青天葵NfD53基因的克隆及生物信息学分析

本研究克隆青天葵NfD53基因并分析其序列特征,可为今后深入研究NfD53蛋白功能及独脚金内酯(SLs)对植物侧枝生长发育的影响提供参考依据.以濒危药用植物青天葵叶片为实验材料,利用RT-PCR及RACE技术克隆获得2条NfD53基因,分别命名为NfD53-1和NfD53-2,应用生物信息学软件分析其序列并进行功能预测.结果 显示:NfD53-1基因全长3523 bp,编码974个氨基酸;NfD53-2全长3916 bp,编码1190个氨基酸.两者等电点分别为:8.34、6.33;亲水性平均数为:-0.250、-0.263;均为不稳定的亲水性蛋白,无信号肽及切割位点,定位于叶绿体.两蛋白都具有ClpA超家族核心序列,而NfD53-1蛋白还具有ClpC超家族核心序列,并含有3个非特异性位点(ClpA,ClpC和AAA_2),NfD53-2蛋白含有1个ClpA非特异性位点.蛋白的二级结构中均以无规则卷曲占比最高,分别占47.16％和51.21％;其次是α-螺旋,占38.70％和31.69％.系统进化分析表明,NfD53-1与番茄等聚为一支,但氨基酸相似度较低(20.27％),NfD53-2与铁皮石斛聚为一支,同源性较高(66.49％).本研究为后续进一步探讨该基因的功能及SLs在植物生长发育中的作用提供数据参考.     

水稻葡聚糖内切酶新基因OsGLU16的克隆及特性研究

β-1,4-葡聚糖内切酶(endo-β-1,4-glucanase,EGase)在植物的组织生长、开花、果实成熟、器官脱落等方面都具有非常重要的作用。本研究克隆了一个新的水稻β-1,4-葡聚糖内切酶家族基因Os GLU16。序列分析表明该基因位于水稻第8号染色体,c DNA全长1 827 bp,包含一个1 572 bp的开放阅读框,编码523个氨基酸残基。蛋白质同源性分析表明,Os GLU16基因编码的蛋白在第54~第516个氨基酸残基区域具有高度保守的GH9催化结构域。本研究成功构建了Os GLU16基因过表达载体p Ca MV35S-Os GLU16-EGFP,并获得了转基因水稻植株。植株表型分析表明,与野生型相比,过表达株系表现为株高和穗长显著下降。本研究初步探明了Os GLU16基因过表达转基因水稻植株的表型特征,这为进一步丰富β-1,4-葡聚糖内切酶基因家族在水稻生长发育过程中的作用提供理论基础。     

马铃薯StKNOX1基因的克隆及表达分析

KNOX基因是一类转录调控因子,其和植物的生长发育密切相关。为了探讨同源异型盒(KNOX)基因在马铃薯(Solanum tuberosum L.)块茎发育中的作用,采用RT-PCR和RACE技术,克隆了1个KNOX同源基因,且根据属性情况命名为StKNOX1,其属于KNOX家族I类蛋白,编码含344个氨基酸的蛋白,包含KNOX1、KNOX2、ELK等结构域。根据相关生物信息研究结果,该基因编码的蛋白与番茄中KNOX1的一致性最高(93.7%)。StKNOX1基因在马铃薯的各组织器官中均有表达,在根和块茎中的表达水平显著高于其他部位的表达水平。本研究将为进一步开展基因功能研究奠定理论基础,为马铃薯生物技术育种提供基因资源。     

玉米ZmBRI1基因的克隆、表达及功能分析

油菜素内酯(BRs)是一种重要的甾族类激素,在植物的生长过程中起着重要的作用。本研究利用同源克隆的方法,从玉米B73自交系中获得了一个新的油菜素内酯受体蛋白编码基因ZmBRI1,该基因全长为3369 bp,编码1122个氨基酸。亚细胞定位分析表明,ZmBRI1蛋白定位于细胞膜上,而且ZmBRI1在各个玉米的各个组织器官中都有表达,其中在幼嫩的组织中表达最高。利用转基因技术将ZmBRI1导入BR不敏感突变体bri1-5中,获得的转基因植株修复了其表型,特别是植株高度、叶片形态和果荚大小。与突变体bri1-5比较,油菜素内酯(brassinolide,BL)处理显著抑制转基因株系根系生长;丙环唑(propiconazole,Pcz)处理显著抑制了下胚轴生长;同时转基因株系DWF4和CPD基因的表达量下降。此外,在野生型(Ws)中过表达ZmBRI1,显著提高了拟南芥在ABA抑制条件下的种子萌发率和植株生长,下调了ABA响应基因RD29A、RD29B、ABI5和RAB18的表达。因此,ZmBRI1不仅参与了植物的形态建成和BR的信号传导,而且参与调控了植物对ABA信号的响应。     

拟南芥花组织高表达TCP家族转录因子At1g30210的克隆与转录激活活性鉴定

本文从拟南芥中克隆到基因At1g30210的全长读码框。多序列比对结果表明,在推导的氨基酸水平上,该基因编码的蛋白含有特征性的TCP保守结构域,与已知的玉米、金鱼草和水稻的TCP家族基因中都存在显著性的保守性。另外通过酵母单杂交实验证明该基因编码的蛋白在酵母体内具有转录激活功能。同时RT-PCR实验结果表明,该基因在拟南芥花组织中表达量相对较高,具有明显的组织表达特异性。花器官高表达转录因子的克隆将为研究TCP基因调控植物花发育和花形态建成提供新的物质基础。     

