大麦HVA1基因和LEA蛋白与植物抗旱性的研究

干旱胁迫下,植物体内会积累多种蛋白以保护细胞免受脱水伤害,其中包括Lea蛋白。LEA蛋白在植物耐寒、耐盐碱、耐干旱性方面起重要作用。大麦HVA1基因编码的蛋白即属于第三组LEA蛋白,国内外学者对该基因的结构与功能进行了深入的研究。根据近年研究结果,本文对LEA蛋白的结构与功能,大麦HVA1基因的表达与调控,大麦HVA1基因高同源性序列的克隆以及转基因植物对HVA1基因抗旱性功能验证等方面进行综述。     

六棱大麦HVA1基因在烟草中遗传转化的研究

本研究依据HVA1基因序列克隆六棱大麦HVA1基因cDNA片段,构建Ubiquitin启动子驱动下的植物表达载体pCAMBIA1300-HVA1。然后通过三亲杂交法将重组质粒PCAMBIA1300-HVA1转入农杆菌LBA4404,并采用农杆菌介导法转化烟草。经PCR,PCR-Southern blotting和RT-PCR检测表明HVA1基因已整合进烟草基因组,并在转录水平上获得表达。功能验证的结果显示,转基因植株叶片的保水率提高了近1倍,暗示转基因烟草具有一定的抗旱潜力。     

Two New Group 3 LEA Genes of Wheat and Their Functional Analysis in Yeast

The group 3 late embryogenesis abundant （LEA） proteins are thought to protect cells from stresses associated with dehydration during periods of water deficit. To investigate the functions of different members of the group 3 LEA genes, we isolated and characterized two new group 3 LEA genes, namely TaLEA2 and TaLEA3, from wheat （Triticum aestivum L.） and introduced TaLEA2 and TaLEA3 into Saccharmyces cerevisiae to examine the effect of these genes on yeast cell tolerance to osmotic, salt, and cold stresses. The TaLEA2 gene encoded a protein of 211 amino acids and possessed five repeats of 11-mer amino acid motifs. The TaLEA3 gene encoded a polypeptide of 211 amino acids with nine repeated units. Overexpression of TaLEA2 and TaLEA3 improved stress tolerance in transgenic yeast cells when cultured in medium containing sorbitol, salt and-20℃ freezing treatments respectively. However, the yeast transformants with TaLEA2 seemed to be more tolerant to hyperosmotic and freezing stress than transformants with TaLEA3. This implies that a close relationship exists between function and the number of repeats of the 11- mer amino acid motif in the group 3 LEA protein.     

The Pattern of Changes in Photosynthetic Apparatus in Response to Cold Acclimation and De-acclimation in Two Contrasting Cultivars of Oilseed Rape

In spring and winter cultivars of oilseed rape (Brassica napus var. oleifera), acclimation of photosynthetic apparatus to cold was connected with the increase in activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) and sucrose-phosphate synthase (SPS). Conversely, cold de-acclimation entailed the decline of RuBPCO and SPS activities. The rate of this photosynthetic de-acclimation might depend on day temperature. On the other hand, temperature rise during de-acclimation (identical during the day and night) resulted in the improvement of photosynthetic activity measured by means of chlorophyll fluorescence. An increase in SPS activity (and even transitory increase in RuBPCO activity) was observed when the elongation growth rate (EGR) accelerated during de-acclimation. Throughout re-acclimation, plants with high EGR were unable to maintain or recover higher photosynthetic capacity, despite the fact that SPS activity remained high or even increased during re-acclimation.     

