotting analysis showed that the target gene was integrated into the Medicago genome. Reverse transcription-PCR indicated that gene TaNHX2 was expressed at the transcriptional level. The relative electrical conductivity in the T2 transgenic plants was lower and the osmotic potential was higher compared to the wild-type plants under salt stress conditions. The tonoplast H+-ATPase, H+-pyrophosphatase (PPase) hydrolysis activities and ATP-dependent proton pump activities in transgenic plants were all higher than those of wild-type plants, and the enzyme activities could be induced by salt stress. The PPi-dependent proton pump activities decreased when NaCl concentrations increased from 100mM to 200mM, especially in transgenic plants. The vacuolar Na+-H+ antiport activities of transgenic plants were 2-3 times higher than those of the wild -type plants under 0mM and 100mM NaCl stress. Na+-H+ antiport activity was not detectable for wild-type plants under 200mM NaCl, but for transgenic plants, it was further increased with an increment in salt stress intensity. These results demonstrated that expression of the foreign TaNHX2 gene enhanced salt tolerance in transgenic alfalfa.

Expression of maize heat shock transcription factor gene ZmHsf06 enhances the thermotolerance and drought-stress tolerance of transgenic Arabidopsis

Based on the information of 25 heat shock transcription factor (Hsf) homologues in maize according to a genome-wide analysis, ZmHsf06 was cloned from maize leaves and transformed into Arabidopsis thaliana (L. Heynh.) (ecotype, Col-0). Three transgenic positive lines were selected to assess the basic and acquired thermotolerance and drought-stress tolerance under stresses and for some physiological assays. The sequence analysis indicates that ZmHsf06 contained the characteristic domains of class A type plant Hsfs. The results of qRT-PCR showed that the expression levels of ZmHsf06 were elevated by heat shock and drought stress to different extents in three transgenic lines. Phenotypic observation shows that compared with the Wt (wild-type) controls, the overexpressing ZmHsf06 of Arabidopsis plants have enhanced basal and acquired thermotolerance, stronger drought-stress tolerance and growth advantages under mild heat stress conditions. These results are further confirmed by physiological and biochemical evidence that transgenic Arabidopsis plants exhibit higher seed germination rate, longer axial-root length, higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), higher leaf chlorophyll content, but lower relative electrical conductivity (REC), malondialdehyde (MDA) and osmotic potential (OP) than the Wt controls after heat shock and drought treatments. ZmHsf06 may be a central representative of maize Hsfs and could be useful in molecular breeding of maize or other crops for enhanced tolerances, particularly during terminal heat and drought stresses.

Improvement of soybean transformation via Agrobacterium tumefaciens methods involving α-aminooxyacetic acid and sonication treatments enlightened by gene expression profile analysis

Key messageAntagonists and sonication treatment relieved the structural barriers ofAgrobacteriumentering into cells; hindered signal perception and transmission; alleviated defense responses and increased cell susceptibility toAgrobacteriuminfection.AbstractSoybean gene expression analysis was performed to elucidate the general response of soybean plant to Agrobacterium at an early stage of infection. Agrobacterium infection stimulated the PAMPs-triggered immunity (BRI1, BAK1, BZR1, FLS2 and EFR) and effector-triggered immunity (RPM1, RPS2, RPS5, RIN4, and PBS1); up-regulated the transcript factors (WRKY25, WRKY29, MEKK1P, MKK4/5P and MYC2) in MAPK pathway; strengthened the biosynthesis of flavonoid and isoflavonoid in the second metabolism; finally led to a fierce defense response of soybean to Agrobacterium infection and thereby lower transformation efficiency. To overcome it, antagonist α-aminooxyacetic acid (AOA) and sonication treatment along with Agrobacterium infection were applied. This novel method dramatically decreased the expression of genes coding for F3′H, HCT, β-glucosidase and IF7GT, etc., which are important for isoflavone biosynthesis or the interconversion of aglycones and glycon; genes coding for peroxidase, FLS2, PBS1 and transcription factor MYC2, etc., which are important components in plant–pathogen interaction; and genes coding for GPAT and α-l-fucosidase, which are important in polyesters formation in cell membrane and the degradation of fucose-containing glycoproteins and glycolipids on the external surface of cell membrane, respectively. This analysis implied that AOA and sonication treatment not only relieved the structural membrane barriers of Agrobacterium entering into cells, but also hindered the perception of ‘invasion’ signal on cell membrane and intercellular signal transmission, thus effectively alleviated the defense responses and increased the cell susceptibility to Agrobacterium infection. All these factors benefit the transformation process; other measures should also be further explored to improve soybean transformation.

