小麦丙氨酸-乙醛酸转氨酶基因（ TaAGT2）的克隆、生物信息学预测及表达分析

光呼吸是植物细胞H_2O_2的重要来源,也是维持细胞氧化还原的重要成分,影响植物对生物和非生物逆境的应答,因此,挖掘与光呼吸有关的基因对提高植物的抗逆性具有重要意义。本研究以耐旱小麦品种青麦6号转录组数据为基础,通过RACE技术克隆得到小麦 AGT2基因的全长cDNA,将其命名为 TaAGT2,并对其进行了生物信息学预测及表达模式分析。结果表明, TaAGT2开放阅读框长1 434bp,编码477个氨基酸。该蛋白属于亲水性稳定蛋白,定位于线粒体中,二级结构以α-螺旋(42.14%)和无规则卷曲(32.91%)为主。 TaAGT2基因包含AAT-I基因族保守区域,与二穗短柄草 AGT2的相似性高达97%。通过qRT-PCR对基因 TaAGT2在不同组织中(根、茎和叶)及4种非生物胁迫(低温、ABA、干旱、高盐)下的表达特性进行分析,结果表明, TaAGT2在根、茎和叶中均有表达,茎中的表达显著高于根和叶,在不同胁迫条件下上调表达,表明该基因可能参与调控植物的抗逆反应。     

普通小麦TaADF8基因的克隆及表达分析

肌动蛋白解聚合因子(actin-depolymerizing factor,ADF)普遍存在于真核细胞中,为低分子量的肌动蛋白结合蛋白,在调控细胞内肌动蛋白纤维的聚合和解聚中起关键作用。为给深入研究TaADF8基因在小麦中的功能机理奠定基础,并为进一步丰富小麦ADF基因研究内容提供理论参考,本研究利用电子克隆策略从小麦品种CP53中克隆出TaADF8基因(GenBank登录号为KJ864962)后对其进行序列分析,并进一步采用荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术对其在小麦不同组织间的表达差异及不同非生物胁迫下的表达模式进行分析。核酸序列分析表明,该基因全长695bp,拥有完整的ORF,编码142个氨基酸。氨基酸序列分析表明,该蛋白含有保守的ADF同源区和PIP2结合结构域,且在氨基端有核定位信号。进化和聚类分析表明,小麦TaADF8基因与大麦HvADF2基因、HvADF3基因和水稻OsADF3基因亲缘关系较近,蛋白相似度分别为75.35%、93.66%和67.86%。qRT-PCR表达特性分析显示,该基因为组成型表达,在根、茎、叶、颖壳和雄蕊中均表达,且在根、叶和雄蕊中表达量较高;该基因表达受低温的强烈诱导,同时也受水分、高盐和外源脱落酸胁迫诱导。     

不同发育特性小麦品种春化基因 VRN2的序列分析

为了明确小麦春化基因VRN2与春化发育表现型的关系,以6个不同春化发育特性的普通小麦品种为试验材料,采用PCR技术对春化基因VRN2的CCT保守区中43个氨基酸序列进行了分析。结果表明,不同品种间ZCCT-A1的CCT功能域的序列存在差异,肥麦有R35W突变,另外5个品种均有R39C突变;ZCCT-A2均存在R16C突变;B和D基因组中均未发现突变。这表明VRN2基因编码区的等位变异主要出现在A基因组上,而B和D基因组中的VRN2基因在目前大面积主栽品种中均为显性。     

玉米脂肪酸α-双加氧酶cDNA克隆与表达分析

以玉米自交系丹340为实验材料,同源克隆到玉米脂肪酸α-双加氧酶基因(ZmDOX)cDNA序列,并利用半定量与定量RT-PCR分析其在不同组织和PEG胁迫下的表达特征。序列分析结果表明,该cDNA序列包含1 857 bp完整的开放阅读框,编码619个氨基酸残基的蛋白产物。玉米、水稻、小麦中DOX氨基酸序列高度同源,聚集在同一进化枝,而双子叶植物存在两类DOX蛋白。组织表达特征分析发现,ZmDOX在根、茎、叶中均有表达,其中在根中表达量最高。PEG胁迫条件下,根中ZmDOX对胁迫的响应较快且增加幅度较大,而在叶中表达量有降低的趋势。     

Larvicidal property of essential oils against Culex quinquefasciatus Say (Diptera: Culicidae)

