牡丹PsELF1基因的克隆及表达分析

本研究以已获得的早花基因的EST序列为模板设计引物,通过RACE技术扩增全长,使用生物学软件和q RT-PCR技术分别进行生物信息学分析和表达模式分析,获得牡丹早花基因Ps ELF1的全长及其表达模式,为后续研究其功能提供条件。结果显示,扩增得到7 107 bp的Ps ELF1全长cDNA,其中,包括3'UTR485 bp,5'UTR 464 bp和6 258 bp的编码区,共编码2 085个氨基酸,分子量为238.43 kD。Ps ELF1包括4个可能的磷酸化位点,且均位于N端。二级结构预测显示,Ps ELF1含α-螺旋和无规卷曲较多。同源性分析结果表明,Ps ELF1与杨树ELF1同源性最高。表达特性分析表明,Ps ELF1在花芽休眠解除过程中Ps ELF1的表达水平在0～14 d下调,在14～21 d上调,然后在21～28 d下调。而在低温21 d移入温室后的开花过程中呈下调模式。本研究结果对培育牡丹早花或晚花品种、延长牡丹的观赏期、提高牡丹观赏质量和经济价值具有重要意义。     

牡丹PsPIP2基因的克隆及其蛋白结构预测

质膜内在蛋白作为水通道蛋白的一种存在类型,在跨膜水分运输中起着重要作用。本研究以本实验室获得的水通道蛋白基因的EST序列为模板设计引物,利用RACE技术扩增得到1 174 bp的PsPIP2全长cDNA,其中,包括3'UTR 191 bp,5'UTR 137 bp和846 bp的编码区,共编码281个氨基酸,分子量为29.79 kD。PsPIP2包括8个可能的磷酸化位点,其中7个位于丝氨酸(S),1个位于酪氨酸(Y),推测这些磷酸化修饰位点可能与PsPIP2蛋白通道的开启与闭合相关。二级结构预测显示,PsPIP2含无规卷曲和α-螺旋较多。同源性分析结果表明,PsPIP2与木薯PIP2同源性最高。本研究结果可为研究该基因的生物学功能和牡丹花衰老调控机制提供理论基础。     

花生长链酰基辅酶A合成酶1基因(LACS1)的克隆、鉴定与组织表达

长链酰基辅酶A合成酶(long chain acyl-Co A synthetase,LACS)是油脂代谢的重要催化酶。本研究采用RT-PCR技术,从花生(Arachis hypogaea L.)克隆到LACS1(Gen Bank登录号:KT932703),分析了该基因的结构组成,预测编码氨基酸与其他植物的同源性,采用实时Real-Time PCR技术对LACS1的组织表达进行研究。结果显示,花生LACS1基因全长2 219 bp,包含1 992 bp的ORF,编码663个氨基酸,有22个外显子和21个内含子。氨基酸序列比对显示花生LACS1有真核生物酰基辅酶A合成酶保守结构域,并含有保守的激活位点和绑定位点。同源性分析发现花生LACS1与大豆、野生大豆、鹰嘴豆、绿豆、甜橙等15种物种的氨基酸序列同源性在68%~86%之间,进化树分析显示,花生LACS1与鹰嘴豆等豆科植物亲缘较近。实时荧光PCR分析表明,花生LACS1在花生根、茎、叶、针、仁和花等组织均有表达,但差异明显,其中花的表达量最高,表达量大小顺序为花针叶茎根仁,地上组织表达量高于地下组织。花生LACS1可能参与花生角质层的脂质合成。本研究结果为揭示植物脂肪酸代谢提供理论依据。     

