Overexpression of phospholipase Dα gene enhances drought and salt tolerance of Populus tomentosa

The cDNA of AtPLDα (Arabidopsis thaliana Phospholipase Dα) gene was introduced into P. tomentosa (Populus tomentosa) under the control of the Cauliflower mosaic virus 35S promoter. Southern and Northern blot analyses suggested that the AtPLDα gene has been transferred into the P. tomentosa genome. No obvious morphological or developmental difference was observed between the transgenic and wild-type (WT) plants. Drought and salt tolerance and gene expression of seedlings of several transgenic lines and WT plants (control) were studied. The results showed that the rhizogenesis rate and the average root-length of transgenic lines were significantly higher than WT plants after mannitol and NaCl treatment under the same growth conditions. Northern blot analysis indicated that the higher the PLDα expression in the transgenic plants, the more tolerant the transgenic plants are to drought and salt treatment. Meanwhile, another group of these transgenic lines and WT plants (control) were treated with PEG6000 and NaCl separately. The contents of chlorophylls and the activities of some anti-oxidant enzymes (superoxide dismutase, guaiacol peroxidase and catalase) as well as malondialde-hyde and relative electrical conductivity were analyzed. Altogether, our results demonstrated that overexpression of the PLDα gene can enhance the drought and salt tolerance in transgenic P. tomen-tosa plants.     

Overexpression of phospholipase <Emphasis Type="Italic">D</Emphasis>α gene enhances drought and salt tolerance of <Emphasis Type="Italic">Populus tomentosa</Emphasis>

The cDNA of AtPLDa （Arabidopsis thaliana Phospholipase Da） gene was introduced into P. tomentosa （Populus tomentosa） under the control of the Cauliflower mosaic virus 35S promoter. Southern and Northern blot analyses suggested that the AtPLDa gene has been transferred into the P. tomentosa genome. No obvious morphological or developmental difference was observed between the transgenic and wild-type （WT） plants. Drought and salt tolerance and gene expression of seedlings of several transgenic lines and WT plants （control） were studied. The results showed that the rhizogenesis rate and the average root-length of transgenic lines were significantly higher than WT plants after mannitol and NaCI treatment under the same growth conditions. Northern blot analysis indicated that the higher the PLDa expression in the transgenic plants, the more tolerant the transgenic plants are to drought and salt treatment. Meanwhile, another group of these transgenic lines and WT plants （control） were treated with PEG6000 and NaCI separately. The contents of chlorophylls and the activities of some anti- oxidant enzymes （superoxide dismutase, guaiacol peroxidase and catalase） as well as malondialdehyde and relative electrical conductivity were analyzed. Altogether, our results demonstrated that overexpression of the PLDa gene can enhance the drought and salt tolerance in transgenic P. tomentosa plants.     

Molecular characterization of GmNHX2, a Na＋/H＋ antiporter gene homolog from soybean,and its heterolosous expression to improve salt tolerance in Arabidopsis

Na＋/H＋ antiporters have been well documented to enhance plant salt tolerance by regulating cellular ion homeostasis. Here, a putative Na＋/H＋ antiporter gene homolog GmNHX2 from soybean was cloned and predicted to encode a protein of 534 amino acids with 10 putative transmembrane domains. GmNHX2 was expressed in all soybean plant tissues but enriched in roots and its expression was induced by NaCI and polyethylene glycol （PEG） treatments. GmNHX2 exhibits greater sequence similarity with LeNHX2 and AtNHX6 than that of AtNHX1 and AtSOS1. Although phylogenetic analysis clustered GmNHX2 with organellar （tonoplast and vesicles） antiporters, the GmNHX2-EGFP （enhanced green fluorescent protein） fusion protein was possibly localized in the plasma membrane or organelle membrane of transgenic plant cells, Furthermore, transgenic Arabidopsis plants expressing GmNHX2 were more tolerant to high NaCl concentrations during germination and seedling stages when compared with wild-type plants. These results suggest that GmNHX2 is a membrane Na＋/H＋ antiporter and may function to regulate ion homeostasis under salt stress.     

