大豆2-甲基-6-植基-1,4-苯醌甲基转移酶（GmVTE3）对转基因拟南芥生育酚组成的影响

摘要：2-甲基-6-植基-1,4-苯醌甲基转移酶（MPBQ-MT,VTE3）催化2-甲基-6-植基-1,4-苯醌（MPBQ）的甲基化,生成2,3-二甲基-6-叶绿基-1,4-苯醌（DMPBQ）,是维生素E合成过程中的关键酶之一。本研究利用已克隆的大豆2-甲基-6-植基-1,4-苯醌甲基转移酶基因(GmVTE3)构建了植物表达载体,转化模式植物拟南芥。利用HPLC对转基因拟南芥种子和叶片进行了维生素E含量测定,结果表明转基因植株种子中γ-生育酚/δ-生育酚比值比野生型提高1.7倍,而叶片中δ-生育酚则全部转化为γ-生育酚。表明GmVTE3可以有效的把δ-生育酚转化成活性较高的γ-生育酚,从而提高植物中高活性的维生素E的含量。     

花生γ-生育酚甲基转移酶基因(γ-TMT)的克隆及序列分析

γ-生育酚甲基转移酶(γ-TMT)催化γ-生育酚转变为生物活性最高的α-生育酚,是决定植物中维生素E成分和活性的关键酶。本研究采用电子克隆与PCR相结合的方法获得了花生６个栽培品种(A. hypogaea L.)和花生区组4个二倍体野生种中γ-TMT的全长DNA序列,定名为AhgTMT;从栽培品种丰花2号中获得γ-TMT的cDNA序列,定名为AhrTMT。同一栽培品种的AhgTMT与AhrTMT的序列完全对应,说明该基因无内含子。AhrTMT编码区长1 059 bp,编码352个氨基酸残基的AhTMT蛋白。AhTMT分子量为39.09 kD,等电点6.72,总平均亲水性–0.12;预测的二级结构中α-螺旋占55.40%, β-折叠占10.51%, 无规则卷曲占34.09%;被定位于叶绿体,含有甲基转移酶保守的SAM结构域。AhTMT与已报道植物γ-TMT氨基酸序列的相似性为61.75%~72.80%。栽培品种的AhgTMT与A. ipaensis (BB)、A. batizocoi (BB)、A. duranensis (AA)、A. kuhlmannii (AA) 4个野生种的核苷酸序列同源性分别是100%、99.91%、99.74%、95.63%。     

水稻籽粒维生素E及组分在品种间的变异与分布

维生素E是人与动物必须从食物中摄取的微量营养元素之一,根据侧链的饱和度可将其分为生育酚和三烯生育酚,水稻种子中均含二者。本研究利用反相高效液相色谱法测定了18份籼稻和16份粳稻品种糙米中维生素E各异构体的含量。结果表明,种子中各异构体的含量在籼稻与粳稻间存在显著差异,粳稻种子中的生育酚和维生素E含量一般要高于籼稻;籼稻种子中三烯生育酚与生育酚的比值(1.61)极显著高于粳稻(0.95),籼稻中以γ-三烯生育酚为主,而粳稻中主要含α-生育酚。水稻种子中同种类型异构体(即α-型间或γ-型间)含量间具有显著正相关,但γ-型与α-型异构体间呈显著负相关。这些数据为进一步研究水稻种子中维生素E的代谢调控和营养品质改良提供了有益的表型数据。     

高效液相色谱法测定黑龙江省南部地区大豆维生素E含量的研究

维生素E是大豆的重要功能性成分之一,其含量与组成随品种和生长环境的不同而存在差异。本研究采用高效液相色谱法,以黑龙江省品种合丰25和加拿大品种Bayfield为材料,利用硅胶柱为固定相,甲醇:水(90:10,V/V)为流动相,测定900份黑龙江省哈尔滨、绥化、呼兰三个地区大豆资源中的维生素E含量,使用荧光检测器检测(Ex=295nm,Em=330nm),以α-生育酚、γ-生育酚,β-生育酚为标准品,分析了不同地区大豆中维生素E含量中各个成分的差异。结果显示,哈尔滨地区大豆品种中维生素E的含量最高,最适宜种植大豆维生素E品种。     