水稻中大麦Mlo和玉米Hm1抗病基因同源序列的分析和定位

大麦抗病基因Mlo和玉米抗病基因Hm1编码的产物不具有绝大多数植物抗病基因产物所含有的保守结构域。这两个抗病基因的作用机理也不符合基因对基因学说。从水稻中分离克隆了Mlo基因的同源序列OsMlo-1和玉米Hm1基因的同源序列DFR－1。利用水稻分子标记遗传连锁图,将OsMlo-1定位于水稻第六染色体的两俱RZ667和RG424之间;Osmlo-1距离这两个分子标记分别为20．6和6．0cM(centi-Morgan)。将DFR－1定位于水稻第一染色体两个分子标记R2635和RG462之间;DFR－1距离这两个分子标记分别为11．3和23．9cM。参照已发表的水稻分子标记连锁图,发现OsMlo-1和DFR－1的染色体位点分别与两个报道的水稻抗稻瘟病数量性状位点（QTL）有较好的对应关系。结果提示,水稻中与大麦Mlo 和玉米Hml同源的基因可能也参于抗病反应的调控。     

Regulation of RNA Synthesis by DNA-Dependent RNA Polymerases and RNases during Cold Acclimation in Winter and Spring Wheat

Chromatin DNA-dependent RNA polymerases and RNases activities were measured in winter and spring varieties to understand the overall regulation of RNA synthesis during cold acclimation. We found that total RNA polymerase activities were significantly higher in chromatin isolated from winter wheat compared to the spring wheat during the acclimation period. This increase was parallel to the increase in protein and RNA contents during hardening. The ratio of RNA polymerase I to RNA polymerase II activity was higher than 2 in winter wheat after 30 days of hardening compared, to a ratio of 0.90 under the nonhardening conditions. The increase in activity and the ratio of polymerase I to polymerase II was maintained after the separation of the enzymes from the template, suggesting that RNA synthesis is regulated in part at the enzyme level. On the other hand, the chromatin associated RNase activity decreased in both varieties during acclimation, indicating a nonspecific inhibition caused by low temperature rather than a selective genetic response associated with cold acclimation.     

Detection of Two Membrane Polypeptides Induced by Abscisic Acid and Cold Acclimation: Possible Role in Freezing Tolerance

Membrane proteins labeled in vivo from cold-acclimated and ABA-treatedalfalfa seedlings of two cultivars differing in cold-tolerancehave been compared by SDS polyacrylamide gel electrophoresisand fluorography. Results thus obtained indicate that severalqualitative changes occur in the membrane protein-profile specificallyin response to cold acclimation or ABA treatment. While somepolypeptides disappear from the non-acclimated protein patterns,others specifically appear in response to acclimation. Separationby two-dimensional gel electrophoresis and fluorography hasconfirmed the above and has enabled us to detect two proteinsof Mr 42 kDa and 120 kDa that are induced by both acclimationand ABA treatment in the freezing tolerant cultivar. (Received November 30, 1987; Accepted February 22, 1988)     

Expression of a Late Embryogenesis Abundant Protein Gene,HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice

A late embryogenesis abundant (LEA) protein gene, HVA1, from barley (Hordeum vulgare L.) was introduced into rice suspension cells using the Biolistic-mediated transformation method, and a large number of independent transgenic rice (Oryza sativa L.) plants were generated. Expression of the barley HVA1 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic rice plants. Second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity. Transgenic rice plants maintained higher growth rates than nontransformed control plants under stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions. We also found that the extent of increased stress tolerance correlated with the level of the HVA1 protein accumulated in the transgenic rice plants. Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under water-or salt-stress conditions. Thus, LEA genes hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.     

Overexpression of WsSGTL1 Gene of Withania somnifera Enhances Salt Tolerance,Heat Tolerance and Cold Acclimation Ability in Transgenic Arabidopsis Plants

Background

Sterol glycosyltrnasferases (SGT) are enzymes that glycosylate sterols which play important role in plant adaptation to stress and are medicinally important in plants like Withania somnifera. The present study aims to find the role of WsSGTL1 which is a sterol glycosyltransferase from W. somnifera, in plant’s adaptation to abiotic stress.

Methodology

The WsSGTL1 gene was transformed in Arabidopsis thaliana through Agrobacterium mediated transformation, using the binary vector pBI121, by floral dip method. The phenotypic and physiological parameters like germination, root length, shoot weight, relative electrolyte conductivity, MDA content, SOD levels, relative electrolyte leakage and chlorophyll measurements were compared between transgenic and wild type Arabidopsis plants under different abiotic stresses - salt, heat and cold. Biochemical analysis was done by HPLC-TLC and radiolabelled enzyme assay. The promoter of the WsSGTL1 gene was cloned by using Genome Walker kit (Clontech, USA) and the 3D structures were predicted by using Discovery Studio Ver. 2.5.

