白花虎眼万年青绿原酸合成酶基因QtHQT的克隆及表达分析

羟基肉桂酰辅酶A奎尼羟基肉桂转移酶(hydroxycinnamoyl-Co A quinate hydroxycinamoyl transferase,HQT)是植物绿原酸合成过程中的关键酶。为研究HQT基因在白花虎眼万年青绿原酸合成中的分子机理,根据已获得的白花虎眼万年青转录组数据中的HQT基因序列,设计引物,利用RT-PCR方法获得白花虎眼万年青HQT基因的完整编码区序列;对该基因进行了生物信息学分析,并采用相对定量PCR方法分析了其在不同器官中的表达特性。研究结果表明,分离出的白花虎眼万年青HQT基因长1 284 bp,编码427个氨基酸并包括应有的保守区域,命名为Qt HQT;Qt HQT基因在白花虎眼万年青的鳞茎中表达量最高,珠芽其次,叶片中最低。本研究从白花虎眼万年青中克隆了Qt HQT基因,并对其进行了生物信息学和相对表达量分析,为后续研究奠定了基础。     

茉莉酸甲酯对中国红豆杉细胞基因表达的mRNA差异显示研究

茉莉酸甲酯是一种环戊烷衍生物型植物激素,在植物次生代谢物质生物合成中起着重要作用。我们以中国红豆杉悬浮培养细胞为材料,设两组平行实验,一组为阴性对照组,一组以茉莉酸甲酯进行诱导,所得细胞利用mRNA差异显示技术进行分析,研究了茉莉酸甲酯对红豆杉细胞mRNA表达的差异,最终共获得34个差异表达片段。经二次扩增和单引物阴性对照实验及序列测定,共获得9个阳性差异表达片段。与GenBank数据库比对,结果表明其中2个片段与已知基因有较高的同源性,与欧洲赤松（Pinus sylvestris）cDNA克隆的同源性为88％,与南方红豆杉（Taxus mairei）18SrRNA基因的同源性为93％,其余7个差异表达片段与GenBank中所有基因的同源性都非常低,可能代表了红豆杉细胞中的受茉莉酸甲酯诱导的未知基因序列。     

白花虎眼万年青QtNPY4组成型表达对烟草叶片形态的影响

白花虎眼万年青的离体叶片在细胞分裂素作用下叶片表面产生多个珠芽,即"叶上珠芽",其再生机制尚需阐明。白花虎眼万年青叶上珠芽转录组分析表明,NPY基因在该过程中发挥了重要作用;NPY基因是BTB基因家族的重要成员,在生长素调控下参与了器官发生。为了探析白花虎眼万年青叶上珠芽的发生机制,我们将来自白花虎眼万年青的叶上珠芽的QtNPY4基因置于泛素启动下在烟草中持续表达,在细胞分裂素调控下转基因烟草出现了叶片浅裂、倒卵形甚至叶脉长芽等多种表型。以上研究说明QtNPY4基因具有调控维管束发育和茎尖分生组织活性的功能,能提高植物的再生能力,能赋予或提高叶表维管束分化和再生能力并且受细胞分裂素调控;可能在白花虎眼万年青的叶上珠芽的形成和发育过程中发挥重要作用,在生物技术中具有潜在的应用价值。     

白花虎眼万年青QtWOX8基因的克隆及特性分析

Wuschel-related homeobox(WOX)基因是植物特有的同源异型盒转录因子,在调控干细胞活性和器官发育中发挥重要作用,然而在百合科植物等鳞茎球根花卉中中尚未见到报道。本研究首次从百合科植物白花虎眼万年青叶上珠芽中分离出WOX基因家族成员,进行氨基酸保守序列比对及系统进化分析后,将其命名为QtWOX8基因。将其置于UBI启动子下在烟草中超量表达,转基因烟草的叶片形状、叶脉等发生了明显地改变,结果表明QtWOX8基因对植物干细胞具有明显调控作用,可能在白花虎眼万年青的珠芽和叶片等器官发育过程中发挥着重要作用。     