Essential oils from 22 aromatic plant species were tested for mortality of the mosquito larvae Culex quinquefasciatus. Lethal concentrations were determined for individual essential oils. Essential oils obtained from Thymus vulgaris, Satureja hortensis and Thymus satureioides plants showed the highest effect, with LC₅₀ found lower than 50 μg/ml (33, 36 and 44 μg/ml, respectively). Analyses showed that majority substances for T. vulgaris were thymol and p-cymene (60.3 and 10.1%, respectively); carvacrol and γ-terpinene for S. hortensis (48.1 and 36.7%, respectively), and borneol and thymol for T. satureioides (30.3 and 32.5%, respectively).The selected essential oils also showed very good effectiveness with respect to mortality and percentage of adult emergence upon short-term exposure in water contaminated with lethal doses of individual oils. While there was 77% adult emergence from the larvae in the control, in T. vulgaris, T. satureoides and S. hortensis there was only 12.3, 15.3 and 16.0% adult emergence, respectively. High antioviposition effectiveness was found in all selected oils. Almost 100% deterrence of female oviposition was determined for all oils in concentrations of 0.02%. Significant differences were seen with tested concentrations of 0.01 and 0.005%, where the oil of T. vulgaris proved most effective (repellency about 99.8 and 62.3%, respectively).     

普通小麦TaTPR1基因的克隆及表达分析

具有TPR基序的蛋白质被认为能够介导蛋白质间的相互作用,并且参与多种生物学过程,如细胞周期调控、转录调控、过氧化物酶等蛋白质的运输、信号传导和蛋白质折叠等。为了在小麦分子育种研究中获得更多有价值的候选基因,本研究采用电子克隆和RT-PCR方法从小麦中克隆得到1个TPR类基因,命名为TaTPR1。该基因ORF长度972bp,推测编码包含323个氨基酸残基的蛋白,相对分子质量34.9kD,理论等电点为5.89。氨基酸序列分析表明,该蛋白在142~207区和207~274区分别含有TPR类基因家族特有的保守结构域(TPR_16和TPR_11)。进化和聚类分析表明,小麦TaTPR1基因与粗山羊草AtTPR15基因、乌拉尔图小麦TuTPR15基因的亲缘关系较近,蛋白相似度分别为91.28%和91.55%。Real-time PCR表达特性分析显示,该基因为组成型表达,在根、茎、叶中均表达;幼苗期茎中表达量较高,随幼苗的生长,茎中表达量上调显著;该基因表达受高盐的强烈诱导,也受水分、低温和外源ABA胁迫诱导。     

Changes in essential oil and fatty acid composition in coriander (Coriandrum sativum L.) leaves under saline conditions

The influence of salinity on essential oil and fatty acid composition of Tunisian coriander (Coriandrum sativum L.) leaves grown in hydroponic culture was investigated. Volatile constituents and fatty acid of leaves were analyzed. The essential oil yield increased significantly up to 18 and 43% with 25 and 50 mM NaCl, respectively and decreased significantly under high salinity. The major volatile compound in leaves was (E)-2-decenal with 52% of total essential oil constituents, followed by decanal, dodecanal, (E)-2-tridecenal and (E)-2-dodecenal. Further, the content of these compounds was affected differently by the treatment level.Salinity decreased significantly the total fatty acid content of the upper and basal leaves. α-Linolenic (C18:3n3) was the main compound of both organs, followed by linoleic (C18:2n6), heptadecenoïc (C17:1n7) and palmitic (C16:0) acids. Raising NaCl concentrations lead to an important decrease in the unsaturated to saturated fatty acid ratio stimulating the formation of more rigid membrane.     

小麦β-酮脂酰CoA合成酶基因KCS的克隆与酵母表达

超长链脂肪酸是生物体内众多重要物质的合成底物。KCS基因编码β-酮脂酰CoA合成酶,该酶具有底物特异性,参与超长链脂肪酸延伸的缩合反应,是超长链脂肪酸合成的限速步骤。为了探究小麦KCS基因在超长链脂肪酸合成中的功能,采用同源克隆的方法从小麦(Triticum aestivum L.)中克隆出KCS基因后,利用生物信息学对其编码序列进行分析,并在酿酒酵母(Saccharomyces cerevisiae)中对其进行真核表达。结果表明,小麦TaKCS6基因的开放阅读框为1 287bp,编码428个氨基酸残基。结构域预测结果显示,TaKCS6蛋白含有III型聚酮合酶脂肪酸延伸酶和C末端3-酰基ACP合酶III结构域,属于KCSs蛋白家族。序列比对分析结果显示,TaKCS6氨基酸序列与拟南芥及其他植物的KCS6氨基酸序列在两个功能结构域上和活性位点保守。酵母表达结果显示,TaKCS6基因编码的蛋白参与C24以上超长链脂肪酸的延伸。     