转基因水稻中过量表达OsHPT提高胚乳中生育酚含量

为了提高稻米中生育酚的含量,本研究经农杆菌介导将由CaMV35S启动子驱动的水稻尿黑酸植基转移酶基因(OsHPT)导入粳稻品种武育粳3号,经RT-PCR等分析证明导入基因能在水稻胚乳中表达。HPLC测定结果表明,水稻中过表达OsHPT后,转基因稻米胚乳中总生育酚的含量极显著高于未转化对照,且主要表现为α-生育酚含量的提高,总生育酚和总维生素E含量最高可分别提高1.5和0.8倍。     

大豆BIEX群体维生素E含量的遗传分析

大豆高维生素E含量专用品种的选育是现代大豆品质育种的重要方向,为给大豆高维生素E含量专用品种选育提供遗传学依据,通过对大豆重组自交系群体2008-2009年的维生素E含量进行相关的遗传分析,以Essex×ZDD2315衍生的208个重组自交家系群体BIEX为材料,用主基因+多基因混合遗传模型,分析大豆α-、γ-、δ-生育酚含量以及VE总含量的遗传机制。结果表明,α-生育酚含量、γ-生育酚含量以及VE总含量均属于2对主基因+多基因遗传模型,主基因遗传率分别为59.62%,75.99%,77.05%,多基因遗传率分别为35.90%,13.96%,15.05%;δ-生育酚含量属于1对主基因+多基因遗传模型,主基因遗传率为50.39%,多基因遗传率为38.11%。维生素E及其组分含量遗传涉及主基因和多基因,主基因遗传率在50%以上,多基因遗传率在10%以上,建议育种中要兼顾主基因和微效多基因的利用。     

大豆籽粒维生素E含量的QTL分析

维生素E(VE)具有提高人体免疫力、抗癌、预防心血管疾病等保健作用,从大豆中提取的VE安全性更高。本研究采用高效液相色谱技术(HPLC)检测大豆BIEX群体(Essex×ZDD2315)维生素E的α-生育酚、γ-生育酚和δ-生育酚含量。应用QTLNetwork 2.1软件分别检测到8个和12对控制大豆维生素E及组分含量的加性和互作QTL。α-生育酚含量加性和互作QTL累计贡献值分别为8.68%(2个)和15.57%(4对),γ-生育酚含量加性和互作QTL累计贡献值分别为8.59%(2个)和11.57%(2对),δ-生育酚含量加性和互作QTL累计贡献值分别为5.44%(1个)和17.61%(3对),维生素E总含量的加性和互作QTL累计贡献值分别为11.39%(3个)和9.48%(3对)。未检测到维生素E及组分含量和环境互作的QTL。未定位到的微效QTL累计贡献值为66.16%~75.32%,说明未定位到的微效基因的变异占2/3以上。各性状的遗传构成中,未检测出的微效QTL份额最大,加性QTL和互作QTL贡献相差不大。在育种中应考虑常规方法聚合微效QTL与标记辅助方法聚合主要QTL相结合。     

油料作物种子维生素E基因工程研究进展

摘要 维生素E具有重要的营养价值,是人类和动物维持生殖系统等正常代谢所必需的微量营养素,故称“生育酚”。维生素E具有很强的抗氧化能力,以及营养保健功能。食用植物油是维生素E主要来源,维生素E作为有效的天然抗氧化剂,能保护不饱和脂质免受超氧阴离子等自由基氧化,从而抑制油脂的自动氧化和酸败,延长食用植物油的货架期。通过对维生素E代谢研究的不断深入,人类已经基本了解植物体合成的基本途径。同时也使得利用基因工程改造维生素E成为可能。维生素E基因工程主要有两个策略,一是提高维生素E总量,二是提高其中活性最高α-生育酚所占比重。围绕这两个策略采取了不同解决途径。     