白花虎眼万年青QtCIGR1基因的克隆及功能分析

CIGR(Chitin-inducible gibberellin-responsive gene)基因是植物GRAS基因家族的重要成员,在植物器官发育中发挥重要作用,然而在百合科植物等鳞茎球根花卉中尚未见到报道。本研究从百合科植物白花虎眼万年青叶上珠芽中分离出CIGR基因及其基因家族成员,进行氨基酸保守序列比对及系统进化分析后,将其命名为QtCIGR1基因;将其置于UBI启动子下在烟草中超量表达,转基因烟草的节间显著伸长及直径明显增粗,幼叶形状也发生了明显的变化,表明QtCIGR1基因对植物的节间伸长和直径增粗具有明显调控作用,可能在白花虎眼万年青的珠芽、节间和花茎等器官发育过程中发挥着重要作用。     

白花虎眼万年青QtKN1基因的克隆及功能分析

在离体条件下,白花虎眼万年青的叶片表面可以产生多个珠芽,即叶上珠芽。KNOXI基因是真核生物中高度保守的同源盒转录因子,在植物的珠芽等器官发生中起着重要作用。为了探析白花虎眼万年青叶上珠芽的发生机制,我们从中克隆出了其同源基因,通过对其编码的蛋白质序列比对和系统发育分析,认为其属于KNOXI在白花虎眼万年青中的同源基因,将其命名为Qt KNI基因。将其转入烟草后,导致叶片边缘浅裂、边缘缺失、形状卵形且叶脉紊乱等多种表型。以上研究说明Qt KNI基因可能在白花虎眼万年青的珠芽等器官发育中起着重要作用,并且在生物技术中具有潜在的应用价值。     

扁蓿豆DNA甲基转移酶基因MrMET1的克隆及表达分析

DNA甲基化是一类重要的表观遗传修饰,在植物对非生物胁迫的响应中发挥重要作用。DNA的甲基化需要DNA甲基转移酶催化。为挖掘扁蓿豆中DNA甲基转移酶基因,解析其是否参与扁蓿豆抗逆性应答,本研究利用RT-PCR技术从扁蓿豆中克隆到1个胞嘧啶5-甲基DNA转移酶基因,该基因含1 071 bp的开放阅读框,编码357个氨基酸。生物信息学分析确定该基因为DNA甲基转移酶1 MrMET1基因。实时荧光定量PCR显示,MrMET1基因在不同非生物胁迫(低温,干旱, NaCl)及ABA处理下,不同时间表达量呈现不同的变化趋势,推测该基因参与了扁蓿豆对非生物胁迫的响应。本研究结果对深入解析METs基因的功能,理解牧草对非生物逆境胁迫响应机制具有一定的参考意义。     

白花虎眼万年青叶上珠芽的转录组分析

白花虎眼万年青可在离体条件下以叶片表面上产生多个珠芽方式实现高效再生,"叶上生根,落地成苗",为解决植物困难再生的问题提供了新的思路和途径,但是其形成机制尚不清楚。本研究以其叶片及其表面产生的珠芽为研究材料,进行Illumina Hi Seq 2000测序分析。共获得原始数据9.13 G,经过数据过滤后得到有效数据7.67 G,及178 385条Unigene。研究表明,叶片产生珠芽过程中有2 397条Unigene上调,1 593条Unigene下调;EMB基因、TATA结合蛋白、BTB/POZ锚蛋白重复序列、TOR调控蛋白、FKBP蛋白等基因的表达发生了明显地变化,可能参与了珠芽的形成。本研究首次获得了白花虎眼万年青叶上珠芽的转录组数据,分析了叶片中可能参与形成珠芽的重要功能基因,为探讨叶上珠芽发育的分子机制及进行重要功能基因的挖掘提供了分子数据。     

