116 resultados para Gibberellin
Resumo:
The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.
Resumo:
Dear Editor, Phytohormones are essential regulators of plant development, but their role in the signaling processes between plants and fungi during arbuscular mycorrhizal (AM) establishment is far from being understood (Ludwig-Müller, 2010). AM colonization leads to extensive effects on host metabolism, as revealed by transcriptome studies of AM plants (Hogekamp et al., 2011). Some genes have been specified as an AM core set, since they are mycorrhizal-responsive, irrespective of the identity of the plant, of the fungus, and of the investigated organ. These data support the idea that, on colonization, plants activate a wide reprogramming of their major regulatory networks and argue that mobile factors of fungal or plant origin are involved in such generalized metabolic changes. In this context, hormones may be good candidates (Bonfante and Genre, 2010). However, the emerging picture of the interaction between phytohormones and AMs is very patchy, and information on gibberellin (GA) involvement is still more limited (García-Garrido et al., 2010). The role of GA during nodulation is instead known to control the nodulation signaling pathway (Ferguson et al., 2011).
Resumo:
Effects of plant height on Fusarium crown rot (FCR) disease severity were investigated using 12 pairs of near-isogenic lines (NILs) for six different reduced height (Rht) genes in wheat. The dwarf isolines all gave better FCR resistance when compared with their respective tall counterparts, although the Rht genes involved in these NILs are located on several different chromosomes. Treating plants with exogenous gibberellin increased FCR severity as well as seedling lengths in all of the isolines tested. Analysis of the expression of several defense genes with known correlation with resistance to FCR pathogens between the Rht isolines following FCR inoculation indicated that the better resistance of the dwarf isolines was not due to enhanced defense gene induction. These results suggested that the difference in FCR severity between the tall and dwarf isolines is likely due to their height difference per se or to some physiological and structural consequences of reduced height. Thus, caution should be taken when considering to exploit any FCR locus located near a height gene.
Resumo:
Two genes encoding polyphenol oxidase (PPO) were isolated from pineapple (Ananas comosus[L.] Merr. cv. Smooth Cayenne). Sequence analyses showed that both contained a single intron and encoded typical chloroplast-localized PPO proteins, the sequences of which corresponded to two pineapple PPO cDNAs, PINPPO1 and PINPPO2, recently described by Stewart et al. (2001). Southern blot analyses suggested that pineapple contained only two PPO genes. Analysis of expression of PINPPO1 promoter GUS fusion constructs showed this promoter had a low basal activity and was cold- and wound-inducible, consistent with known mRNA expression profiles. Striking homologies to gibberellin response complexes (GARC) were observed in sequences of both the PINPPO1 and PINPPO2 promoters. Transient assays in mature pineapple fruit and stable expression in transgenic tobacco showed that PINPPO1 promoter-GUS fusions were indeed gibberellin (GA) responsive. A role for the element within the putative GARCs in mediating GA-responsiveness of the PINPPO1 promoter was confirmed by mutational analysis. PINPPO2 was also shown to be GA-responsive by RT-PCR analysis. Mutant PINPPO1 promoter-GUS fusion constructs, which were no longer GA-inducible, showed a delayed response to cold induction in pineapple fruit in transient assays, suggesting a role for GA in blackheart development. This was supported by observations that exogenous GA3 treatment induced blackheart in the absence of chilling. Sequences showing homology to GARCs are also present in some PPO promoters in tomato, suggesting that GA regulates PPO expression in diverse species.