Results

The WsSGTL1 transgenic plants were confirmed to be single copy by Southern and homozygous by segregation analysis. As compared to WT, the transgenic plants showed better germination, salt tolerance, heat and cold tolerance. The level of the transgene WsSGTL1 was elevated in heat, cold and salt stress along with other marker genes such as HSP70, HSP90, RD29, SOS3 and LEA4-5. Biochemical analysis showed the formation of sterol glycosides and increase in enzyme activity. When the promoter of WsSGTL1 gene was cloned from W. somnifera and sequenced, it contained stress responsive elements. Bioinformatics analysis of the 3D structure of the WsSGTL1 protein showed functional similarity with sterol glycosyltransferase AtSGT of A. thaliana.

Conclusions

Transformation of WsSGTL1 gene in A. thaliana conferred abiotic stress tolerance. The promoter of the gene in W.somnifera was found to have stress responsive elements. The 3D structure showed functional similarity with sterol glycosyltransferases.     

Regulation of Adipogenesis and Key Adipogenic Gene Expression by 1, 25-Dihydroxyvitamin D in 3T3-L1 Cells

The functions of 1, 25-dihydroxyvitamin D (1, 25-(OH)₂D₃) in regulating adipogenesis, adipocyte differentiation and key adipogenic gene expression were studied in 3T3-L1 preadipocytes. Five concentrations (0.01, 0.1, 1, 10, 100nM) of 1, 25-(OH)₂D₃ were studied and lipid accumulation measured by Oil Red O staining and expression of adipogenic genes quantified using quantitative real-time PCR. Adipogenic responses to 1, 25-(OH)₂D₃ were determined on 6, and 12 h, and days 1-10 after induction of adipogenesis by a hormonal cocktail with or without 1, 25-(OH)₂D₃. In response to 1, 25-(OH)₂D₃ (1, 10, and 100 nM), lipid accumulation and the expression of PPARγ, C/EBPα, FABP4 and SCD-1 were inhibited through day 10, and vitamin D receptor expression was inhibited in the early time points. The greatest inhibitory effect was upon expression of FABP4. Expression of SREBP-1c was only affected on day 2. The lowest concentrations of 1, 25-(OH)₂D₃ tested did not affect adipocyte differentiation or adipogenic gene expression. The C/EBPα promoter activity response to 1, 25-(OH)₂D₃ was also tested, with no effect detected. These results indicate that 1, 25-(OH)₂D₃ inhibited adipogenesis via suppressing adipogenic-specific genes, and is invoked either during PPARγ activation or immediately up-stream thereof. Gene expression down-stream of PPARγ especially FABP4 was strongly inhibited, and we suggest that the role of 1, 25-(OH)₂D₃ in regulating adipogenesis will be informed by further studies of adipogenic-specific gene promoter activity.     

RsICE1基因表达和转基因水稻抗冷通路研究

以萝卜幼苗和水稻T1代转RsICE1基因(从抗寒萝卜植株中分离的一个编码碱性螺旋-环-螺旋低温胁迫转录因子,HQ891287)株系为材料,通过半定量RT-PCR及实时定量PCR等方法,分析RsICE1基因的表达、水稻转基因株系中RsICE1基因的遗传及冷诱导基因的表达情况.结果显示:(1)半定量RT PCR分析表明,RsICE1基因在萝卜的根、茎、叶中为组成型表达,在幼苗根和茎中表达量较强;RsICE1基因的表达水平能够被冷处理和NaCl处理诱导上调表达,但ABA和脱水处理无上调作用.(2)卡方测验表明,水稻转基因T1代潮霉素抗性发生了3∶1分离模式;Southern和Northern杂交结果表明,4个抗冷转基因株系中RsICE1基因均以单拷贝、单位点整合到水稻基因组并正常表达.(3)实时定量PCR分析表明,冷胁迫下,RsICE1基因超表达但对水稻OsDREB1A(AF300970)、OsDREB1B(AF300972)、OsDREB1F(AY785897)基因表达没有影响,表明RsICE1基因对转基因水稻抗寒性的影响不依赖OsDERB1冷反应通路.     