甘蔗丙二烯氧化物合成酶ScAOS的克隆与表达分析

茉莉酸类物质(JAs)作为植物体内的内源生长调节物质,其包括了茉莉酸、茉莉酸甲酯、茉莉酸异亮氨酸及其相关衍生物质,对植物生长发育及抗性防御机制发挥着重要作用。丙二烯氧化物合成酶(allene oxide synthase, AOS)是植物体内茉莉酸生物合成途径的关键酶,影响着茉莉酸类物质的生物合成,进而调节植物的形态建成和防御反应。本研究基于甘蔗转录组数据库,成功克隆到甘蔗丙二烯氧化物合成酶ScAOS的ORF全长序列,利用生物信息学方法了解该基因基本理化性质,并通过qRT-PCR实验分析不同组织和不同胁迫下ScAOS的表达水平差异。生物信息学结果表明,ScAOS基因开放阅读框为1 450 bp,编码482个氨基酸,等电点为6.30,不稳定系数为28.4,平均疏水性值为-0.12,预测ScAOS基因编码的蛋白为稳定酸性亲水蛋白。二级结构三级结构预测其元件结构主要为α-螺旋结构和无规则卷曲,该基因含有P450超家族的PLN02648的结构域,属于细胞色素P450基因家族中的CYP74A蛋白家族成员。系统进化树结果显示,ScAOS与高粱处在同一分支上,一致性高达90.4%。qRT-PCR结果显示,ScAOS在甘蔗根茎叶中均有表达,其中在茎中的表达量最高,在叶中的表达量最低。不同胁迫处理的表达差异结果显示,ScAOS均能响应盐害、干旱、植物激素、病虫害等环境胁迫。其中在NaCl、PEG、MeJA、病原菌和黏虫取食胁迫下表达水平均表现持续上升趋势,而在ABA处理下则表现下调趋势。本研究成功克隆到甘蔗ScAOS基因,并通过表达分析发现ScAOS在植物抗性防御方面作用显著。     

广藿香PatTIFY6b基因克隆与功能分析

茉莉酸甲酯能诱导广藿香叶片中百秋李醇含量升高,但是其中的分子机制不清楚。本研究从茉莉酸甲酯处理的广藿香叶片转录组数据中筛选到一个表达量显著增加的基因,经过序列比对发现该基因与唇形科丹参转录因子Sm TIFY6b具有高度同源性,因此将其命名为PatTIFY6b。通过生物信息学对PatTIFY6b编码蛋白的结构和功能进行分析和预测,并以广藿香cDNA为模板,克隆得到PatTIFY6b。PatTIFY6b定位于细胞核,在广藿香不同组织中均有表达,叶片中表达量最高。茉莉酸甲酯处理下,PatTIFY6b和百秋李醇合成途径基因具有相似的表达模式,说明茉莉酸甲酯可能通过PatTIFY6b调控百秋李醇的合成。利用原核表达系统,成功表达了PatTIFY6b蛋白。将PatTIFY6b过表达在双子叶模式植物拟南芥中,筛选到纯合株系PatTIFY6b-ox-17。本研究首次克隆广藿香PatTIFY6b基因并对其功能进行初步探究,为后期研究广藿香百秋李醇生物合成的分子调控机制提供理论基础。     

In-vitro Fertilization of Ovules of Some Species of Brassicaceae*

The method of in-vitro fertilization of ovules can be successfully applied to various species of Brassincaceae. Mature embryos and plants were obtained after in-vitro pollinating the ovules of Arabis caucasica, Brassica napus, B. oleracea var. sabellica (kale), B. oleracea var. italica (broccoli), Diplotaxis tenuifolia, Moricandia arvensis and Sisymbrium Loeselli. In the case of Sinapis alba fertilization and embryo development did not occur. The same method has been successfully used for obtaining hybrid immature embryos at different stages of development from crosses between B. napus X D. tenuifolia, B. napus X M. arvensis, B. oleracea var. italica X D. tenuifolia, D. tenuifolia x B. napus, D. tenuifolia. X M. arvensis and D. tenuifolia X S. Loeselli. The present findings show that in-vitro pollination of ovules of various species of Brassicaceae makes it possible to (a) perform the whole process of fertilization and embryogenesis; and (b) obtain intergeneric hybrid embryos.     