小麦CO-like基因TaCO9的克隆及分析

CO蛋白(CONSTANS)是光周期途径中重要的调控因子,为了从普通小麦中进一步挖掘COlike基因,利用同源克隆的方法得到与大麦HvCO9基因同源的小麦TaCO9基因。结果表明,在冬性品种西农889中,初步克隆得到TaCO9基因的三个同源序列,分别命名为TaCO9-1、TaCO9-2、TaCO9-3。其cDNA序列全长均为870bp,开放阅读框为1 977bp,编码289个氨基酸,含有CO-like蛋白家族典型的CCT结构域,但不含B-box结构域;而在春性品种中发现TaCO9-1序列的第二外显子区域有6个碱基的插入,利用中国春缺-四体材料将该序列定位于小麦的1A染色体,命名为TaCO9-1A。系统发育分析表明,TaCO9蛋白与水稻Ghd7及大麦HvCO9位于同一分支。空间结构分析表明,其CCT结构域的NF-YA2区域较为保守,而该区域与CCAAT box互作相关。本研究克隆得到的TaCO9基因可能是小麦CO-like基因家族的新成员,与大麦HvCO9基因的结构相似,可作为新的小麦光周期候选基因加以研究利用。     

青稞法尼基转移酶β亚基编码基因HbERA1的克隆及表达分析

ERA1(Enhanced response to ABA)基因编码法尼基转移酶(Farnesyl transferase)β亚基,该酶在干旱胁迫下对ABA信号负向调控因子的修饰起着关键作用。本研究以青稞(Hordeum vulgare subsp.vulgare)抗旱品种喜马拉雅10号为材料,利用RT-PCR技术克隆获得了ERA1基因全长cDNA序列,命名为HbERA1(登录号:KJ699392)。生物信息学分析表明,该基因全长1 401bp,可编码466个氨基酸序列,蛋白分子量为51.14kD,等电点为5.00。Prosite Scan分析结果表明,HbERA1含有多个干旱胁迫响应蛋白的作用位点,如酪蛋白激酶Ⅱ磷酸化位点、N-糖基化位点、蛋白激酶C磷酸化位点及N-豆蔻酰化位点。利用实时定量PCR方法研究了HbERA1在干旱胁迫条件下及复水后不同时间点的表达情况,发现在水分过剩处理下(土壤绝对含水量15.5%),HbERA1在土壤绝对含水量为33.4%时表达量最高,并随着土壤绝对含水量的下降而下调表达;进行干旱胁迫后(15.5%)基因表达量也明显下调表达;复水后表达逐渐恢复,复水8h时超过正常表达水平,表明HbERA1基因可能参与调控水涝和干旱胁迫双重信号传导。     

Effect of imazamox plus bentazon on dry bean (Phaseolus vulgaris L.)

Imazamox plus bentazon has the potential to provide broad spectrum weed control in dry bean (Phaseolus vulgaris L.). Field trials were conducted in Ontario over a two year period (2003 and 2006) to evaluate the effect of imazamox plus bentazon applied post-emergence at 25 + 600 and 50 + 1200 g ai ha⁻¹ on black, brown, cranberry, kidney, otebo, pinto, white and yellow eye beans. Treatments included a non-treated check. All treatments were maintained weed free during the growing season. The eight market classes responded similarly to imazamox plus bentazon. Imazamox plus bentazon applied post-emergence caused less than 4% visible injury at 7, 14 and 28 days after treatment. Imazamox plus bentazon applied post-emergence did not have any effect on plant height, shoot dry weight, seed moisture content and yield of dry bean. Based on these results, imazamox plus bentazon at the proposed maximum use dose can be safely used for weed management in black, brown, cranberry, kidney, otebo, pinto, white and yellow eye bean production in Ontario. Additional research is needed to determine if cultivars within a market class of dry bean differ in their response to imazamox plus bentazon.     