拟南芥γ-生育酚甲基转移酶启动子在转基因烟草中的表达特性分析

γ-生育酚甲基转移酶（γ-TMT）是维生素E生物合成途径中的关键酶之一,催化γ、δ-生育酚甲基化,生成活性较高的α、β-生育酚。研究构建了含有γ-TMT启动子和GUS报告基因的植物表达载体,通过农杆菌介导转化烟草,筛选出PCR阳性植株,进行GUS组织化学染色,结果表明,在γ-TMT启动子的驱动下,报告基因GUS在烟草的根、茎、叶中均有表达。     

大豆籽粒生育酚及其组分含量鉴定与优异种质筛选

生育酚（维生素E）具有防止人体动脉硬化、降低胆固醇、增强血液循环和防治心血管疾病等多种功效。大豆较其他植物含有更为丰富的生育酚,但目前大规模筛选优异种质的工作开展较少且研究不够深入。鉴于此,利用299份大豆品种资源,通过高效液相色谱法测定其籽粒生育酚及其组分含量,明确其遗传变异及相关性,并筛选生育酚含量高的特异种质。结果表明,供试大豆品种生育酚及其组分含量存在丰富的遗传变异,其δ-、γ-、α-生育酚及总生育酚平均含量分别为44.15、143.05、66.81及254.01μg/g,变异系数在15.37%~38.04%之间,α-生育酚变异系数最高;3种成分占总含量比值分别为17.47%、56.57%和25.96%,变异系数为11.78%~32.21%,α-生育酚比值变异系数最高;同时发现,总含量与3种组分含量间的相关系数均达到极显著水平,δ-生育酚与γ-及α-生育酚的相关系数也达极显著水平;基于各供试品种生育酚及其组分含量,将其分为3类,并筛选出13份高生育酚含量特异种质,其中包括黑河44、合丰50、合农75和绥农28等,为今后通过常规及分子育种方法实现生育酚及其组分含量遗传改良奠定了材料基础。     

外源生育酚对棉花(Gossypium spp)纤维分化发育的影响

为探究生育酚的非抗氧化功能及其在棉纤维分化发育中的作用,本研究以陆地棉标准系TM-1的胚珠为材料,在BT培养基中加入不同种类和浓度的生育酚,通过胚珠离体培养诱导棉纤维分化发育,同时使用电子显微成像技术,研究外源生育酚对棉纤维分化发育的影响。胚珠离体培养和电镜扫描试验结果表明:胚珠经离体培养3 d后,4种外源生育酚对纤维细胞分化和发育均有促进作用;α-生育酚能显著地促进胚珠表皮纤维突起数量的增加,而γ-生育酚则促进纤维细胞的伸长;胚珠在离体培养条件下,浓度为1.0μmol/L的α-和γ-生育酚对棉纤维的分化发育的促进作用最为显著。本研究将为丰富生育酚的非抗氧化功能和验证生育酚在纤维的分化发育阶段所起的作用提供了理论依据。     

拟南芥尿黑酸叶绿醇转移酶(HPT)基因启动子的分离及表达特性分析

尿黑酸叶绿醇转移酶（HPT）是催化生育酚生物合成分支途径中的关键酶,催化叶绿醇焦磷酸（phytyl-PP）与尿黑酸（HGA）缩合生成生育酚的前体。从拟南芥中分离HPT基因的启动子序列946 bp,构建了该启动子与GUS嵌合的重组载体,通过农杆菌介导转化拟南芥,取正常生长条件下的拟南芥转基因植株进行GUS组织化学染色,结果表明,在HPT启动子的驱动下,GUS基因主要在转基因拟南芥的根中高表达,在成熟叶片、花丝、雌蕊中也有一些表达,而在茎、根尖、花药、种荚及发育的种子中则未表达,可见该启动子为根优势表达启动子。     