Disturbances in the Biosynthesis or Signalling of Brassinosteroids That Are Caused by Mutations in the HvDWARF,HvCPD and HvBRI1 Genes Increase the Tolerance of Barley to the Deacclimation Process

Journal of Plant Growth Regulation - Tolerance to deacclimation is an important physiological feature in plants in the face of global warming, which is resulting in incidents of increases in winter...     

Genome-Wide SNP Identification and Characterization in Two Soybean Cultivars with Contrasting Mungbean Yellow Mosaic India Virus Disease Resistance Traits

Mungbean yellow mosaic India virus (MYMIV) is a bipartite Geminivirus, which causes severe yield loss in soybean (Glycine max). Considering this, the present study was conducted to develop large-scale genome-wide single nucleotide polymorphism (SNP) markers and identify potential markers linked with known disease resistance loci for their effective use in genomics-assisted breeding to impart durable MYMIV tolerance. The whole-genome re-sequencing of MYMIV resistant cultivar ‘UPSM-534’ and susceptible Indian cultivar ‘JS-335’ was performed to identify high-quality SNPs and InDels (insertion and deletions). Approximately 234 and 255 million of 100-bp paired-end reads were generated from UPSM-534 and JS-335, respectively, which provided ~98% coverage of reference soybean genome. A total of 3083987 SNPs (1559556 in UPSM-534 and 1524431 in JS-335) and 562858 InDels (281958 in UPSM-534 and 280900 in JS-335) were identified. Of these, 1514 SNPs were found to be present in 564 candidate disease resistance genes. Among these, 829 non-synonymous and 671 synonymous SNPs were detected in 266 and 286 defence-related genes, respectively. Noteworthy, a non-synonymous SNP (in chromosome 18, named 18-1861613) at the 149^th base-pair of LEUCINE-RICH REPEAT RECEPTOR-LIKE PROTEIN KINASE gene responsible for a G/C transversion [proline (CCC) to alanine(GCC)] was identified and validated in a set of 12 soybean cultivars. Taken together, the present study generated a large-scale genomic resource such as, SNPs and InDels at a genome-wide scale that will facilitate the dissection of various complex traits through construction of high-density linkage maps and fine mapping. In the present scenario, these markers can be effectively used to design high-density SNP arrays for their large-scale validation and high-throughput genotyping in diverse natural and mapping populations, which could accelerate genomics-assisted MYMIV disease resistance breeding in soybean.     

Group 1 and 2 LEA protein expression correlates with a decrease in water stress induced protein aggregation in horsegram during germination and seedling growth

Plants produce an array of proteins as a part of a global response to protect the cell metabolism when they grow under environmental conditions such as drought and salinity that generate reduced water potential. The synthesis of hydrophilic proteins is a major part of the response to water deficit conditions. An increased expression of LEA proteins is thought to be one of the primary lines of defense to prevent the loss of intercellular water during adverse conditions. These LEA proteins are known to prevent aggregation of a wide range of other proteins. In this study we report the water stress induced protein aggregation and its abrogation followed by expression of group 1 and group 2 LEA proteins of water soluble proteomes in horsegram. Water stress caused an increased protein aggregation with magnitude and duration of stress in horsegram seedlings. Tissue-specific expression of LEA 1 protein decreased in the embryonic axis when compared to cotyledons in 24 h stressed seedlings. We found no cross reaction of LEA 1 with proteome of 48 h stressed embryonic axis and 72 h stressed root and shoot samples. However, LEA 2 antibodies were cross reacted with four polypeptides with different molecular mass in shoot tissue samples and found no reaction with root proteome as evidenced from immuno-blot analysis. The role of LEA proteins in relation to protein aggregation during water stressed conditions was discussed.