(Prunussalicina)WRKY 33的克隆与表达分析

WRKY类转录因子是植物防御反应信号转导的重要组成部分,本研究为了探讨WRKY33基因的功能,以嫩叶为材料,采用RT-PCR和RACE技术相结合的方法,克隆得到WRKY33全长c DNA(命名为Ps WRKY33),对其进行生物信息学分析。以基因组DNA为模板,对该基因的ORF区域进行PCR扩增,获得WRKY33的g DNA全长。采用荧光定量PCR技术检测该基因在水杨酸处理下的表达模式。试验结果表明:Ps WRKY33 c DNA全长为2 113 bp,开放阅读框长为1 770 bp,编码589个氨基酸;Ps WRKY33蛋白含有两个WRKY保守结构域,与Pm WRKY33蛋白同源性最高。Ps WRKY33基因组全长为2 844 bp,存在3个内含子和4个外显子。荧光定量结果表明:Ps WRKY33在1 mmol/L水杨酸处理后,30 h表达量最高,呈先上升后下降的趋势。研究结果为深入研究WRKY33基因与水杨酸诱导植物抗病性的关系提供一定的参考价值。     

夜来香LIS基因的克隆与表达分析

为了研究夜来香生物钟和花香代谢机制,本研究以夜来香花瓣为材料,利用PCR技术,从中获得了夜来香LIS基因的全长cDNA,并命名为CnLIS。生物信息学分析结果表明:该cDNA序列全长为1 800 bp,编码560个氨基酸,其中ORF为1 683 bp,5'UTR为56 bp,3'UTR为61 bp;该序列编码的氨基酸序列与软枣猕猴桃、温州蜜柑LIS基因的同源性分别为55%、50%,属于类异戊二烯合成C1超级家族。实时荧光定量PCR分析结果表明,0~12 h CnLIS表达水平较低,16 h后表达量明显上调,20 h表达量达最大,随后表达量急剧下降,其表达量随花瓣开放与闭合呈节律性变化。推测CnLIS可能参与夜来香香气形成,调控花香形成过程,同时,该基因的表达可能受生物钟基因所调控。     

花园君子兰叶绿体基因组序列

花园君子兰(Clivia gardenii)是君子兰属著名的常绿草本植物。本试验首次研究了花园君子兰的叶绿体基因组。其叶绿体基因组的长度为158 156 bp,包括86 307 bp的长单拷贝区域(LSC),18 231 bp的短单拷贝区域(SSC)和26 809 bp的反向重复区域(IRs)。总的GC含量为37.96%。注释了130个基因,包括86个编码基因,36个tRNA基因和8个rRNA基因。其中20个基因位于IR重复区,包括7个编码基因,9个tRNA基因和4个rRNA基因。选择石蒜科的12个物种进行系统发育树分析,采用全长基因组序列构建了系统发育树,结果显示C. gardenii和C. miniata形成一个单系群,表明花园君子兰和大花君子兰亲缘关系最近;并且和Hippeastrum、Leucojum、Narcissus、Lycoris等属形成了姐妹群。这些物种也形成了一个单系群,代表了石蒜亚科。本研究结果为君子兰的育种及叶绿体基因工程等研究提供了数据基础。     

牡丹PsAP1基因的表达分析及功能鉴定

为进一步探讨MADS-box基因家族在牡丹花器官发育和开花时间调控中的功能提供理论依据,为培育早花品种提供候选基因。以RACE扩增获得Ps AP1基因的全长c DNA为基础,分析了其表达模式和功能,以牡丹课题组利用454测序得到的类AP1基因的部分c DNA序列为基础,利用RACE扩增方法获得1 130 bp牡丹Ps AP1基因全长c DNA,同源性分析表明,牡丹Ps AP1与葡萄Vv AP1的相似性最高,为80.4%。实时定量PCR分析了其初花期的表达模式,结果表明,Ps AP1基因在萼片中转录量最高,其次为花瓣,在雄蕊中表达量最低。将Ps AP1基因在拟南芥中异源表达,分析鉴定了3个转基因株系的表型,结果发现异源表达Ps AP1拟南芥开花时间较野生型提前了约3 d。     