Over-expression of an Arabi-dopsisd δ-OAT gene enhances salt and drought tolerance in transgenic rice

δ-OAT, ornithine-δ-aminotransferase, is the key enzyme involved in proline biosynthesis. In this study the Arabidopsis δ-OAT gene was transferred into rice (Oryza sativa L. ssp japonica cv. Zhongzuo 321), whose successful integration was demonstrated by PCR and Southern blot analysis. The over-expression of the gene in transgenic rice was also confirmed. Biochemical analysis showed that, under salt or drought stress conditions, proline contents in the leaves and roots in transgenic rice plants were 5- to 15-fold of those in non-transgenic controls. Under stress conditions, germinating rate of transgenic lines is higher than that of controls. Although the growth of rice plants tested were more and more retarded with the increasing of NaCI concentration, the transgenic plants grow faster compared to the controls under the same stress condition. Meanwhile, the resistance to KCl and MgSO4 stresses was also found enhanced in transgenie rice. Furthermore, the over-expression of δ-OAT also improved the yield of transgenic plants under stress conditions. The average yield per plant of transgenic lines increases about 12%--41% more than that of control line sunder 0.1 mol/L NaCI stress. These data indicated that the over-expression of δ-OAT, with the accumulation of proline, resulted in the enhancement of salt and drought tolerance and an increase of rice yield, which is of significance in agriculture.     

Disease-tolerance of transgenic tobacco plants expressing Ah-AMP gene of Amaranthus hypochondriacus

An antimicrobial peptide gene from Amaranthus hypochondriacus, Ah-AMP, was amplified by PCR and cloned. Sequence analysis results revealed that this gene is 261 bp in length encoding a precursor polypeptide of 87 amino acid residues. Ah-AMP gene was inserted in the binary vector pBin438 to construct a plant expression vector pBinAH916. Leave explants of Nicotiana tabacum var. SR1 were transformed with Agrobacterium tumefaciens LBA4404 harboring the above expression vector. Results from PCR, Southern and Northern blot analyses confirmed that the Ah-AMP gene had been integrated into the tobacco genome and was transcribed at mRNA level. Two bacterial-resistant transgenic plants were selected by inoculating the plants with Pseudomonas solanacearum and statistic analysis of two T1 lines showed that the resistance increased by 2.24 and 1.62 grade and the disease index decreased by 49.6% and 37.3% respectively when compared with the non-transformed control plants SR1. The results from challenging the plants with inoculums of Phytophthora parasitica showed that the symptom development was delayed and disease index was significantly reduced. These results suggest that Ah-AMP gene may be a potentially valuable gene for genetic engineering of plant for disease-resistance.     

Molecular mechanism of dehydrin in response to environmental stress in plant

Dehydrins, known as the D-11 subgroup of late embryogenesis abundant (LEA) protein, are an immunologically distinct family of proteins, which typically accumulate in desiccation-tolerant seed embryo or in vegetative tissues in response to various environmental stresses such as drought, salinity and freezing. The existence of conservative sequences designated as K, S, and Y segments is a structural feature of dehydrins, and the K segment found in all dehydrins represents a highly conserved 15 amino acid motif (EKKGIMDKIKEKLPG) and forms an amphiphilicα-helix. According to the arrangement of these domains and clustering analysis, dehydrins are subdivided into 5 subtypes: YnSK, Kn, KnS, SKn and YnK. Different types of dehydrins are induced by different environmental stress in plants. Study results showed that dehydrins might play important protective roles under abiotic stress via a number of different mechanisms, including improving or protecting enzyme activities by the cryoprotective activity in responding to freeze/thaw or dehydration; stabilizing vesicles or other endomembrane structures by function as the membrane stabilizer during freeze induced dehydration, and preventing the membrane system from the oxidative damage induced by reactive oxygen radicals as the radical scavenger. Here, the gene expression and molecular mechanisms of dehydrin in response to stress in plants are discussed.     

Molecular mechanism of dehydrin in response to environmental stress in plant

Dehydrins， known as the D-11 subgroup of late embryogenesis abundant (LEA) protein, are an immunologically distinct family of proteins, which typically accumulate in desiccation-tolerant seed embryo or in vegetative tissues in response to various environmental stresses such as drought, salinity and freezing. The existence of conservative sequences designated as K, S, and Y segments is a structural feature of dehydrins, and the K segment found in all dehydrins represents a highly conserved 15 amino acid motif (EKKGIMDKIKEKLPG) and forms an amphiphilic α-helix. According to the arrangement of these domains and clustering analysis, dehydrins are subdivided into 5 subtypes: YnSK, Kn, KnS, SKn and YnK. Different types of dehydrins are induced by different environmental stress in plants. Study results showed that dehydrins might play important protective roles under abiotic stress via a number of different mechanisms, including improving or protecting enzyme activities by the cryoprotective activity in responding to freeze/thaw or dehydration; stabilizing vesicles or other endomembrane structures by function as the membrane stabilizer during freeze induced dehydration, and preventing the membrane system from the oxidative damage induced by reactive oxygen radicals as the radical scavenger. Here, the gene expression and molecular mechanisms of dehydrin in response to stress in plants are discussed.     