Resumo:
Strawberries (Fragaria sp.) are adapted to diverse environmental conditions from the tropics to about 70ºN, so different responses to environmental conditions can be found. Most genotypes of garden strawberry (F. x ananassa Duch.) and woodland strawberry (F. vesca L.) are short-day (SD) plants that are induced to flowering by photoperiods under a critical limit, but also various photoperiod x temperature interactions can be found. In addition, continuously flowering everbearing (EB) genotypes are found. In addition to flowering, axillary bud differentiation in strawberry is regulated by photoperiod. In SD conditions, axillary buds differentiate to rosette-like structures called "branch crowns", whereas in long-day conditions (LD) they form runners, branches with 2 long internodes followed by a daughter plant (leaf rosette). The number of crown branches determines the yield of the plant, since inflorescences are formed from the apical meristems of the crown. Although axillary bud differentiation is an important developmental process in strawberries, its environmental and hormonal regulation has not been characterized in detail. Moreover, the genetic mechanisms underlying axillary bud differentiation and regulation of flowering time in these species are almost completely unresolved. These topics have been studied in this thesis in order to enhance strawberry research, cultivation and breeding. The results showed that 8-12 SD cycles suppressed runner initiation from the axillary buds of the garden strawberry cv. Korona with the concomitant induction of crown branching, and 3 weeks of SD was sufficient for the induction of flowering in the main crown. Furthermore, a second SD treatment given a few weeks after the first SD period can be used to induce flowering in the primary branch crowns and to induce the formation of secondary branches. Thus, artificial SD treatments effectively stimulate crown branching, providing one means for the increase of cropping (yield) potential in strawberry. It was also shown by growth regulation applications, quantitave hormone analysis and gene expression analysis that gibberellin (GA) is one of the key signals involved in the photoperiod control of shoot differentiation. The results indicate that photoperiod controls GA activity specifically in axillary buds, thereby determining bud fate. It was further shown that chemical control of GA biosynthesis by prohexadione-calcium can be utilized to prevent excessive runner formation and induce crown branching in strawberry fields. Moreover, ProCa increased berry yield up to 50%, showing that it is an easier and more applicable alternative to artificial SD treatments for controlling strawberry crown development and yield. Finally, flowering gene pathways in Fragaria were explored by searching for homologs of 118 Arabidopsis thaliana flowering-time genes. In total, 66 gene homologs were identified, and they distributed to all known flowering pathways, suggesting the presence of these pathways also in strawberry. Expression analysis of selected genes revealed that the mRNA of putative floral identity gene APETALA1 accumulated in the shoot apex of the EB genotype after the induction of flowering, whereas it was absent in vegetative SD genotype, indicating the usefulness of this gene product as the marker of floral initiation. The present data enables the further exploration of strawberry flowering pathways with genetic transformation, gene mapping and transcriptomics methods.
Resumo:
Gibberellic acid (GA3) induced a marked elongation of 2.5-centimeter shoot tips of Cuscuta chinensis Lamk. cultured in vitro. In terms of the absolute amount of elongation, this growth may be the largest reported for an isolated plant system. The response to hormone was dependent on an exogenous carbohydrate supply. The hormone-stimulated growth was due to both cell division and cell elongation. The growth response progressively decreased if GA3 was given at increasingly later times after culturing, but the decreased growth response could be restored by the application of indole-3-acetic acid (IAA) to the apex. Explants deprived of GA3 gradually lost their ability to transport IAA basipetally, but this ability was also restored by auxin application. The observations are explained on the basis that: (a) the growth of Cuscuta shoot tip in vitro requires, at least, both an auxin and a gibberellin; and (b) in the absence of gibberellin the cultured shoot tip explants lose the ability to produce and/or transport auxin.
Resumo:
Cytokinins induced haustoria formation in excised 10-mm segments ofCuscuta vine, the subapical 25-to-50-mm region being most responsive, producing a mean of 4–6 haustoria per segment. The order of effectiveness of cytokinins continuously applied (72 h) was 6-benzylaminopurine (BA) ges isopentenyladenine (iP) Gt zeatin (Z). Ribosides of BA and Z were as effective as the bases, whereas riboside of iP ([9R]iP) was half as effective as iP. Haustoria induction was influenced by weather and seasonal conditions at the time of vine collection; materials obtained on warm, sunny days responded better than those obtained on rainy, cloudy, or cool days. Haustoria were induced equally well all around the segment, and no thigmostimulus was needed for induction. p ]A 10-min pulse of 100 mgrM BA induced half as many haustoria as a 60-min pulse or continuous application of BA. White light inhibited haustoria induction elicited by a short (30-min) pulse of BA, whereas a longer (120-min) BA application overcame this light inhibition. Auxins (IAA or NAA, 1–10 mgrM), gibberellin (GA3, 1–10 mgrM), ethylene (as ethrel, 10–100 mgrM), and abscisic acid (ABA, 100 mgrM) were individually inhibitory (60–80%) with respect to haustoria induction when given continuously with 50 mgrM BA. A 60-min pulse of auxins (10 mgrM), GA3 (100 mgrM), or ethrel (10 mgrM), given at various time intervals during or after a 60-min pulse of 100 mgrM BA, showed that inhibition was maximal (70–95%) between 4 and 16 h of BA application and negligible (GA3) or much reduced (auxin, ethrel) at 20 h, indicating a ldquocommitmentrdquo to haustoria formation by this time.