The potential role of PR-8 gene of apple fruit in the mode of action of the yeast antagonist,Candida oleophila,in postharvest biocontrol of Botrytis cinerea

Several pathogenesis-related (PR) genes of apple have been cloned and their response to different pathogens has been studied. Different PR genes, however, may have a variable response depending on the specific organ or tissue as well as microbe. The objective of the current study was to characterize the expression of specific apple PR genes in fruit tissues in response to the antagonistic yeast, Candida oleophila (a common postharvest biocontrol agent), and the fungal pathogen, Botrytis cinerea (a major postharvest pathogen). Apple PR-5 and PR-8 gene expression was characterized in fruit in response to C. oleophila and B. cinerea. Results indicated that PR-8 expression was significantly elevated in response to both fungi. In contrast, neither C. oleophila nor B. cinerea treatment markedly affected PR-5 expression. The PR-8 gene was then synthesized and cloned into a Pichia pastoris expression system to study the antifungal activity of the PR-8 protein against B. cinerea both in vitro and in vivo. Collectively, the PR-8 gene of apple is associated with the response to B. cinerea infection, and may play a role in the mechanism by which C. oleophila effectively inhibits B. cinerea disease in apple fruit, namely by the induction of this specific host PR gene.     

灰分对煤自燃特性影响的实验研究

为了研究灰分对煤自燃能力的影响作用,利用绝热氧化实验装置对不同灰分含量煤样进行升温氧化实验,采用R₇₀、T_CPT、B 3种指标表征灰分含量对煤样自发氧化过程的影响。结果表明：1）灰分含量越大,煤样低温氧化阶段温升速率越小,温升加速点温度越高,煤样的自发氧化过程越慢,煤越不易自燃;灰分含量大于40%后,煤自燃倾向性快速减弱。温升加速点是反应微观信息的零活化能温度的宏观累计结果,具有直观且滞后的特点。灰分越大,滞后越明显,温差越大。2）R₇₀、T_CPT、B 3种指标与灰分关系表现为二次函数。R₇₀和T_CPT两种指标显示灰分越大,自燃倾向性越弱,与实践经验相符。受水分权重影响,B指标显示煤样在灰分小于40%时,灰分越大,煤样自燃倾向性越强,这与实践经验相悖。因此,B在判定灰分对煤样自燃倾向性的影响时具有一定的局限性。     

Yield Performance of Wheat Isolines with Different Dosages of the Short Arm of Rye Chromosome 1

Translocations of the short arm of rye (Secale cereale L.) chromosome 1 (1RS) in wheat (Triticum aestivum L. cv. Pavon 76) are known to increase root biomass. Such an increase enhances water and nutrient uptake and may improve grain yield. Two greenhouse experiments and a field experiment were carried out at the University of California, Riverside, in 2012 and 2013 under well‐watered and terminal drought treatments to evaluate phenotypic characters associated with varying dosages of 1RS, including grain yield. The genotypes used were cultivar Pavon 76 (R₀), Pavon 76/Pavon1RS.1AL (F₁ hybrid) with a single dosage of 1RS (R₁A), Pavon 1RS.1AL with two dosages of 1RS (R₂A), Pavon 1RS.1DL (R₂D) also with two dosages of 1RS and Pavon 1RS.1AL‐1RS.1DL (R₄AD) with four dosages of 1RS. There was a significant positive correlation between number of dosages of 1RS and root biomass. However, no correlation was found between root biomass and grain yield per plant. Drought in the field experiment reduced grain yield significantly. Under well‐watered field conditions, grain yield of R₂A (215.9 g plant^?1) was significantly greater than those of R₂D (191.8 g plant^?1) and R₄AD (161.7 g plant^?1). Also, grain yield of R₄AD was significantly less than those of F₁, Pavon 76 and R₂D under well‐watered conditions. Under drought field conditions, no significant differences were found among the genotypes for grain yield was found between F₁ (14.7 g plant^?1) and R₄AD (12.4 g plant^?1). Harvest index was significantly greater in well‐watered (44.2 %) than in drought (34.6 %) field conditions. On average, genotypes F₁ (42.3 %) and R₂A (40.6 %) had higher harvest index than R₂D (38.3 %) and R₄AD (35.5 %) in the field. Also, Pavon 76 (40.2) and R₂D (38.3) had higher harvest index than R₄AD. Drought tolerance was lowest for R₄AD due to its relatively lower grain yield potential. In general, Pavon 1RS.1AL carrying two dosages of 1RS showed higher grain yield under wet treatments. Pavon 1RS.1AL‐1RS.1DL carrying four dosages of 1RS produced the largest shoot and root biomasses, but the least grain yield.     