Isolation of cDNAs Enconding Two Distinct Transcription Factors Binding to DRE cis-acting Element Involved in Cold- and drought-induced Expression of Arabidopsis rd29A Gene

ＩｎｔｒｏｄｕｃｔｉｏｎＳｅｖｅｒｅｅｎｖｉｒｏｎｍｅｎｔａｌｃｈａｎｇｅｓ ,ｓｕｃｈａｓｌｏｗ ｔｅｍｐｅｒａｔｕｒｅ ,ｄｒｏｕｇｈｔａｎｄｈｉｇｈ ｓａｌｔ ,ａｆｆｅｃｔｔｈｅｇｒｏｗｔｈａｎｄｄｅｖｅｌｏｐｍｅｎｔｏｆ ｐｌａｎｔｓａｎｄｔｈｅｐｒｏｄｕｃｔｉｖｉｔｙｏｆｃｒｏｐｓ .Ｐｌａｎｔｃｅｌｌｃａｎｎｏｔｏｂｔａｉｎｗａｔｅｒｗｈｅｎｓｕｂｊｅｃｔｅｄｔｏｄｒｏｕｇｈｔｏｒｈｉｇｈ ｓａｌｔｃｏｎｄｉｔｉｏｎ .Ｌｏｗ ｔｅｍｐｅｒａｔｕｒｅａｌｓｏｒｅｄｕｃｅｓｗａｔｅｒｓｔａｔｅｏｆ …     

拟南芥NCED3基因在水稻中的过量表达可以提高水稻的干旱胁迫耐受性

9-顺-环氧类胡萝卜素双加氧酶(NCED)是高等植物脱落酸(ABA)生物合成途径中的关键酶,其催化的裂解反应直接生成ABA的前体物质.本文应用拟南芥rd29A诱导型启动子和35S组成型启动子成功地将AtNCED3基因在水稻中过量表达,通过耐旱性筛选实验证明转基因水稻对干旱胁迫的耐受性有了显著提高,并且该优良性状在T2代中得到稳定遗传.进一步的分析表明,过量表达AtNCED3可以促进转基因水稻种子的休眠;在含rd29A诱导型启动子的转基因水稻中检测到ABA下游基因OsBZ8的表达;推测 AtNCED3在水稻中的过量表达可能会提高水稻中内源 ABA的水平,从而提高植株的耐旱性.     

白桦BpGRAS1基因的克隆及耐盐功能分析

【目的】 GRAS转录因子是植物特有的转录因子家族之一,在植物响应盐、干旱等非生物胁迫中发挥重要的调控作用。从白桦(Betula platyphylla )中克隆GRAS转录因子基因,研究其耐盐功能,为研究木本植物GRAS转录因子的抗逆机制奠定理论基础。【方法】 在白桦转录组数据库中获得一个GRAS转录因子基因,命名为BpGRAS1 (GenBank 登录号: MN117546.1)。利用生物信息学进行多序列比对、构建进化树。分别构建植物过表达(pROKⅡ-BpGRAS1) 及抑制表达(pFGC5941-BpGRAS1) 载体。利用农杆菌介导高效瞬时遗传转化体系获得BpGRAS1基因瞬时过表达(OE)、抑制表达(IE) 及对照 (WT) 白桦植株。通过实时荧光定量RT-PCR(qRT-PCR) 技术分析盐胁迫下OE、IE及WT植株中BpGRAS1基因的表达情况,鉴定转基因植株中BpGRAS1的表达效率是否响应盐胁迫。在盐胁迫下比较了BpGRAS1基因瞬时过表达、抑制表达及对照白桦植株的电解质渗透率、失水率、丙二醛(MDA) 含量、过氧化物酶 (POD) 和超氧化物歧化酶 (SOD) 活性。【结果】 BpGRAS1基因的开放阅读框为1 425 bp,编码 474个氨基酸。BpGRAS1具有GRAS家族的序列特征,在C端的氨基酸序列相似度较高,与AtSHR亲缘关系较近。盐胁迫处理下,BpGRAS1的表达量升高,过表达植株中表达量高于对照,抑制表达植株中表达量低于对照,说明BpGRAS1受盐胁迫诱导,成功获得过表达及抑制表达植株。过表达BpGRAS1基因能降低白桦在盐胁迫下的电解质渗透率、失水率及 MDA 的积累,并显著增强了 POD 和 SOD 酶的活性,从而提高转基因植株的耐盐性。【结论】 BpGRAS1基因响应盐胁迫,过表达BpGRAS1基因降低了盐胁迫下植株细胞受损程度,通过增强POD 和 SOD 活性提高白桦的耐盐能力。     