Resumo:
The potential of mefluidide (N-(2,4-dimethyl-5[[trifluromethyl) sulfonyl] amino] phenol) acetamide) to act as a submersed aquatic plant growth regulator was evaluated using a laboratory bioassay system. Main stem elongation of hydrilla (Hydrilla verticillata (L.f.) Royle) and Eurasian watermilfoil (Myriophyllum spicatum L.) was effectively reduced by mefluidide at low concentrations. The lowest effective concentration of mefluidide that reduced stem length in Eurasian watermilfoil (100 yg a.i./L) was 5 times lower than that for hydrilla (500 yg a.i./L). Short-term net photosynthetic rates of these plants were not affected by mefluidide at concentrations as high as 1000 yg a.i./L. The minimum exposure time required to maintain an inhibitory effect for at least 28 days at a concentration of 500 yg ai.i./L was 3 to 7 days for Eurasian watermilfoil and 7 to 14 days for hydrilla. The results suggest that mefluidide is a more effective growth regulator for Eurasian watermilfoil than hydrilla. Exogenously applied gibberellic acid (GA) did not completely overcome the inhibitory effect of mefluidide even when GA was added at a high concentration (10-5 M). In addition, the internodal lengths of stems treated with mefluidide were not reduced as they were when treated with gibberellin synthesis inhibitors. The reduction of main stem elongation by mefluidide appeared to be due to the inhibition of new cell and tissue development at the stem tip rather than from inhibition of GA biosynthesis.
Resumo:
It was shown, with the aid of osmotic inhibition of germination, that the action of the far-red-absorbing form of phytochrome (Pf) in promoting germination can be completed even if the seed is held under conditions where germination is not possible. An effect of the continuing action of Pf beyond the point of complete germination promotion was demonstrated by enhancement of germination rate after removal of the osmotically active solute.
Previous reports that the rate of growth in water of seeds freed from the expansion-restricting endosperm is independent of the state of phytochrome were confirmed. However, a marked, phytochrome-mediated enhancement of the growth potential of such seeds was demonstrated through restricting water uptake by incubation in an osmoticum.
An experimental system, utilizing the appearance of a geotropic curvature in the radicle of the excised axial portion of the seed, was developed for more detailed studies of the phytochrome-enhanced growth potential. It was possible to demonstrate the light effect in water as well as in osmotica; this apparently is not possible with de-endospermed entire seeds. As in intact seeds, the effect of the continuing action of Pf is to enhance the rate of the response. Secretion of a chemical inhibitor of growth by the endosperm as a possible mechanism of induction of light sensitivity has been ruled out.
The phytochrome-dependent rate of appearance of geotropic curvature in osmotica is paralleled in time by a similar dependence of the rate of early extension growth of the embryonic axis. Only the first small increment of growth is a differentially responsive to red (R) and far-red (F); the rate of later increase in length is independent of the light regime.
It was shown that the high concentrations of gibberellic acid required for germination promotion in the intact seed are due at least in part to a diffusion barrier in the endosperm, and that the occasional reports in the literature of the ineffectiveness of kinetin are probably due to the same phenomenon. It was shown that gibberellin, like red light, enhances the growth potential of the axis, but kinetin does not. The difference in rates of response obtained after R-irradiation or gibberellin treatment, together with other results reported in the literature, strongly suggests that gibberellic acid and red light promote germination by different means. The idea that kinetin promotes germination by yet another mechanism, probably operating in the cotyledons, was supported through two different experimental approaches.
The phenomenon of temperature-dependent dark germination was examined in detail, using a wide range of both temperatures and incubation times. With the aid of the half-seed system, it was demonstrated that the promotive effect of low temperature on germination could not be due to a low optimum temperature for early growth of the radicle, since the rate of that process increased with increasing temperature, up to the highest temperature used.
It was shown that phytochrome does not function at high temperatures. This fact is of considerable importance in interpreting the phenomenon of thermodormancy, since in the literature only a small part of the effect of high temperature has been ascribed to an effect on phytochrome, and at that, only to an acceleration of dark reversion of Pf to the red-absorbing form of phytochrome (Pr). Partial denaturation of phytochrome may also make some contribution.
It was shown that the germination-promoting effect of low temperature depends on the presence of Pf, and concluded that low temperatures act by delaying or preventing transformation of Pf. Support for the assumption that Pf, not Pr, is the active form of phytochrome in lettuce seeds was drawn from the same evidence.