Evolutionary studies of the soybean: the frequency and distribution of alleles among collections of Glycine Max and G. Soja of various origin

Summary Allelic frequencies at 10 loci common throughout the genus Glycine subgenus Soja were determined for 27 geographical area-species samples. The samples included 366 accessions of Glycine soja, the wild soybean, and 193 accessions of G. max, the domesticated soybean, from the USDA Soybean Germplasm Collection. The data indicate that the alleles for grey pubescence (t ₁), low seed coat peroxidase level (ep), and blunt pubescence tip (pb) probably arose as mutations during the domestication of G. max. The remaining seven loci studied(Fr, Pa ₁, Pa ₂, Fg ₁, Fg ₂, Fg ₃and Fg ₄)are polymorphic throughout the subgenus Soja; differences among collections of G. max and G. soja seem to be the result of differing selection pressures. A cluster analysis of allelic frequencies reveals two distinct groups within the subgenus corresponding to G. soja and G. max, Semi-wild accessions of G. max, while morphologically more similar to cultivated plants, clustered with samples of G. soja. The semi-wild accessions examined are thought to have arisen via hybridization between G. soja and G. max.Joint contribution: North Central Region, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, and Journal Paper No. J-9868 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011; Project 2107.     

Genetic control of crossability of triticale with rye

Limited genetic knowledge is available regarding crossability between hexaploid triticale (2n= 6x= 42, 21″, AABBRR, amphiploid Triticum turgidum L.‐Secale cereale L.) and rye (2n= 14, 7″, RR). Our objectives were to determine (1) the crossability between triticales and rye and (2) the inheritance of crossability between F₂ progeny from intertriticale crosses and rye. First, ‘8F/Corgo’, a hexaploid triticale, was crossed as a female with two landrace ryes, ‘Gimonde’ and, ‘Vila Pouca’ and two derived north European cultivars, ‘Pluto’ and ‘Breno’. These crosses produced 21.7, 20.9, 5.9, and 5.6%, seed‐set or crossability, respectively, showing that the landrace ryes produced higher seed‐set than the cultivars. Second, ‘Gimonde’ rye was crossed as a male with four triticales for 3 years. The control cross, ‘Chinese Spring’ wheat × rye, produced 80‐90% seed‐set. Of the four triticales, ‘Beagle’ produced 35.7‐56.8% seed‐set. The other three triticales produced less than 20% seed‐set, showing that the triticales differ in crossability with ‘Gimonde’ rye. Third, six FiS from intertriticale crosses (‘8F/Corgo’בBeagle’, ‘Beagle’בCachirulo’, ‘Lasko’בBeagle’, ‘8F/Corgo’בCachirulo’, ‘Lasko’בCachirulo’, ‘Lasko’ב8F/Corgo’) were crossed to ‘Gimonde’ rye. Results indicated that lower crossability trait was partially dominant in the two F₁S from crosses involving ‘Beagle’(high crossability) with‘8F/Corgo’ and ‘Cachirulo’(low crossability) and completely dominant in the ‘Beagle’בLasko’ cross, as it happens in wheat. Fourth, segregants in four F₂ populations (‘Lasko’בBeagle’, ‘8F/Corgo’בBeagle’, ‘Lasko’ב8F/Corgo’, and‘8F/Corgo’בCachirulo’) were crossed with rye. Segregation for crossability was observed, although distinct segregation classes were blurred by environmental and perhaps other factors, such as self‐incompatibility alleles in rye. Segregation patterns showed that ‘Beagle’, with high crossability to rye, carries either Kr1 or Kr2. The three triticales with low crossability with rye were most likely homozygous for Kr1 and Kr2. Therefore, it is likely that the Kr loci from A and B genomes acting in wheat also play a role in triticale × rye crosses.     

Analysis of mode of inheritance of Fusarium wilt resistance in castor (Ricinus communis L.)