Ethanol vapor and saprophytic yeast treatments reduce decay and maintain quality of intact and fresh-cut sweet cherries

The objective of this study was to evaluate the use of an ethanol vapor release pad and a saprophytic yeast Cryptococcus infirmo-miniatum (CIM) to reduce decay and maintain postharvest quality of intact or fresh-cut sweet cherries (Prunus avium) cv. Lapins and Bing. Intact or fresh-cut fruit were packed in perforated clamshells (capacity 454 g) and stored at 1, 10 or 20 °C for up to 21, 14 and 8 d, respectively. For ethanol treatment, a pad made with silica gel powder containing 10 g ethanol and covered with perforated film, which allows ethanol vapor to diffuse gradually, was attached to the upper lid of the clamshells. Ethanol treatment caused accumulation of ethanol in the packaging headspace, about 10 μL L⁻¹ with little change within 14 d at 1 °C, 23 μL L⁻¹ at d 1 and decreased to 15 μL L⁻¹ at d 10 at 10 °C, and 26 μL L⁻¹ at d 1 and decreased to 13 μL L⁻¹ at d 3 at 20 °C. Ethanol content in fruit was less than 9 mg kg⁻¹ in all the control fruit, and increased to 16, 34 and 43 mg kg⁻¹ in ethanol-treated fruit at 1, 10 and 20 °C, respectively. Nonetheless, a sensory taste panel did not perceive any flavor difference from the ethanol treatment. The ethanol treatment retarded softening, darkening, and acid decrease in fruit as well as discoloration of the stems, and extended shelf-life of intact cherries. Ethanol reduced brown rot (Monilinia fructicola) in fresh-cut cherries stored at 20 °C, but not at 1 and 10 °C. A pre-packaging dip in CIM completely controlled brown rot in inoculated fresh-cut cherries stored at 1 °C, and in naturally infected cherries at 20 °C.     

膜下滴灌条件下高产甜菜灌溉的生理指标

甜菜是我国重要的糖料作物,其生物产量高,需水量大,合理灌溉是节约用水、提高产量的有效措施之一。本试验连续两年研究了内蒙古半干旱地区膜下滴灌条件下,不同灌水量甜菜块根产量与叶面积指数、净光合速率、蒸腾速率、叶水势、土壤含水量和耗水量之间的关系,以及不同灌水量对甜菜产量和水分利用效率的影响。结果表明,高产甜菜的叶面积指数在叶丛快速生长期大于7.37,在块根糖分增长期和糖分积累期分别为6.08~6.51和4.19~5.57,在叶丛快速生长期、块根糖分增长期和糖分积累期叶水势分别为–0.09~–0.22、–0.18~–0.39和–0.26~–0.48 MPa,净光合速率分别为21.28~28.23、21.90~28.75和22.06~26.58μmol m–2 s–1,蒸腾速率在叶丛快速生长期和块根糖分增长期分别为9.36~10.21 mmol m–2 s–1和6.37~7.73 mmol m–2 s–1,在糖分积累期大于4.69 mmol m–2 s–1,耗水量分别为140.15~312.78、44.93~200.45和56.32~113.06 mm。甜菜产量、产糖量、水分利用效率均高的合理灌溉量,在丰雨年份(生育期降雨量500 mm)为1350 m3 hm–2,在少雨年份(生育期降雨量300 mm)为1800 m3 hm–2,为甜菜节水灌溉提供了理论依据和生理指标。     

The effect of temperature and other factors on chlorophyll a fluorescence and the lower oxygen limit in apples (Malus domestica)