Attempts to stimulate germination by repeated irradiation with F over relatively prolonged incubation times resulted in failure, as have similar attempts reported in the literature. However, an enhancement of growth potential in the half-seed system by the maintenance of a small amount of Pf over long periods at ordinary temperatures by repeated irradiation with F was demonstrated.
It was observed that cold storage of the dry seed prevents or delays loss of dark dormancy during post-harvest storage. No change in the response of the half-seed in osmoticum to R and F was observed in seeds that has lost dark dormancy; that is, no internal change took place to measurably increase the growth potential of the embryonic axis. This suggests that the endosperm is the seat of changes responsible for after-ripening of photoblastic lettuce seed.
Resumo:
预计到本世纪末,大气CO2浓度将会增加到540~970ppm,大气CO2浓度升高所引起的全球气候变化已经受到广泛的关注。植物生长依赖CO2,并且对大气CO2浓度升高在结构和生理上产生响应。目前已有大量报道,从生态系统、群落、种群、个体、器官、组织、生理以及生化等水平上研究高浓度CO2所对植物产生的影响。但是有关高浓度CO2对植物有性生殖影响的报道却很少,同时多数实验均建立在短期的生殖响应,忽视了植物在长期高CO2浓度下具有的反馈作用和CO2浓度变化对植物的驯化作用。植物有性生殖与其生态适应性和农作物籽粒产量的关系极为密切;同时,植物有性生殖特性的变化,也可作为预测植物对全球气候变化响应的重要指标之一。为此,利用高浓度CO2对植物进行长期选择实验将很有必要。研究结果将为预测未来大气CO2浓度增加的条件下陆地生态系统的演变趋势、全球变化对植物有性生殖响应的方式和机制提供新的思路和有效方法。 在本研究中,我们以模式植物拟南芥(Arabidopsis thaliana)作为实验材料,利用370和700ppm CO2对其进行连续8个世代处理,首先研究高浓度CO2对每一个世代的拟南芥有性生殖特性的影响,然后比较各个世代中各种生殖特性指标变化的规律,从细胞、组织和个体尺度上揭示拟南芥有性生殖对全球变化的响应模式。此外,在700ppm CO2处理下,我们对拟南芥叶片生理、生化以及结构的变化进行了相关研究。两部分研究结果及主要结论如下: 首先,在每一个世代中,与370ppm CO2相比较,700ppm CO2处理显著促进了拟南芥开花,缩短生长周期,增加花、角果及种子等生殖的产量,降低种子N含量,提高种子C/N比、种子千粒重以及生殖生物量所占总生物量的比例等,而对种子萌发率、角果所含种子数目以及角果长度则无显著影响。但是, 通过对相同CO2浓度处理条件下,不同世代之间的研究结果比较发现,不同世代之间相关的生殖生物学指标并无显著差异。 其次,高浓度CO2显著降低叶片气孔密度、气孔指数、气孔导度以及蒸腾速率。在高浓度CO2处理下,叶肉细胞中叶绿体数目、叶绿体宽度和表观面积、淀粉粒大小和数量、叶片和细胞壁厚度等都显著增加,但是基粒内囊体膜的数量却显著下降。叶片中碳水化合物如可溶性总糖、淀粉以及纤维素含量在高浓度CO2下分别显著增加71.9%、78.7% 和 22.3%。此外,在高浓度CO2处理下,叶片中多数激素如如吲哚乙酸(indole-3-acetic acid, IAA)、赤霉素(gibberellin, GA)、玉米素核苷(zeatin riboside, ZR)、二氢玉米素核苷(dihydrozeatin riboside, DHZR)和异戊烯基腺苷(isopentenyl adenosine, iPA)均都显著地增加,而脱落酸(abscisic acid, ABA)含量却有所下降。最后,叶片中各种矿物质元素含量如N、P、K、Ca和Mg等含量在高浓度CO2处理下也都显著下降,而C/N比增加24.8%。 以上结果表明: (1) 在每一个世代中,700ppm CO2处理对拟南芥各种有性生殖特性具有显著的影响,但是高浓度CO2处理对植物所引起的效应在多个世代以内并不能够传递给后代,所以在多个有性生殖世代内,高浓度CO2处理对植物生长、生殖没有驯化作用。 (2) 在高浓度CO2处理下,拟南芥叶片中叶绿体超微结构的变化,可能主要是由于叶绿体中淀粉粒数量和体积大小显著增加而引起。 (3) 在高浓度CO2处理下,由于拟南芥叶片内与促进细胞分裂与伸长的激素含量显著增加,从而对拟南芥植株生长发育速率的提高起了重要的作用。 (4) 拟南芥生长在高浓度CO2条件下,其叶片中各种矿质元素含量(如N、P、K、Ca和Mg)均显著降低,究其原因可能是,第一由于叶片中碳水化合物含量的显著增加而对矿物质元素具有稀释作用;第二由于蒸腾速率下降,引起矿质元素从根部随着蒸腾流运输到地上部分的含量相应减少。
Resumo:
株高是农作物的重要农艺性状之一,适度矮化有利于农作物的耐肥、抗倒、高产等。20世纪50年代,以日本的赤小麦为矮源的半矮秆小麦的培育和推广,使得世界粮食产量显著增长,被誉为“绿色革命”。迄今为止,已报到的麦类矮秆、半矮秆基因已达70多个,但由于某些矮源极度矮化或者矮化的同时伴随不利的农艺性状,使得真正运用于育种实践的矮源较少。因此,发掘和鉴定新的控制麦类作物株高的基因,开展株高基因定位、克隆及作用机理等方面的研究,对实现麦类作物株高的定向改良,具有重要的理论意义和应用价值。簇毛麦(Dasypyrum villosum,2n=14,VV)是禾本科簇毛麦属一年生二倍体异花授粉植物,为栽培小麦的近缘属。本课题组在不同来源的簇毛麦杂交后代中发现了一株自然突变产生的矮秆突变体。观察分析了该突变体的生物学特性,对矮秆性状进行了遗传分析,对茎节细胞长度、花粉的活力进行了细胞学观察,考察了该突变体内源赤霉素含量及不同浓度外施赤霉素对突变体的作用,分析了赤霉素生物合成途径中的内根贝壳杉烯氧化酶(KO)和赤霉素20氧化酶(GA20ox)的转录水平,对赤霉素20氧化酶和赤霉素3-β羟化酶(GA3ox)进行了克隆和序列分析,并对GA20ox进行了原核表达和表达的组织特异性研究。