AT-hook基因及其在植物开花调控中的研究进展

AT-hook基因是能够编码与双链DNA小沟中富含AT碱基的序列特异性结合的一类基因,其所编码的蛋白含有以甘氨酸-精氨酸-脯氨酸(GRP)三个氨基酸残基为中心的DNA结合蛋白基序。笔者通过对AT-hook基因的特点和功能及其在拟南芥及水稻等高等植物开花中的调控作用综述。AT-hook基因不仅参与植物的生长发育及逆境胁迫与激素信号应答,同时在花器官的形成以及植物开花中也起着重要的调控作用。该基因在花器官组织中的表达量最高,影响成花素基因的表达,且其编码蛋白能够通过改变染色质状态或招募蛋白复合体在表观水平上调控植物开花相关基因的转录,从而影响植物开花。该基因可能为植物开花的表观遗传调控提供了新的途径。     

阳春砂WRKY40的克隆、分析及原核表达

转录因子可有效调控植物次生代谢产物的生物合成,本研究从姜科药用植物阳春砂(Amomum villosum Lour.)中克隆获得两个WRKY转录因子基因,命名为AvWRKY 40-1和AvWRKY 40-2。两个基因的编码框分别为852 bp和885 bp,分别编码283和294个氨基酸。Av WRKY 40-1和Av WRKY 40-2编码的蛋白序列一致性为75%,在GenBank中均比对上拟南芥的WRKY 40,且均含有WRKY转录因子家族所共有的WRKKYGQK七肽序列及CX5CX23HNH锌指结构域。系统发育进化树表明:Av WRKY 40-1和Av WRKY 40-2与菠萝(Ananas comosus)的AcWRKY 71亲缘关系最近,且与单子叶植物的WRKY聚为一支。两个基因在阳春砂果皮和种子团中的表达有差异,并且与阳春砂转录组中筛选出来的萜类合酶基因表达相关性也存在差异,AvWRKY 40-1表现出与萜类合酶基因更高的相关性。成功构建了两个基因的重组表达载体pDEST 17-Av WRKY 40-1和pDEST 17-Av WRKY 40-2,经诱导表达得到其融合蛋白。本研究首次克隆阳春砂的WRKY类转录因子基因并获得其融合蛋白,为后续Av WRKY 40-1和Av WRKY 40-2的功能鉴定及其对萜类的代谢调控研究提供基础。     

甘薯扩展蛋白IbEXPA2基因克隆和表达分析

扩展蛋白(Expansin)是细胞壁的重要组成成分,参与细胞壁的舒张,与植物器官生长发育密切相关。甘薯是一种块根类作物,其块根不仅是繁殖器官,也是储能器官,因此,开展甘薯块根生长膨大机理研究具有重要意义。本研究在前期转录组学研究基础上,克隆到一个可能与块根膨大相关的扩展蛋白基因IbEXPA2,并进行生物信息学和qPCR表达分析。生物信息分析表明,IbEXPA2编码253个氨基酸,包含DPBB＿1和Pollen＿allerg＿1结构域,具有一段信号肽及23个氨基酸的跨膜结构域。该基因编码的蛋白与三裂叶薯expansin-A4蛋白(登录号:XP＿031109781.1)的同源性最高,为98.03%;时空表达分析表明该基因在甘薯品种‘西瓜红’和‘桂经薯8号’块根中表达显著高于纤维根,‘西瓜红’生长的各个时期块根中表达均显著高于茎和叶,在移栽50 d时表达量最高,为对照纤维根的8.49倍;‘桂经薯8号’生长多数时期在块根中的表达也显著高于茎和叶,在移栽35 d时表达量最高,为对照的8.11倍。推测该基因参与甘薯块根生长膨大调控。     

拟南芥花组织高表达TCP家族转录因子Atlg30210的克隆与转录激活活性鉴定

本文从拟南芥中克隆到基因Atlg30210的全长读码框。多序列比对结果表明，在推导的氨基酸水平上，该基因编码的蛋白含有特征性的TCP保守结构域，与已知的玉米、金鱼草和水稻的TCP家族基因中都存在显著性的保守性。另外通过酵母单杂交实验证明该基因编码的蛋白在酵母体内具有转录激活功能。同时RT—PCR实验结果表明，该基因在拟南芥花组织中表达量相对较高，具有明显的组织表达特异性。花器官高表达转录因子的克隆将为研究TCP基因调控植物花发育和花形态建成提供新的物质基础。     

马铃薯青薯九号甜菜碱醛脱氢酶基因(StBADH)克隆及进化分析

本研究利用同源克隆,从马铃薯青薯9号中克隆到2个甜菜碱醛脱氢酶基因,分别命名为St BADH-504和St BADH-505。St BADH-504为1 631 bp,CDS为1 515 bp,编码504个氨基酸蛋白,编码蛋白亚细胞定位于线粒体;St BADH-505为1 709 bp,CDS为1 518 bp,编码505个氨基酸蛋白,编码蛋白亚细胞定位于叶绿体或过氧化物酶体,二个蛋白都有醛脱氢酶高度保守的十肽(VSLELGGKSP)结构。进化分析表明,St BADH-504和St BADH-505蛋白与茄科植物中BADH蛋白聚为一类,但二者分属不同分支。盐胁迫表达特征分析表明,马铃薯叶片中St BADH-504表达受盐胁迫的诱导,而St BADH-505为组成型表达,不受盐胁迫诱导。本研究有助于了解马铃薯耐盐机理,为耐盐马铃薯资源创制提供理论参考。     

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.     

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.     

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...     

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.     

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.