The effects of temperature, scan interval and rate of oxygen (O₂) decline on pulse frequency modulation (PFM)-based minimum fluorescence (F_α) and the F_α-based lower oxygen limit (LOL) were investigated using ‘Honeycrisp’ apples (Malus × domestica Borkh). The effects of temperature and hypoxic stress on pulse amplitude modulation (PAM) fluorescence parameters were also investigated. The PFM scan interval had no effect on the F_α baseline, but increases in the scan interval decreased the low-O₂-induced fluorescence spike intensity (ΔF_α). Temperature negatively correlated with the F_α baseline while ΔF_α °C⁻¹ was greater at lower than at higher temperatures. When using a PAM fluorometer, the minimum fluorescence (F_o), and to a lesser extent the maximum fluorescence (F_m), were similarly affected by temperature as was F_α. Temperature altered the LOL in fruit. Apples stored at 20, 10, 3.5 and 0 °C spiked at 0.72, 0.33, 0.22 and 0.08 kPa O₂, respectively, under a rapid O₂ decline (i.e., 20.9 to <1 kPa O₂ in ≈5–6 h). Although the low-O₂ F_α spike apex values did not change with temperature, the spike intensity increased with temperature due to a reduced fluorescence baseline. A slower O₂ decline rate produced slightly higher LOL and lower spike intensity values. In conclusion, temperature and rate of O₂ decline affected the low-O₂-induced PFM fluorescence spike intensity as well as the LOL, while the PFM scan interval affected the spike intensity.     

Effect of two wheat genotypes and Swedish environment on falling number, amylase activities, and protein concentration and composition

Variation in falling number, amylase activities, protein concentration and composition were investigated in two wheat cultivars grown in Sweden over two seasons, in four locations, with four N fertilizer rates, with and without fungicide treatment. The results showed that;

Cultivar differences in gaseous 1-methylcyclopropene accumulation in whole and fresh-cut apple fruit

A number of studies have shown that responses of apple fruit to 1-methylcyclopropene (1-MCP) vary considerably among cultivars. This study was designed to determine if cultivars show differences in accumulation of gaseous 1-MCP. Apple fruit were placed in 1.76 L jars that were sealed and injected with 20 μL L⁻¹ 1-MCP. After 12 h, samples of intercellular atmosphere were removed and analyzed for 1-MCP concentration. Accumulation of internal gaseous 1-MCP varied markedly among cultivars, ranging from 0.14 ± 0.06, 0.22 ± 0.03, and 0.77 ± 0.30 in ‘Redcort’, ‘McIntosh’, and ‘Empire’, respectively, to 2.10 ± 0.28, 3.33 ± 0.13, and 6.93 ± 0.35 μL L⁻¹ in ‘Gala’, ‘Cameo’, and ‘Honeycrisp’, respectively. Accumulation of gaseous 1-MCP was reduced an average of 51% in fruit treated with Sta-Fresh 8711 fruit wax. The role of the epidermis in modulating 1-MCP ingress was determined by measuring gaseous 1-MCP accumulation in fresh-cut tissue. Fresh-cut cortical tissue rapidly depleted headspace 1-MCP (>95%) over a 1-h exposure yet accumulated negligible quantities of internal gaseous 1-MCP. By contrast, cortical tissue treated with ascorbic acid or hypotaurine, or aged for several hours prior to exposure to 1-MCP, showed reduced consumption of headspace 1-MCP and high accumulation of internal gaseous 1-MCP. Levels of internal 1-MCP in cortical tissue from the cultivars generally paralleled those for intact fruit, ranging from 0.23 ± 0.07, 0.37 ± 0.18 and 1.09 ± 0.14 μL L⁻¹ in ‘Empire’, ‘McIntosh’ and ‘Redcort’, respectively, to 2.40 ± 0.71, 4.55 ± 0.15, and 6.24 ± 0.85 in Gala’, ‘Cameo’, and ‘Honeycrisp’, respectively. Although commercial fruit wax influences gaseous 1-MCP accumulation, the comparable accumulation patterns in unwaxed whole and fresh-cut apple fruit suggest that epidermal tissue/native waxes alone do not account for cultivar differences.