小麦细胞色素P450(TaP450)基因的克隆与序列分析

细胞色素P450是一种多功能氧化酶,在植物体内担当着生物合成、代谢解毒以及抗逆等重要功能。本研究采用RACE方法从普通小麦中同源克隆到一个新的P450基因,命名为TaP450(Genbank No.KJ541960)。该基因基因组序列全长为2 643bp,含有2个外显子和1个内含子,cDNA全长为2 033bp,含有一个1 557bp的开放读码框,推导蛋白含518个氨基酸;序列分析发现其具有保守的P450结构域,但该蛋白与已报道的小麦P450蛋白序列有较大差异,表明它是小麦P450家族的新成员;蛋白结构特征分析发现,该蛋白的分子量为57.092kD,等电点为8.63,N端具有一个含29个氨基酸的跨膜结构和一个含22个氨基酸的信号肽,亚细胞定位分析表明该蛋白属于分泌蛋白。其在小麦叶片、茎、根与种子中均有表达,但在叶片和茎中表达量最高;在胁迫处理下,该基因的表达量均上调,且在盐胁迫处理下上调尤为显著,表明该基因与盐胁迫密切相关。     

Identification of resistance to Uromyces pisi (Pers.) Wint. in Pisum spp. germplasm

A germplasm collection of 2759 pea accessions was screened for resistance against Uromyces pisi (Pers.) Wint. All accessions displayed a compatible interaction (high infection type) both in adult plants under field conditions and in seedlings under growth chamber conditions, but with varying levels of disease reduction. The identified resistance was based on reduction of disease severity with no associated host cell necrosis, which fits the definition of Partial Resistance. No complete resistance or incomplete resistance based on hypersensitivity was observed.     

小麦耐冷相关基因TaCTR的克隆及其表达特性分析

低温冷害是小麦生长发育过程中面临的重要非生物逆境因素。为了挖掘小麦耐冷功能基因,本研究采用同源克隆的方法从普通小麦品种小偃22中分离到一个耐冷相关基因TaCTR,该基因序列全长2 192 bp,含有12个外显子、11个内含子,编码区全长为1 407 bp,编码468个氨基酸,分子量约为53.43 kDa;系统进化树分析表明,该基因在进化关系上与山羊草最近;亚细胞定位结果显示,该基因编码的蛋白为膜蛋白;实时荧光定量PCR（RT-PCR）结果表明,TaCTR在不同品种、不同组织和不同发育阶段低温特异表达;在干旱、高盐及GA处理下,TaCTR的表达量显著上升,说明该基因可能参与调控小麦的抗逆反应。     

Field trials of Metarhizium anisopliae var. acridum (Ascomycota: Hypocreales) against oriental migratory locusts, Locusta migratoria manilensis (Meyen) in Northern China

During 2002–2006, nymph bands of Locusta migratoria manilensis (Meyen) were treated by ground and aerial applications in 6000 ha of grasslands and the nearby beach of Yellow river using a soybean oil miscible suspension ULV formulation of Metarhizium anisopliae var. acridum isolate CQMa102. The formulation was also applied in Tianjin, Henan, Hebei, Shandong and Shanxi provinces of Northern China by ground and aerial applications. During field studies, cage tests were carried out in corresponding field plots in order to estimate the mortality accurately. Doses of 3.3×10¹² and 5.0×10¹² conidia ha⁻¹ were equally effective and caused 90% mortality 9–13 days after treatment. In the ground spray trial, 3.3×10¹² conidia ha⁻¹ killed >90% of L. migratoria manilensis 11–15 days after treatment in a wide variety of vegetation and weather conditions. The decline of locust populations was slower where vegetation was taller and denser. In the aerial spray treatment, the final percent survival of locusts was lowered to 10% at 11 and 14 days in the field cage and open field locusts, respectively. Furthermore, the M. anisopliae oil miscible suspension formulation did not appear to harm natural enemies of locusts in the field.     

小麦隐性核不育保持系表达载体的构建及拟南芥遗传转化

利用小麦杂种优势能够创制稳定的雄性不育系。相比于细胞质雄性不育系和光温敏雄性不育系,细胞核雄性不育具有育性稳定、易恢复、不受环境影响等特点。小麦隐性核不育基因 ms1的克隆以及杂交制种技术(hybrid seed production technology, SPT)的开发,为雄性不育的保持和杂种优势利用奠定了基础。本研究将小麦花粉育性恢复基因 Ms1、花粉致死基因 ZmAA1、红色荧光蛋白基因 DsRed2及其特异性启动子和终止子连接到表达载体pGEII上,转化野生型拟南芥,得到6株转基因拟南芥。通过荧光显微镜观察拟南芥种子,发现野生型拟南芥种子表面平滑,不能发出红色荧光;而转基因种子表面呈现颗粒状,能够发出红色荧光。这说明表达载体构建成功,并能够在拟南芥中成功表达。这为创制人工小麦隐性核不育系奠定了理论和材料基础。     

Essential oil composition of Hypericum perfoliatum L. and Hypericum tomentosum L. growing wild in Tunisia

