转GsPPCK1基因苜蓿植株的获得及其耐碱性分析

磷酸烯醇式丙酮酸羧化酶激酶(phosphoenolpyruvate carboxylase kinase, PPCK)是一种钙不依赖的丝氨酸/苏氨酸(Ser/Thr)类蛋白激酶, 参与碳氮代谢等多个生物学过程, 然而其在碱胁迫反应中的作用尚未见报道。本研究在前期野生大豆碱胁迫基因表达谱数据基础上, 采用同源克隆的方法分离野生大豆(Glycine soja)PPCK1基因, 该基因与大豆(Glycine max) PPCK1基因(AY374445)具有99%的相似性, 被命名为GsPPCK1。在50 mmol L^–1 NaHCO₃ 胁迫处理3 h内, 根和叶中GsPPCK1基因上调表达, 属碱胁迫早期应答基因。通过农杆菌介导法对肇东苜蓿进行遗传转化, 并对RT-PCR阳性的超表达转基因株系进行耐碱性分析表明, 在100 mmol L^–1 NaHCO₃处理15 d后转基因株系生长状态良好, 而非转基因对照株系明显萎蔫、失绿、甚至死亡; 转基因株系的丙二醛含量和相对质膜透性显著低于非转基因株系(P<0.05), 而叶绿素含量和根系活力显著高于非转基因对照(P<0.05), 说明超量表达GsPPCK1基因增强了苜蓿的耐碱能力。以上结果表明, GsPPCK1参于植物耐碱胁迫反应过程, 在碱胁迫基因工程研究领域具有良好的理论和实际应用意义。     

The Suaeda liaotungensis kitag betaine aldehyde dehydrogenase gene improves salt tolerance of transgenic maize mediated with minimum linear length of DNA fragment

In this research we established a particular vector-free and marker-free plant transformation system of maize to overcome the obstacles of biosafety limits. The BADH gene was introduced into maize by pollen-tube pathway, using the principle of minimum linear length of the transformation element, which was composed of only the BADH gene, expression regulatory sequence (35S CAMV promoter, NOS terminator), and T-DNA border sequence at both sides. Twenty-seven of 2076 transformed samples were positive in PCR amplification and the PCR positive rate of T₁ generation was 1.3%. Further Southern blotting results indicated that the BADH gene was integrated into maize genome. Transgenic lines of progeny were examined for tolerance to NaCl by induced salt stress with 250 mM NaCl Hoagland solution. After 15 days of treatment, 73.9–100% of the transgenic seedlings survived and grew well, whereas most wild-type seedlings wilted and showed loss of chlorophyll. Only 8.9% of the wild-type plants survived but gradually died after salt stress. The electrical conductivity of the transgenic line of progeny after salt stress was lower than wild type. The transgenic progeny had higher glycinebetaine and Chlorophyll content than wild type after salt stress.     

Genetic transformation of lipid transfer protein encoding gene in <Emphasis Type="Italic">Phalaenopsis amabilis</Emphasis> to enhance cold resistance

Embryogenic callus of Phalaenopsis amabilis derived from leaf tissue was cocultivated with Agrobacterium tumefaciens strain LBA4404 harboring a plant cloning vector. The vector carried the lipid transfer protein (LTP) encoding gene cloned from cold tolerant Brazilian upland rice cv. IAPAR 9. The highest transformation efficiency (12.16%) was obtained when 1–2 mm calli were infected and cocultivated with 0.4 (OD₆₀₀) A. tumefaciens for 20 min. Transgene integration of kan-resistant plants was confirmed through polymerase chain reaction analysis and Southern hybridization. Four hundred seventy transgenic plants, each derived from an independent protocorm-like body, were obtained. The expression of rice cold-inducible LTP gene in transgenic P. amabilis improved its adaptive responses to cold stress. The examination of transgenic plants revealed that enhanced cold tolerance was most likely due to the increased accumulation of several compatible solutes such as total soluble sugars, proline, antioxidant superoxide dismutase, decreased accumulation of malondialdehyde, and maintained electrolytes within the membrane compared with controls.     