转蔗糖: 蔗糖-1-果糖基转移酶基因提高烟草的耐旱性

蔗糖: 蔗糖-1-果糖基转移酶（sucrose: sucrose 1-fructosyltransferase, 1-SST）以蔗糖为底物催化生成蔗果三糖等低聚合度的果聚糖.将从莴苣中克隆的1-SST基因重组到pCAMBIA1300-als中，构建了在CaMV 35S启动子调控下的植物表达载体，利用农杆菌介导的叶盘转化法将1-SST基因导入烟草中，PCR和Southern杂交检测表明获得了转基因植株，RT-PCR结果表明该基因在烟草中正常表达. 对T₀代转基因烟草进行的耐旱性分析结果表明，干旱胁迫6d的转基因植株丙二醛含量和电解质渗漏率显著低于未转基因对照，叶片相对含水量下降速度也明显比对照慢. 对转基因植株叶片糖分分析表明，转基因烟草植株积累果聚糖，并在干旱胁迫后含量明显增加，而未转基因对照植株不积累果聚糖. 在14%PEG溶液中未转基因烟草种子的萌发率仅为转基因烟草种子的一半；在附加200mmol/L甘露醇的培养基中未转基因烟草种子根的生长明显受到抑制，而转基因烟草根的生长发育正常. 以上研究结果表明，转1-SST基因烟草植株耐旱性的提高可能与该基因的表达有关.     

一种带有表型标记基因的诱导型抗旱植物表达载体的构建

为了克服组成型表达转录因子基因影响转基因植物性状的缺点,并构建一种具有级联放大作用并带有表型标记的诱导型植物双价表达载体。研究采用PCR方法从拟南芥克隆获得冷诱导转录因子CBF3基因,蜡质合成相关WIN1基因,干旱诱导RD29A基因启动子和冷诱导的LEA14基因启动子,并用CBF3转录因子所调控的下游RD29A基因启动子和LEA14基因启动子分别驱动CBF3基因和W1N1基因表达,构建了双价植物表达载体RD29AP-CBF3／LEA14P—WIN1／pcAMBIA2201。我们预测在转基因植物中,该表达系统可在干旱等逆境信号存在条件下,通过级联放大的方式诱导表达,在增加植物抗逆性的同时,增加叶片表层蜡质的积累,从而易于表型识别。本研究为利用花粉管通道法转化棉花,提高抗逆转基因棉花田间筛选的效率奠定了基础。     

单子叶植物的FLP/frt位点特异性重组系统的构建

来自于啤酒酵母2?μm质粒的FLP/frt位点特异性重组系统可用于转基因植物（细胞）筛选完成后去除选择标记基因.水稻肌动蛋白actin基因启动子和玉米泛素ubiquitin基因启动子可有效驱动单子叶植物中外源基因的转录.为培育去除选择标记基因的抗盐耐旱单子叶转基因植物，构建了适合于单子叶植物的FLP/frt位点特异性重组系统. 该系统由含有FLP重组酶基因的植物表达载体pCAMBIA3300 Ubi FLP bar和含有frt位点的植物双抗（抗除草剂、抗盐耐旱）表达载体pCAMBIA1300 Ubi TsPPase frt als frtm以及pCAMBIA1300 Actin1 AtNHX1 frt als frtm组成.     

Expression of the SOD gene from Trichoderma harzianum in Saccharomyces cerevisiae

Superoxide dismutases are metalloproteins which play a major role in defense against oxygen radicalmediated toxicity in aerobic organisms. Such proteins are important endogeneity cytoprotection factor involving defence. A 751-bp full-length cDNA sequence of an SOD gene was isolated from the Trichoderma harzianum. The full-length cDNA of the SOD gene consists of one 465-bp open reading frame nucleotide, which encodes a 15.7-kDa polypeptide consisting of 154 amino acid residues. Sequence analysis revealed that SOD gene has more than 72%-86% amino acid sequence homology with those of other fungi. The SOD gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2-SOD. SOD expressed by pYES2-SOD was induced by galactose. We test whether SOD could offer abiotic stress resistance when it was introduced into yeast ceils. A transgenic yeast harboring T. harzianum SOD was generated under the control of a constitutively expressed GAL promoter. The results indicated that SOD yeast transformants had significantly higher resistance to salt and drought stress.