海岛棉GbWRKY40基因的克隆及特征分析

【目的】WRKY转录因子调控多种生物学进程,包括植物生长发育和应答多种环境胁迫。本研究旨在分析WRKY转录因子在海岛棉纤维发育中的功能。【方法】从海岛棉中克隆了1个WRKY转录因子基因GbWRKY40,进行同源性分析、多序列比对,利用荧光定量聚合酶链式反应分析其表达模式,通过构建酵母表达载体并转化酵母菌株AH109研究其转录激活活性。【结果】该基因c DNA全长1713 bp,5'非编码区长261 bp,3'非编码区长510 bp;开放阅读框长942 bp,编码313个氨基酸,预测相对分子质量约为34.138×103,等电点为8.46,包含5个外显子和4个内含子;其编码蛋白含有1个WRKY保守区(WRKYGQK)和1个锌指基序(C-X5-C-X23-H-X1-H),属于WRKY家族第Ⅱ类a组,包含3个核定位信号区,与陆地棉GhWRKY40同源性最高。GbWRKY40在根和开花后25 d纤维中表达量高,而且不具有转录激活活性。【结论】GbWRKY40可能参与调控棉纤维次生壁发育。     

山杏LACS基因家族生物信息学及表达分析

在植物脂肪酸的代谢途径中,长链酰基辅酶A合成酶把游离脂肪酸活化成酰基辅酶A,是调控脂肪酸代谢的重要酶。依据山杏(Prunus sibirica)转录组数据,开展LACS保守域比对分析,共确定6个具有完整ORF的LACS基因,其长度为2 360~2 900 bp,编码氨基酸为654~730 aa。开展LACS蛋白保守域及进化树分析,确定了山杏LACS具有AMP绑定域,说明其在物种间的高度保守性。同时,对不同发育期山杏杏仁的LACS基因开展q RT-PCR表达谱分析。为探究杏仁发育过程中油脂的累积模式,以山杏为试材,采用索氏提取法对其油脂含量进行测定,确定杏仁油的主要累积期在20~50 DAF。结合油脂含量的检测结果,对蛋白互作网络和基因表达等方面加以分析,确定山杏LACS9与油脂累积密切相关。本研究结果可为后续探究LACS基因家族在山杏杏仁发育及油脂累积中的调控机制奠定基础。     

芥菜型油菜PAP1基因的克隆与表达分析

花青素是导致芥菜型油菜叶片颜色差异的重要物质,PAP1基因是花青素合成途径中的一个关键转录调控基因.本研究利用同源克隆技术,以不同叶色芥菜型油菜为材料,根据同源性较高的白菜PAP1基因序列设计引物,克隆芥菜型油菜PAP1基因序列.芥菜型油菜PAP1基因的基因长度在1348～1669 bp,编码序列长744～753 bp,包括3个外显子和2个内含子区域.PAP1蛋白包括两个MYB结合域,分别位于第9～59和62～110氨基酸.进化分析表明芥菜型油菜PAP1基因与白菜和芜菁具有较高的同源性,与拟南芥亲缘关系较远.对比不同叶色芥菜型油菜基因序列发现,紫叶芥和红叶芥PAP1基因的编码区序列无差异,但编码的蛋白质与绿叶芥有22个氨基酸差异,定量PCR分析表明PAP1基因及其调控的下游基因如DFR、TT19等在绿叶芥油菜中表达水平较低,上述差异可能导致了芥菜型油菜叶色的差异.本研究为探究不同叶色芥菜型油菜的可能形成机制及遗传改良提供参考.     

Biological Activity and Quantification of Suspected Allelochemicals from Alfalfa Plant Parts

Autotoxicity restricts reseeding of alfalfa (Medicago sativa L.) after alfalfa until autotoxic chemical(s) breaks down or is dispersed into external environments. A series of aqueous extracts from leaves, stems, roots and seeds of alfalfa ‘Vernal’ were bioassayed against alfalfa seedlings of the same cultivar to determine their autotoxicity. The highest inhibition was found in the extracts from the leaves. Extracts at 40 g dry tissue l^?1 from alfalfa leaves were 15.4, 17.5 and 28.7 times more toxic to alfalfa root growth than were those from roots, stems and seeds, respectively. A high‐performance liquid chromatography (HPLC) analysis with nine standard compounds showed that the concentrations and compositions of allelopathic compounds depended on the plant parts. In leaf extracts that showed the most inhibitory effect on root growth, the highest amounts of allelochemicals were detected. Among nine phenolic compounds assayed for their phytotoxicity on root growth of alfalfa, coumarin, trans‐cinnamic acid and o‐coumaric acid at 10^?3 m were most inhibitory. The type and amount of causative allelochemicals found in alfalfa plant parts were highly correlated with the results of the bioassay, indicating that the autotoxic effects of alfalfa plant parts significantly differed.     