主要研究结果如下:1. 该突变体与对照植株在苗期无差异,在拔节后期才表现出植株矮小,相对对照植株,节间伸长明显受到抑制,叶鞘长度基本不变。在成熟期,对照植株的平均株高为110cm,而突变株的平均株高为32cm,仅为对照植株的1/3 左右。除了株高变矮以外,在成熟后期,突变株还表现一定程度的早衰和雄性不育。I2-KI染色法观察花粉活力结果表明,对照植株花粉90%以上都是有活力的,而突变植株的花粉仅20%左右有活力。2. 突变株与对照植株的杂交F1代均表现正常株高,表明该突变性状为隐性突变。F1代植株相互授粉得到的168株F2代植株中,株高出现分离,正常株高(株高高于80cm)与矮秆植株(株高矮于40cm)的株数比为130:38,经卡方检验,其分离比符合3:1的分离比,因此推测该突变体属于单基因的隐性突变。3. 用ELISA方法检测突变株和对照植株的幼嫩种子中内源性生物活性赤霉素(GA1+3)含量,结果表明突变株的赤霉素含量为36 ng/ml,而对照植株的赤霉素含量为900 ng/ml。对突变株外施赤霉素,发现矮秆突变株的株高和花粉育性均可得到恢复。这些结果表明该突变株为赤霉素缺陷型突变。4. 用荧光定量PCR方法比较突变株与对照植株中内根贝壳杉烯氧化酶和赤霉素20氧化酶的转录水平,结果表明突变株的KO转录水平比对照植株分别提高了6倍(苗期)和16倍(成熟期),突变株的GA20ox转录水平与对照植株在苗期无明显差异,在成熟期突变株较对照植株则提高了10倍左右。这些结果表明该矮秆突变体与赤霉素的生物合成途径密切相关,而且极有可能在赤霉素的生物合成途径早期就发生了改变。5. 以簇毛麦总基因组为模板,同源克隆了GenBank登录号为EU142950,RT-PCR分离克隆了簇毛麦的GA3ox基因cDNA全长序列,分析结果表明该cDNA全长1206bp,含完整编码区1104bp,推测该序列编码蛋白含368个氨基酸残基,分子量为40.063KD,等电点为6.27。预测的氨基酸序列含有双加氧酶的活性结构,在酶活性中心2个Fe离子结合的氨基酸残基非常保守。该序列与小麦、大麦和水稻的GA3ox基因一致性分别为98%、96%、86%。基因组序列与cDNA序列在外显子部分一致,在478-715bp和879-1019bp处分别含238bp和140bp的内含子。6. 通过RT-PCR技术克隆了簇毛麦的GA20ox基因全长,命名为DvGA20ox,GenBank登录号为EU142949。该基因全长1080个碱基,编码359个氨基酸,具有典型的植物GA20ox基因结构。该基因编码的蛋白质与小麦、大麦、黑麦草等GA20ox蛋白的同源性分别为98%,97% 和91%。该序列重组到原核表达载体pET-32a(+)上,将获得的重组子pET-32a(+)-DvGA20ox转化大肠杆菌BL21pLysS后用IPTG进行诱导表达。SDS-PAGE分析表明,DvGA20ox基因在大肠杆菌中获得了高效表达,融合蛋白分子量为55kDa。定量PCR分析表明,该基因在簇毛麦不同器官中的表达差异明显:叶片中表达水平最高,根部表达水平次之,茎部和穗中表达较弱。在外施赤霉素后,该基因的表达水平在两小时以后急剧下降,表明该基因的表达受自身的反馈调节。本研究结果认为,(1)该簇毛麦矮秆突变体为单基因的隐性突变;(2)该矮秆突变体为赤霉素敏感突变,内源赤霉素含量检测表明突变体的内源性赤霉素含量仅为对照植株的1/30;(3)荧光定量PCR结果表明突变株的赤霉素生物合成途径的关键酶基因表达水平比对照植株高,而且突变植株的赤霉素生物合成改变很可能发生在赤霉素生物合成途径的早期;(4)GA20ox有表达的组织特异性,且受到自身产物的反馈调节。 Plant height is an impotrant agronomic trait of triticeae crops.Semi-dwarf cropcultivars, including those of wheat, maize and rice, have significantly increased grainproduction that has been known as “green revolution”. The new dwarf varieties couldraise the harvest Index at the expense of straw biomass, and, at the sametime, improvelodging resistance and responsiveness to nitrogen fertilizer. Moreover, dwarf traits ofplant are crucial for elucidating mechanisms for plant growth and development aswell. In many plant species, various dwarf mutants have been isolated and theirmodles of inheritance and physiology also have been widely investigated.The causesfor their dwarf phenotypes were found to be associated with plant hormones,especially, gibberellins GAs.Dasypyrum villosum Candargy (syn.Haynaldia villosa) is a cross-pollinating,diploid (2n = 2x = 14) annual species that belongs to the tribe Triticeae. It is native toSouthern Europe and West Asia, especially the Caucasuses, and grows underconditions unfavorable to most cultivated crops. The genome of D. villosum,designated V by Sears, is considered an important donor of genes to wheat for improving powdery mildew resistance, take-all, eyespot, and plant and seed storageprotein content. A spontaneous dwarf mutant was found in D. villosum populations.The biological character and modles of inheritance of this dwarf mutant are studied.The cell length of stem cell is observed. The