转GsCBRLK/SCMRP双价基因苜蓿耐碱性及氨基酸含量分析

从野生大豆盐碱胁迫基因表达谱中筛选并克隆到GsCBRLK基因, 与人工合成的高甲硫氨酸含量SCMRP基因构建成双价植物表达载体, 将其转入苜蓿, 获得超量表达的转基因苜蓿, 并进行耐碱性分析。结果显示, 经过100、150 mmol L^–1 NaHCO₃处理14 d后, 转基因株系生长状态良好, 而非转基因对照株系萎蔫、失绿、甚至死亡;转基因株系的丙二醛含量和相对质膜透性显著低于非转基因株系(P<0.05), 而SOD酶活性显著高于非转基因对照(P<0.05), 说明超量表达GsCBRLK基因增强了苜蓿的耐碱能力;各转基因株系的甲硫氨酸含量均比对照植株高, 表明SCMRP基因的导入提高了苜蓿叶片甲硫氨酸的含量。     

蛋白激发子Hrip1基因在拟南芥中表达可提高植株的耐盐耐旱能力

Hrip1是从极细链格孢(Alternaria tenuissima)代谢物中分离的一种蛋白激发子。将蛋白激发子基因Hrip1转化到拟南芥,对5个T₁代转基因拟南芥株系进行分子检测, 证明Hrip1基因能够在拟南芥中转录和表达。转基因植株对盐和干旱胁迫的抗性显著增强, 75 mmol L⁻¹ NaCl和50 mmol L⁻¹甘露醇渗透胁迫2 d, 转基因植株种子平均相对发芽率为32.1%和77.9%, 分别比野生型的增加3.72倍和5.61倍; 150 mmol L⁻¹ NaCl和50 mmol L⁻¹甘露醇处理拟南芥幼苗7 d后, 转基因植株平均相对根长为81.79%和93.25%, 分别是野生型的1.53倍和1.34倍。3周龄的转基因植株在250 mmol L⁻¹ NaCl条件下胁迫20 d, 平均存活率为67%, 显著高于野生型(42%)(P<0.05); 干旱胁迫25 d后, 复水5 d转基因植株平均存活率为72%, 而野生型仅为44%。检测结果显示转基因植株叶片的抗氧化酶活性明显高于野生型, 用200 mmol L⁻¹ NaCl和200 mmol L⁻¹甘露醇处理24 h后, POD活性分别比野生型植株提高1.56倍和1.85倍, CAT活性分别比野生型植株提高1.64和1.86倍。说明蛋白激发子Hrip1基因在拟南芥中的表达能够改善和提高植株的耐盐抗旱能力。     

Transgenic soybeans expressing betaine aldehyde dehydrogenase from Atriplex canescens show increased drought tolerance

Betaine aldehyde dehydrogenase (BADH) catalyses the oxidation of betaine aldehyde to glycine betaine. To test whether BADH can increase drought tolerance in soybean (Glycine max), BADH from the drought‐tolerant plant Atriplex canescens (AcBADH) was introduced into the soybean cultivar ‘Jinong 17’ by Agrobacterium‐mediated cotyledon transformation. Eight independent AcBADH transgenic lines were subjected to drought stress. As expected, AcBADH was expressed in transgenic soybean leaves and not in the control. In transgenic plants, AcBADH expression increased following drought treatment. Under osmotic stress, the germination index was 6%–17% higher in the transgenic lines than in the control. Using a randomized block design, we measured drought‐related physiological indices and yield traits. The proline content in AcBADH transgenic soybeans increased by 12.5%–16.6%, peroxidase activity increased by 1%–7%, dry weight of plant increased by 15%–20% and malondialdehyde contents decreased by 1.5%–13%, compared to the control. Under drought conditions, two of the eight transgenic soybean lines had higher yields than the control, with increases of 7.59%–8.84%. Therefore, transgenic expression of AcBADH may provide a promising strategy to engineer drought tolerance without adverse consequences.     

转AtDREB2A基因苜蓿的耐碱性分析

对已获得的转基因苜蓿进行了RT-PCR检测,确定AtDREB2A基因已在转基因苜蓿中超量表达。并研究了转AtDREB2A基因苜蓿在碱胁迫(100mmol/L,pH8.5)下表型和生理生化指标的变化。结果表明,转AtDREB2A基因苜蓿的相对质膜透性及丙二醛含量显著低于野生型株系,株高、叶绿素及根系活力明显高于野生型株系,说明AtDREB2A基因的超量表达提高了转基因苜蓿的耐碱能力。     