Changes in Seed Yield and Quality with Maturity in Onion (Allium cepa L., cv. 'Early Cream Gold')

Development of onion (Allium cepa L., cv. ‘Early Cream Gold’) seed under cool climate conditions in Tasmania, Australia occurred over a longer duration than previously reported, but similar patterns of change in yield components were recorded. In contrast to previous studies, umbel moisture content declined from 85 to 67 % over 57 days while seed moisture content decreased from 85 to 31 %. Seed yield continued to increase over the duration of crop development, with increasing seed weight compensating for seed loss resulting from capsule dehiscence in the later stages of maturation. Germination percentage was high and did not vary significantly from 53 to 77 days after full bloom (DAF), but mean germination time declined and uniformity of germination increased significantly over the same time period. The percentage abnormal seedlings declined with later harvest date, resulting in highest seed quality at 77 DAF. The results of this study suggest that the decision to harvest cool climate onion seed crops before capsule dehiscence will result in a loss of potential seed yield and quality.     

Location of a high-lysine gene and the DDT-resistance gene on barley chromosome 7

Summary The high-lysine gene in Risø mutant 1508 conditions an increased lysine content in the endosperm via a changed protein composition, a decreased seed size, and several other characters of the seed. The designation lys3a, lys3b, and lys3c, is proposed for the allelic high-lysine genes in three Risø mutants, nos 1508, 18, and 19. Linkage studies with translocations locate the lys3 locus in the centromere region of chromosome 7. A linkage study involving the loci lys3 and ddt (resistance to DDT) together with the marker loci fs (fragile stem), s (short rachilla hairs), and r (smooth awn) show that the order of the five loci on chromosome 7 from the long to the short chromosome arm is r, s, fs, lys3, ddt. The distance from locus r to locus ddt is about 100 centimorgans.     

Simultaneous Determination of Four Phenolic Acids in Radix Salviae Miltiorrhizae Formula Granules by QAMS

[Objectives]This study aimed to establish a QAMS(quantitative analysis of multi-components by single-marker)method for simultaneous determination of four phenol...     

Pollen and pollination experiments. III. The viability of apple and pear pollen as affected by irradiation and storage

Summary Apple and pear pollen was irradiated with doses of 0, 50, 100, 250 and 500 krad (gamma rays) and stored at 4°C and 0–10% r.h. From the in-vitro germination percentages an average LD 50 dose of about 220 krad was estimated. For both irradiated and untreated pollen a close and corresponding lineair relationship existed between germination percentage and pollen tube growth.Irradiated pollen was much more sensitive to dry storage conditions than untreated pollen, resulting in less germination and more bursting. Apparently, irradiation caused the pollen cell membrane to lose its flexibility faster than normal. Rehydration of dry-stored, irradiated pollen in water-saturated air restored germination percentages up to their initial levels. The importance of this procedure in germination trials is stressed.     

Water Extraction Process of Chinese Herbal Compound Man Gan Ning

[Objectives]To optimize the water extraction process of Chinese Herbal Compound Man Gan Ning and establish a method for its extraction and content determination...     

Present status and future strategy in breeding faba beans (Vicia Faba L.) for resistance to biotic and abiotic stresses

Progress is being made, mainly by ICARDA but also elsewhere, in breeding for resistance to Botrytis, AScochyta, Uromyces, and Orobanche; and some lines have resistance to more than one pathogen. The strategy is to extend multiple resistance but also to seek new and durable forms of resistance. Internationally coordinated programs are needed to maintain the momentum of this work.Tolerance of abiotic stresses leads to types suited to dry or cold environments rather than broad adaptability, but in this cross-pollinated species, the more hybrid vigor expressed by a cultivar, the more it is likely to tolerate various stresses.     

Optimization of Extraction Technology and Determination of Content of Total Phenols of Leaves in Acanthopanax giraldii Harms

[Objectives] To determine the optimum extraction technology for total phenols of leaves in Acanthopanax giraldii Harms.[Methods]The single factor test and ortho...     