influence of extraneous gibberellin tothe dwarf mutant is also examined; the transcript level of key enzyme of gibberellinbiosynthesis pathway in mutant and control plants is compared. GA3ox and GA20oxare cloned and its expression pattern is researched.1. The dwarf mutant showed no difference with control plants at seedlingstage.At mature stage, the average height of control plants were 110cm and the dwarfplants were 33cm. The height of the mutant plant was only one third of the normalplants due to the shortened internodes. Cytology observation showed that theelongation of stem epidermal and the parenchyma cells were reduced. The dwarfmutant also shows partly male sterile. Pollen viability test indicates that more than80% of the pollen of the mutant is not viable.2. The inheritance modle of this dwarf mutant is studied. All The F1 plantsshowed normal phenotype indicating that the dwarfism is controlled by recessivealleles. Among the 168 F2 plants, there are 130 normal plants and 30 dwarf plants, thesegregation proportion accord with Mendel’s 3:1 segregation. We therefore proposethat this dwarf phenotype is controlled by a single recessive gene.3. Quantitative analyses of endogenous GA1+3 in the young seeds indicated thatthe content of GA1+3 was 36ng/ml in mutant plants and 900ng/ml in normal plants.The endogenous bioactive GA1+3 in mutant plants are only about 1/30 of that innormal plants. In addition, exogenously supplied GA3 could considerably restore themutant plant to normal phenotype. These results showed that this mutant wasdefective in the GA biosynthesis.4. More than ten enzymes are involved in GA biosynthesis. KO catalyzes thefirst cytochrome P450-mediated step in the gibberellin biosynthetic pathway and themutant of KO lead to a gibberellin-responsive dwarf mutant. GA20ox catalyze therate-limited steps so that their transcript level will influence the endogenous GAbiosynthesis and modifies plant architecture. The relative expression levels of genesencoding KO and GA20ox were quantified by real time PCR to assess whether thechanges in GA content correlated with the expression of GA metabolism genes andwhere the mutant occurred during the GA biosynthesis pathway. In mutant plants,the transcript levels of KO increased about 6-fold and 16-fold at the seedling stage and elongating stage respectively comparing with the normal plants. For theseedlings, there was no notable difference in the expression of GA20ox betweenmutant and normal plants. At the elongating stage, GA20ox transcript increased 10times in mutant plants, suggesting that the GA biosynthesis pathway in mutant plantshad changed from the early steps rather than the late steps.5. A full length cDNA of D. villosum gibberellin 3β-hydroxylase homology(designated as DvGA3ox) was isolated and consisted of 1206bp containing an openreading frame of 1104bp encoding 368 predicted amino acid residues. Identityanalysis showed that the gibberellin 3β-hydroxylase nucleotide sequence shared 98%,96% and 86% homology with that of