转PvP5CS1基因拟南芥植株对干旱和盐胁迫的反应

为探索普通菜豆脯氨酸合成酶基因P5CS1在植物渗透胁迫中的作用,本研究应用农杆菌介导法,将PvP5CS1基因转入拟南芥,获得6株阳性转基因株系;通过检测转基因植株与野生型植株在干旱和盐胁迫下种子发芽率,幼苗脯氨酸含量、株系电导率、相对根长和成株死亡率,分析了PvP5CS1基因的表达对改善拟南芥抗渗透胁迫的效应。结果表明,在150 mmol L^-1 NaCl和150 mmol L^-1甘露醇渗透胁迫下,转基因植株平均相对发芽率分别是野生型的1.6倍和1.62倍;150、250 mmol L^-1甘露醇和150 mmol L^-1 NaCl处理下,转基因拟南芥植株平均脯氨酸含量分别是野生型的2.68、1.30和1.30倍;平均相对电导率分别是野生型植株的85%、77%和85%;平均相对根长分别是野生型植株的1.2、1.3和1.2倍;300 mmol L^-1 NaCl处理下,转基因植株的平均死亡率为42%,显著低于野生型(90%)(P<0.05);干旱胁迫下,转基因植株的平均死亡率为56%,显著低于野生型(70%)(P<0.05),说明PvP5CS1基因在拟南芥中的表达明显改善了转基因植株的抗旱性和耐盐性。     

野生大豆GsGST19基因的克隆及其转基因苜蓿的耐盐碱性分析

谷胱甘肽S-转移酶对植物抵御逆境胁迫和解除细胞毒素起着重要作用。本研究从野大豆盐碱胁迫基因表达谱中筛选并克隆得到GsGST19基因,将其转化苜蓿,获得超量表达的转基因苜蓿,并对转基因苜蓿进行耐盐碱性分析。结果显示在正常培养条件下,转基因苜蓿株系19-4和19-9的GST酶活性分别是非转基因株系的1.52倍和1.49倍。在100 mmol L^–1 NaHCO₃处理14 d后转基因株系生长状态良好,而非转基因对照株系明显萎蔫、失绿、甚至死亡;转基因株系的丙二醛含量和相对质膜透性显著低于非转基因株系(P<0.05),而叶绿素含量和根系活力显著高于非转基因对照(P<0.05),说明超量表达GsGST19基因增强了苜蓿的耐盐碱能力。     

Field analyses of horizontal gene flow among Vigna angularis complex plants

Recent studies have indicated that spontaneous interspecies crossing commonly occurs among vascular plants, and therefore that horizontal gene flow from transgenic plants into wild relatives is unavoidable. Few surveys, however, have been conducted to determine an actual flow frequency for individual plant species. For the present estimation of gene flow among Vigna angularis complex (small red bean) plants, randomly amplified polymorphic DNA was analysed, the patterns of which differ between a cultivar, var. angularis and its wild counterpart, var. nipponensis. Cultivars and wild‐type plants were planted alternately and approximately 1% of the screened F₁ beans of wild type were hybrid. These F₁ hybrids were selected, allowed to self to produce F₂ beans, and subsequent crossing between the F₂ plants and wild type resulted in that 3.7% of the F₃ generation possessed DNA fragments specific to the cultivar. These results indicate that gene flow actually occurs among V. angularis complex plants, and that transferred genes might be stably maintained by the offspring. The present observation cannot be directly applied to transgenic plants due to the lack of information as to whether or not artificially introduced genes behave as do native genes after horizontal movement. However, as gene flow is found to be inevitable in this species, specific assessment of whether or not the gene to be introduced confers a significant selective advantage to the host is critical for utilization of genetically modified plants in future.     

转BADH基因玉米植株的获得及其耐盐性分析

采用超声波辅助花粉介导植物转基因方法, 将甜菜碱醛脱氢酶(BADH)基因导入玉米自交系郑58, 获得了耐盐性强的转基因玉米植株。经卡那霉素抗性初筛、PCR扩增、Southern blot杂交分析, 证明BADH基因已导入转化植株并整合到其基因组中。用不同浓度的NaCl溶液对T₂代转基因玉米植株与对照进行盐胁迫处理, 结果表明, 转BADH基因玉米植株表现出一定的抗逆性, 生长状况明显优于对照; 根据非转化苗对NaCl的反应以及生长状况, 确定250 mmol L^-1 NaCl溶液为玉米幼苗耐盐性筛选的适宜浓度; 依据此临界浓度下形态指标和生理生化指标的测定结果, 与对照相比, 转基因植株的株高提高10.94%~25.7%, 鲜重增加8.62%~18.2%, 干重增加9%~18.18%, 相对电导率降低37.21%~58.14%, 叶绿素含量增加15.89%~90.65%, 超氧化物歧化酶(SOD)活性提高64.92%~148.29%, 丙二醛(MDA)含量减少26.97%~48.05%。综上所述, 转入甜菜碱醛脱氢酶(BADH)基因提高了玉米的耐盐性。这是首例将BADH基因导入优良玉米自交系郑58的报道。超声波辅助花粉介导法是一种经济、高效、实用和无基因型依赖性的植物基因转化方法。     