Cloning, Localization and Expression Analysis of ZmHsf-like Gene in Zea mays

Using homology cloning method, a heat shock transcription factor(Hsf) like gene, ZmHsf-like, was cloned from maize(Zea mays) leaves. Sequence analyses showed that the open reading frame(ORF) of the gene ZmHsf-like is 1 404 bp long, encoding 467 amino acids. The sequence of amino acids encoded by ZmHsf-like contains the most conserved and typical DNA-binding domain of Hsf family. By bombardment into onion epidermis, we firstly found that the ZmHsf-like was subcellular-located in nucleus. NucPred analysis revealed there is a classic NLS of KKRR peptide in protein. Real-time PCR showed that ZmHsf-like gene expressed in leaves, stems and roots of maize seedlings under normal growth conditions, and the highest expression level was in roots, lower in leaves and the lowest in stems. The ZmHsf-like gene expression could be up-regulated by heat shock, PEG, ABA, and H2O2 in different degrees, among which the heat shock and ABA worked more efficiently. Obvious differences of the peak value and its corresponding time point of ZmHsf-like gene expression were observed among treatments. Experiments with inhibitor further suggested that the up-regulation ZmHsf-like gene expression of heat shock was H2O2-dependent while the induction of ZmHsf-like with PEG did not depend on the existence of H2O2. These results pointed out that ZmHsf-like gene probably regulates responsive reactions to abiotic stresses especially heat shock and drought through different signal transduction pathways.

Analysis of T-DNA Flanking Sequences and Event Specific Detection of Transgenic Alfalfa with Gene BADH : Analysis of T-DNA Flanking Sequences and Event Specific Detection of Transgenic Alfalfa with Gene BADH

为了从分子水平上鉴别不同的转基因株系，以转 BADH 基因苜蓿的T 0 代基因组DNA为模版，采用热不对称交错PCR(TAIL-PCR)方法分离其外源基因插入位点的侧翼序列，获得了B127株系的左翼序列和右翼序列，以及B125、B138、B295和B196株系的左翼序列。侧翼序列特征分析表明，有的T-DNA边界序列被删除，有的边界序列被保留，并填充了一段未知来源的核苷酸序列。根据侧翼序列中插入载体序列和紧邻插入序列的基因组序列特征，分别设计PCR扩增的上、下游引物，并对获得的42个转 BADH 株系分别进行左、右翼序列的扩增，结果表明，转基因植株B106、B125、B138、B157、B158、B289、B295、B305和B127具有相同的扩增条带，B203、B220、B223和B196具有相同的扩增条带，说明这些株系可能仅来源于2个转化事件。本研究建立的事件特异性检测方法可以准确地将不同的转化株系区别开来。

Analysis of T-DNA Flanking Sequences and Event-Specific Detection of Transgenic Alfalfa with Gene Encoding Betaine Aldehyde Dehydrogenase

The gene encoding betaine aldehyde dehydrogenase (BADH) has been transformed into alfalfa (Medicago sativa L.) and resulted in 42 transgenic plants with improved salt tolerance. However, these transgenic lines were derived from the same transformant vector, which were unable to distinguish them from each other using common methods. For differentiating these transformants at molecular level, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to separate the T-DNA flanking sequences for identifying transgenic plants in event-specific detection. A total of 6 sequences flanking either the left or the right borders of the T-DNA were obtained. The left border sequence of T-DNA was completely deleted from the vector and not integrated into the genome of alfalfa in the transgenic plant B196. Although the left border flanking sequence in the transgenic plant B127 was reserved, it was filled with a DNA sequence of unknown origin. The forward and backward primers for PCR were designed based on the characteristics of the flanking sequences originating from the vector sequence and the alfalfa genomic sequence adjacent to the integrated vector sequence, respectively. According to the result of PCR amplification in the 42 BADH-transgenic lines, plants B106, B125, B127, B138, B157, B158, B289, B295, and B305 presented the same amplification banding pattern. Plants B196, B203, B220, and B223 exhibited the same banding pattern, which was different from that amplified from other plants. These results indicated that the plants with identical amplification banding patterns may come from the same transformation event.