wheat, barley and rice. The predicted peptidecontained the active-site Fe of known gibberellin 3β-hydroxylase and the regionhomologous to wheat, barley and Arabidopsis. The genomic clone of gibberellin3β-hydroxylase has two introns.6. The full-length cDNA of D. villosum gibberellin 20 oxidase (designated asDvGA20ox) was isolated and consisted of 1080-bp and encoded 359 amino acidresidues with a calculated mol wt of 42.46 KD. Comparative and bio-informaticsanalyses revealed that DvGA20ox had close similarity with GA20ox from otherspecies and contained a conserved LPWKET and NYYPXCQKP regions. Tissueexpression pattern analysis revealed DvGA20ox expressed in all the tissues that wereexamined and the highest expression of DvGA20ox in expanding leaves followed byroots. Heterologous expression of this cDNA clone in Escherichia coli gave a fusionprotein that about 55KD. Transcript levels of DvGA20ox dramatically reduced twohours after application of biologically active GA3, suggesting that the biosynthesis ofthis enzymes might be under feedback control.
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赤霉素是一种高效能的广谱植物生长调节剂,为五大植物激素之一,具有重要的生物学功能。目前利用赤霉素突变体研究生物合成途径和信号转导已经成为热点。 GA 20-氧化酶是GA生物合成中的一类关键酶,它位于GA合成途径的中心位置。本研究根据烟草(Nicotiana tabacum)GA 20-氧化酶基因序列,设计2对分别含有特定酶切位点的特异引物,以烟草基因组DNA为模板,扩增目的基因(约250 bp)片段。将正、反向目的片段分别插入中间载体的内含子两侧,再经BamH I和Sac I双酶切回收约700 bp的目的片段,插入到双元载体质粒p2355中,成功构建了含GA 20-氧化酶基因片段反向重复序列的植物表达载体p23700。分别将p2355质粒和p23700质粒导入根癌农杆菌(Agrobacterium tumefaciens)EHA105中并转化烟草叶片细胞,经卡那霉素选择培养,PCR及GUS组织染色鉴定,获得转基因烟草植株。以EHA105-p2355转化的烟草,获得41株转基因植株,均没有矮化表型;而以EHA105-p23700转化的烟草,获得转基因植株14株,其中具有矮化表型的烟草10株,表明反向重复序列转录产物能形成发夹RNA(hpRNA),产生小分子干扰RNA(small interferring RNA,简称siRNA),干扰目的基因的表达。 赤霉素含量测定表明矮化植株中赤霉素合成途径的最终产物GA3总含量明显低于野生型烟草植株。荧光定量PCR结果表明,矮化转基因烟草的GA 20-氧化酶基因表达量受到明显抑制,表达量明显低于野生型对照。同时对上游内根-贝壳杉合成酶(Ent-kaurene synthase,KS)基因,下游的GA-3β羟化酶基因进行了RT-PCR分析,结果显示上游基因的表达没有规律性变化,而下游基因表达量亦降低。上述结果表明,GA 20-氧化酶基因的表达被有效地干扰了,表达受到抑制,从而影响植株体内GA3的合成,影响植株的生长发育,导致植株矮化。并推测,GA 20-氧化酶基因受到抑制,可能影响下游基因的表达。并且通过干旱胁迫测试,发现矮化植株相对于野生型植株及不含干扰片段的转基因植株,对干旱的耐受力有了很大的提高,具有更强的耐受力。 研究结果为进一步进行相关研究奠定基础。 Gibberellin(GA) is an efficient plant growth regulator. As one of five major plant hormones, it plays an important biological function. Using GA mutant for investigating biosynthetic pathways and signal transduction has become high lights. GA 20-oxidase is a crucial enzyme involved in gibberellin biosynthesis. According to tobacco (Nicotiana tabacum) GA 20-oxidase enzyme gene sequence and based on binary vector p2355, we constructed a plant expression vector p23700, which habors an inverted repeat DNA fragment of GA 20-oxidase gene drivered by Cauliflower mosaic virus promtor (CaMV 35Sp). Binary plasmid p2355 had no inverted repeat DNA fragment of GA 20-oxidase gene. The vector p2355 and p23700 were introduced into Agrobacterium tumefaciens EHA105 and tobacco leaf transformation was conducted. After selected by kanamycin and characterized by PCR and GUS hischemical reaction, transsgenic plants were obtained. Fourtheen transgenic plants, which