Production and field performance of transgenic alfalfa (Medicago sativa L.) expressing alpha-amylase and manganese-dependent lignin peroxidase

Summary Transgenic alfalfa plants expressinBacillus licheniformis alpha-amylase and mangaese-dependent lignin peroxidase (Mn-P) from Phanerochaete chrysosporium were produced using the Agrobacterium tumefaciens transformation system. In each case, there was a range of expression of the introduced gene among independent transgenic plants. Plants producing alpha-amylase showed no alteration of phenotype. Production of Mn-P in alfalfa, howeven, in most cases adversely affected plant growth and development. Affected plants were stunted with yellowing foliage, but survived and produced seed. Results from field trials showed that Mn-P production in transgenic alfalfa reduced dry matter yield and plant height. The extent of these symptoms and yield reduction was, for the most part, related to the level of foreign protein production as estimated by Western analysis. Field data from transgenic plants expressing alpha-amylase showed that there was no effect of foreign protein production on plant performance. Expression of Mn-P was shown to segregate in sexual progeny derived from transgenic plants.Abbreviations Mn-P manganese-dependent lignin peroxidase     

Molecular and biological studies on male-sterile cytoplasm in the Cruciferae. IV. Ogura-type cytoplasm found in the wild radish, Raphanus raphanistrum

The orf138 gene, which is specific to Ogura male-sterile cytoplasm, was analysed in mitochondrial DNA (mtDNA) of the wild radish, Raphanus raphanistrum, by polymerase chain reaction (PCR), Southern hybridization and sequencing. The effect of R. raphanistrum cytoplasm on the expression of male sterility was also examined in progeny with R. sativus. A PCR-aided assay and Southern hybridization revealed that three out of six strains analysed included plants with orf138. The sequence of wild type orf138 was same as that of Ogura, except for one or two nucleotide substitutions. Southern hybridization showed a novel mtDNA configuration in R. raphanistrum, in addition to the normal and Ogura types identical to those in R. sativus. Among interspecific hybrids, all the F₁ had normal pollen fertility. In the F₂ progeny between female wild plants having orf138 and the maintainer of Ogura male sterility, male-sterile plants were segregated, fitting the ratio of 3 fertile: 1 sterile plant. R. raphanistrum has cytoplasm that induces male sterility in radishes, and contains a dominant fertility restorer gene.     

水稻蛋白二硫键异构酶基因沉默载体构建及其转基因后代的高温结实特性分析

采用RT-PCR法亚克隆了PDI基因保守区内450 bp的靶标序列作为干扰区段, 构建了含有内含子hpRNA (ihpRNA)的双元表达载体pTCK303-RiOsPDI, 经农杆菌介导转化日本晴, 获得转基因植株; 通过在T₀代对其潮霉素(Hyg)抗性基因的PCR鉴定, 确定携带有干扰片段的T-DNA区已整合到水稻基因组中, 且在转基因T₁代符合3∶1的分离模式。半定量PCR和荧光定量PCR的检测结果表明, PDI基因沉默转基因阳性植株不同器官中的PDI表达量均显著降低, 尤其是其籽粒中表达量较微, 几乎能引起靶基因80%左右沉默。对转基因T₂代植株的高温结实特性和籽粒理化品质的检测结果, PDI基因沉默会引起高温胁迫处理下结实率的大幅度降低, 耐热性显著下降, 但其在常温处理下的结实率与对照之间无显著差异。此外, PDI基因沉默后, 稻米的透明度下降、垩白度增加, 但对籽粒粗蛋白总量和直链淀粉含量的影响不甚明显。     