Castor (Ricinus communis L.) is an important industrial oilseed crop grown worldwide. Wilt caused by Fusarium oxysporum f.sp. ricini is a devastating disease in castor. The inheritance mode of wilt resistance was investigated. The F₁, F₂ and backcross generations of four crosses involving four resistant and three susceptible parents were developed. The role of digenic (R¹ and R²) epistatic interactions on wilt resistance was confirmed. The 15 : 1, 9 : 7 and 13 : 3 ratios indicated duplicate dominant, duplicate recessive and dominant and recessive epistatic interactions, respectively. Castor parents used in the crosses exhibited varied inheritance modes. All generations of a cross exhibited similar inheritance mode when parents were comparable. However, generations varied in inheritance mode when parents were not comparable in inheritance mode. These results would have practical interest when decisions are required regarding the choice of parents and methodology in resistance and hybrid breeding. The results also provided a basis for investigating molecular genetics of wilt resistance mechanisms.     

添加硝酸钙对池塘沉积物中理化因子的影响研究

为减少池塘底泥对养殖水环境的影响,以硝酸钙和养殖池塘底泥沉积物为研究对象,采用模拟试验方法,探讨了添加硝酸钙对池塘沉积物理化因子的影响。结果表明：添加硝酸钙后,表层沉积物间隙水中NH₄⁺-N浓度持续降低,最终降至初始值的14.78%;同时可以显著降低表层和深层沉积物间隙水中的PO₄^3--P的浓度,在表层降低的幅度较大,第7~28天可降低超过85%的PO₄^3--P。试验结果表明,添加硝酸钙可以显著改善池塘底泥沉积物中NH₄⁺-N和PO₄^3--P的含量,研究结果对减少池塘底泥沉积物的修复具有积极的生态学意义。     

Genetic Analysis of Chromosome 2D of Wheat

The Yugoslavian varieties ‘Novosadska Rana 1’ and ‘Sava’ are shown by monosomic comparisons to carry weak height promoters on chromosome 2D characteristic of the ‘Akakomugi’ gene for reduced height, Rht8. Reciprocal monosomic crosses between ‘Bersee’ and ‘Sava’ demonstrate ‘Sava’ chromosome 2D reduces height by about 16 cms, accelerates ear emergence by about 9 days and increases yield through increased grain number and grain size. Recombinant lines developed for chromosome 2D suggest that this chromosome in Mediterranean wheats carries three genes, Rht8, Ppd1 and Yr16, important to their adaptation. Rht8 and Ppd1, a gene for day length insensitivity together reduce height. Ppd1 and, to a minor degree, either yr16, the susceptible allele of a gene for adult plant resistance to yellow rust or a closely linked gene, accelerate time to flowering and thereby avoid desiccating Yugoslavian summer conditions. The same genes reduce spikelets numbers but this is offset by increased floret fertility producing an overall increase in the number of grains per ear. Ppd1 also by avoiding desiccating conditions increases gram size and together with either yr16 or the closely linked fertility gene increases ear and plant yields.     

Polyacrylamide gel electrophoresis of S-RNase fragments for identification of S-genotypes of Japanese pear (Pyrus pyrifolia)

Japanese pear (Pyrus pyrifolia) exhibits gametophytic self-incompatibility (GSI) controlled by a complex and multiallelic S locus. The pistil-part product of the S locus is the polymorphic ribonuclease, S-RNase. Information on S-genotypes is important for the production and breeding of Japanese pears. Molecular analyses of S-genotypes of Japanese pear have been conducted with the CAPS (cleaved amplified polymorphic sequence) system; PCR amplification of S-RNase fragments by a common primer pair followed by digestion with restriction enzymes each of which cleaves a specific S haplotype. Here, we show that the separation of S-RNase fragments by polyacrylamide gel electrophoresis (PAGE) distinguishes four out of nine S haplotypes of Japanese pear without restriction digestion. S³-, S⁵-, S⁶- and S⁸-RNases were identified as distinct bands by PAGE. S³- and S⁵-RNases were separated by PAGE despite their identical fragment sizes. Using this system, three Japanese pear lines with unknown S-genotypes were analyzed. The newly determined S-genotypes of the lines were confirmed by CAPS analysis.