The essential oils obtained by hydrodistillation from the aerial parts of Tunisian native Hypericum perfoliatum L. (sect. Drosocarpium Spach.) and Hypericum tomentosum (sect. Adenosepalum Spach.) were analyzed by GC and GC–MS. Thirty-two compounds were identified in the essential oils of H. perfoliatum with α-pinene (13.1%), allo-aromadendrene (11.4%), germacrene-D (10.6%), n-octane (7.3%), α-selinene (6.5%) and β-selinene (5.5%) as main constituents. Sixty-seven components were identified in the oil of H. tomentosum with menthone (17.0%), n-octane (9.9%), β-caryophyllene (5.3%), α-pinene (5.2%), lauric acid (4.1%) and β-pinene (3.7%) as the most abundant components. Both oils were characterized by the presence of many components which could have numerous applications in food, pharmaceutical and perfume industries.     

Sorghum (Sorghum bicolor L. Moench) contains a XIP-type xylanase inhibitor but none of the TAXI- and TLXI-types

A xylanase inhibitor of the xylanase-inhibiting protein (XIP) type was detected in sorghum (Sorghum bicolor L. Moench) whole meal using Western blot and immunoprobing with polyclonal anti-XIP antibodies. No detectable levels of Triticum aestivum xylanase inhibitor (TAXI) and thaumatin-like xylanase inhibitor (TLXI) type xylanase inhibitors were present. Trichoderma longibrachiatum xylanase affinity chromatography (AC) was used for the purification of sorghum XIP. Biochemical characteristics and protein sequence data show that sorghum XIP strongly corresponds to wheat (T. aestivum L.) XIP-I. Like wheat XIP-I, it inhibits both glycoside hydrolase family (GH) 10 and 11 xylanases, indicating that the XIP-I active site residues are well conserved in sorghum XIP. However, in contrast to wheat XIP-I, the inhibitor is unable to affect Aspergillus niger GH 11 xylanase activity. Its specific inhibition activities towards the other xylanases tested are comparable to those of wheat XIP-I. Based on the available sorghum expressed sequence tag (EST) database, XIP is expressed in sorghum in different tissues and developmental stages. Also expression in the presence of several plant hormones and under biotic as well as abiotic stress conditions is suggested.     

Characteristics, composition and thermal stability of Acacia senegal (L.) Willd. seed oil

The chemical composition, main physicochemical properties and thermal stability of oil extracted from Acacia senegal seeds were evaluated. The oil, moisture and the ash contents of the seeds were 9.80%, 6.92% and 3.82%, respectively. Physicochemical properties of the oil were iodine value, 106.56 g/100 g of oil; saponification value, 190.23 mg KOH/g of oil; refractive index (25 °C), 1.471; unsaponifiable matter, 0.93%; acidity, 6.41% and peroxide value, 5.43 meq. O₂/kg of oil. The main fatty acids in the oil were oleic acid (43.62%) followed by linoleic acid (30.66%) and palmitic acid (11.04%). The triacylglycerols (TAGs) with equivalent carbon number ECN 44 (34.90%) were dominant, followed by TAGs ECN 46 (28.19%), TAGs ECN 42 (16.48%) and TAGs ECN 48 (11.23%). The thermal stability analysed in a normal oxidizing atmosphere showed that the oil decomposition began at 268.6 °C and ended at 618.5 °C, with two stages of decomposition at 401.5 °C and 576.3 °C. According to these results, A. senegal seed oil has physicochemical properties, fatty acids composition and thermal characteristics that may become interesting for specific applications in several segments of food and non-food industries.     

燕麦AsRBP1基因克隆及表达特性分析

为了给AsRBP1基因在小麦抗逆转基因分子育种中的应用奠定理论基础,采用同源克隆方法从燕麦中克隆、获得AsRBP1基因,其cDNA序列全长447 bp,编码148个氨基酸.AsRBP1在氨基端具有典型的RNA识别基序(RNA-Recognition Motif,RRM),在羧基端富含甘氨酸,其含量达35.8％.系统进化树分析表明,AsRBP1与拟南芥AtGR-RBP7亲缘关系很近.实时荧光定量聚合酶链式反应(Real-time PolymeraseChain Reaction,RT-PCR)分析该基因的表达特性表明,AsRBP1明显受到外源脱落酸(Abscisic acid,ABA)与低温的诱导,同时对干旱和高盐胁迫也做出响应.由此,推测该基因属于GR-RBPs基因家族成员,可能参与逆境胁迫应答,在增强植物的抗逆性中发挥着重要的作用,可以为小麦抗逆分子育种提供优良候选抗逆基因资源.