were transformed by EHA105-p23700, were obtained. Among them, 10 were dwarf mutants. However, 41 transgenic plants with the same normal phenotype as wild type,which were transformed by EHA105-p2355, were obtained. Analysis of Gibberellin contents showed that it was lower in dwarf mutants than in normal phenotype plants. Moreover, comparing to normal phenotype plants including wild type and transgenic plants with no interference fragment, the drought tolerance of dwarf plants have greatly increased. And their proline content increased obviously after drought test. Fluorescence quantitative real time PCR (RT-PCR) showed that GA 20-oxidase gene expression was significantly inhibited in dwarf transgenic tobacco. Meanwhile, the expression of the upstream gene ent-kaurene synthase (KS) gene and downstream gene GA-3β hydroxylase gene was also detected by RT-PCR. The results presented that KS gene expression had no regular change while GA-3β hydroxylase gene expression reduced. It implied that inhibiting GA 20-oxidase gene probably reduce the expression of downstream genes. The results showed that the transcriptional products of the foreign inverted repeat fragment can form hairpin RNA (hpRNA) to induce RNAi. It presented that GA 20-oxidase gene expression was effectively interfered, resulting in reducing GA3 synthesis and inhibiting plant growth and development, then dwarf plants were produced. However, the dwarf plants had higher tolerance of drought.
Resumo:
BACKGROUND AND PURPOSE: NF-κB-driven inflammation is negatively regulated by the zinc finger protein A20. Gibberellic acid (GA3 ) is a plant-derived diterpenoid with documented anti-inflammatory activity, which is reported to induce A20-like zinc finger proteins in plants. Here, we sought to investigate the anti-inflammatory effect of GA3 in airway epithelial cells and determine if the anti-inflammatory action relates to A20 induction.
EXPERIMENTAL APPROACH: Primary nasal epithelial cells and a human bronchial epithelial cell line (16HBE14o-) were used. Cells were pre-incubated with GA3 , stimulated with Pseudomonas aeruginosa LPS; IL-6 and IL-8 release, A20, NF-κB and IκBα expression were then evaluated. To determine if any observed anti-inflammatory effect occurred via an A20-dependent mechanism, A20 was silenced using siRNA.
KEY RESULTS: Cells pre-incubated with GA3 had significantly increased levels of A20 mRNA (4 h) and protein (24 h), resulting in a significant reduction in IL-6 and IL-8 release. This effect was mediated via reduced IκBα degradation and reduced NF-κB (p65) expression. Furthermore, the anti-inflammatory action of GA3 was abolished in A20-silenced cells.
CONCLUSIONS AND IMPLICATIONS: We showed that A20 induction by GA3 attenuates inflammation in airway epithelial cells, at least in part through its effect on NF-κB and IκBα. GA3 or gibberellin-derived derivatives could potentially be developed into anti-inflammatory drugs for the treatment of chronic inflammatory diseases associated with A20 dysfunction.