Characterization of transgenic male sterility in alfalfa

Dependable male sterility would help to make hybrid cultivar development a reality in alfalfa once higher levels of heterosis are attained. Alfalfa plants obtained by genetic transformation with a construct containing the Barnase gene under the control of a tobacco anther tapetum specific promoter were studied. Vacuolization and degeneration of the tapetal cell cytoplasm at a premeiotic stage of development were observed in all five transformed plants (T₀)examined, but the severity of the abnormalities varied greatly among pollen sacs of a genotype. During the meiotic stage, some pollen sacs showed reduction in size, and the tapetum generally appeared thinner when compared to those of the non transgenic plants; tapetal cells showed abnormal vacuolization and signs of cytoplasm degeneration. Despite this, some microspores were formed and some pollen grains were shed in all the T₀ plants, but these were highly variable in size and had very low in vitro germinability. Self-fertility was negligible. The T₀ plants were crossed with one or two unrelated non transgenic male-fertile plants. Mendelian segregation was observed with two exceptions. Instability of the trait in F₁ progenies was noticed, varying for different T₀ parents. F₁ plants exhibiting higher sterility than the primary transformants were observed, indicating that it should be possible to obtain good male sterile plants by backcrossing this trait into different genetic backgrounds. The possible use of this transgenic male sterility in alfalfa breeding is briefly discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ectopic expression of GmPAP3 enhances salt tolerance in rice by alleviating oxidative damage

GmPAP3, a purple acid phosphatase from soybean (Glycine max), was previously shown to alleviate salt stress in BY‐2 cells and Arabidopsis thaliana by reducing oxidative damage. To make use of GmPAP3 for crop improvement, we investigated whether the protective function of GmPAP3 is persistent in rice. Compared with the untransformed wild type, the transgenic rice plants exhibited enhanced germination rate, longer shoots and roots, and higher survival rate under salt stress, when compared to the untransformed control. In addition, the transgenic plants also showed increased activity of superoxide dismutase (SOD) and catalase (CAT), proline content, water content and chlorophyll content, but decreased electrolyte leakage and malondialdehyde content in leaves. These results indicate that the expression of GmPAP3 could be used to enhance salt tolerance in rice.     

碱胁迫相关基因GsWRKY15的克隆及其转基因苜蓿的耐碱性分析

WRKY蛋白属于锌指型转录调控因子,能够参与植物多种逆境响应。本研究利用前期野生大豆盐碱胁迫RNA-seq测序数据,从构建的碱胁迫基因调控网络中筛选并克隆到GsWRKY15基因。分析GsWRKY15在碱胁迫下野生大豆根中的表达模式,发现该基因受碱胁迫诱导显著上调表达,且在胁迫后1 h表达量最高。分析GsWRKY15基因在野生大豆各组织中的表达特异性,发现该基因在各组织中均有表达,花中表达量最高。采用根癌农杆菌侵染苜蓿子叶节方法,将GsWRKY15转化肇东苜蓿,获得39株抗性植株。通过PCR、Southern blot和RT-PCR方法分析抗性植株,获得了超量表达GsWRKY15基因的转基因株系并对其进行了耐碱性分析。在150 mmol L–1 Na HCO3处理2周后转基因苜蓿生长状态良好,而非转基因苜蓿出现萎蔫、变黄,甚至死亡;非转基因苜蓿的相对质膜透性和丙二醛含量显著高于转基因苜蓿,而叶绿素含量显著低于转基因苜蓿;同时分析碱胁迫下转基因植株中胁迫相关基因的表达模式,发现H+-Ppase、NADP-ME、KIN1、RD29A基因的表达量高于非转基因苜蓿。结果表明GsWRKY15基因的超量表达能够显著增强苜蓿的耐碱能力。     

转codA基因提高番茄植株的耐热性

以野生型番茄(cv. Moneymaker)和转codA番茄为材料,用不同温度(25、30、35、40、45和50℃)分别处理2 h,测定叶片净光合速率(P_n)、PSII最大光化学效率(F_v/F_m)、过氧化氢(H₂O₂)含量、丙二醛(MDA)含量、相对电导率(REC)和抗氧化酶活性等生理指标;42℃高温处理0、3和6 h后,检测热响应基因的表达量以及D1蛋白的含量,研究高温胁迫对上述参数的影响,探讨转codA基因提高番茄叶片耐热性的机制。。结果表明,高温胁迫下,转codA基因番茄叶片P_n和F_v/F_m的抑制程度明显低于野生型,H₂O₂、MDA的积累量以及REC均低于野生型,而且明显增强了过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)的活性。此外,转codA基因番茄叶片中抗氧化酶基因和热胁迫基因的表达水平均高于野生型,而D1蛋白的降解水平低于野生型。转codA基因番茄体内合成的甜菜碱提高了转基因番茄的耐热性,这与提高和维持较高的抗氧化酶活性、促进热激响应基因的表达及减缓D1蛋白的降解等有关。