Cytoplasmic male sterility,resistance to gall mite and mildew,and season of leafing out in black currants

Summary Cytoplasmic male sterility (cms) is described in the F₁ hybrids Ribes × carrierei (R. glutinosum albidum × R. nigrum) and R. sanguineum × R. nigrum. In backcrosses to R. nigrum, progenies with R. glutinosum cytoplasm were either all male sterile, or segregated for full male fertility (F) and complete (S) and partial (I) male sterility. Ratios of F:I+S suggested that two linked genes controlled cms, F plants being dominant for one (Rf ₁) and recessive for the other (Rf ₂).Segregation for cms in relation to three linded genes, Ce (resistance to the gall mite, Cecidophyopsis ribes), Sph ₃(resistance to American gooseberry mildew, Sphaerotheca mors-uvae) and Lf ₁(one of two dominant additive genes controlling early season leafing out) indicated that Rf ₁and Rf ₂were in this linkage group. The gene order and approximate crossover values appeared to be: % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% aabaqaciGacaGaamqadaabaeaafaaakeaacaWGdbGaamyzamaamaaa% baGaaiiiaiaacccacaGGWaGaaiOlaiaacgdacaGG0aGaaiiiaiaacc% caaaGaaiiiaiaacccacaGGGaGaamOuaiaadAgaliaaigdakmaamaaa% baGaaiiiaiaacccacaGGGaGaaiiiaiaaccdacaGGUaGaaiOmaiaacs% dacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaaaacaWGsbGaamOzaSGa% aGOmaOWaaWaaaeaacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaiaacc% cacaGGGaGaaiiiaiaacccaaaGaamitaiaadAgaliaaigdakmaamaaa% baGaaiiiaiaacccacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaiaacc% cacaGGGaGaaiiiaiaacccacaGGGaaaaiaadofacaWGWbGaamiAaSGa% aG4maaaa!6E4D!\[Ce\underline { 0.14 } Rf1\underline { 0.24 } Rf2\underline { } Lf1\underline { } Sph3\]. Crossover values of 0.36 for Ce-Lf ₁, and 0.15 for Lf ₁-Sph ₃were estimated from the relative mean differences in season of leafing out between seedlings dominant and recessive for Ce and Sph ₃.It is suggested that competitive disadvantage of lf ₁-carrying gametes and/or zygotes at low temperatures may be implicated in the almost invariable deficit of plants dominant for the closely linked mildew resistance allele Sph ₃. Poor performance of lf ₁- (and possibly lf ₂-) carrying gametes and young zygotes during periods of low temperature at flowering might also account for the liability of some late season cultivars and selections to premature fruit drop (running off).     

Screening techniques and sources of resistance to parasitic angiosperms

Parasitic angiosperms cause great losses in many important crops under different climatic conditions and soil types. The most widespread and important parasitic angiosperms belong to the genera Orobanche, Striga, and Cuscuta. The most important economical hosts belong to the Poaceae, Asteraceae, Solanaceae, Cucurbitaceae, and Fabaceae. Although some resistant cultivars have been identified in several crops, great gaps exist in our knowledge of the parasites and the genetic basis of the resistance, as well as the availability of in vitro screening techniques. Screening techniques are based on reactions of the host root or foliage. In vitro or greenhouse screening methods based on the reaction of root and/or foliar tissues are usually superior to field screenings and can be used with many species. To utilize them in plant breeding, it is necessary to demonstrate a strong correlation between in vitro and field data. The correlation should be calculated for every environment in which selection is practiced. Using biochemical analysis as a screening technique has had limited success. The reason seems to be the complex host-parasite interactions which lead to germination, rhizotropism, infection, and growth of the parasite. Germination results from chemicals produced by the host. Resistance is only available in a small group of crops. Resistance has been found in cultivated, primitive and wild forms, depending on the specific host-parasite system. An additional problem is the existence of pathotypes in the parasites. Inheritance of host resistance is usually polygenic and its transfer is slow and tedious. Molecular techniques have yet to be used to locate resistance to parasitic angiosperms. While intensifying the search for genes that control resistance to specific parasitic angiosperms, the best strategy to screen for resistance is to improve the already existing in vitro or greenhouse screening techniques.