拟南芥PRRs基因在红光和蓝光信号转导中的作用初探

以拟南芥为材料,在红光和蓝光下对PRRs(pseudo-response regulators)突变体prr5p、rr7、prr9和toc1及其野生型的下胚轴表型进行比较观察,并采用实时定量PCR方法对突变体中光信号通路相关基因ZTL(zeitlupe)和CO(constans)的节律表达进行分析.结果表明:在红光下,prr5和toc1的下胚轴长度比野生型显著增长,在蓝光下,prr7p、rr9和toc1较野生型短,表明突变体降低了拟南芥对红光的敏感性,却增强了对蓝光的敏感性.红光和蓝光下,PRRs突变体中ZTL和CO的mRNA节律表达与野生型明显不同,其中红光下prr5和prr7、蓝光下prr5和toc1中的ZTLmRNA的表达显著下降且节律消失;红光下prr7和prr9以及蓝光下prr5突变体中的COmRNA表现基本无节律.因此推测,PRRs与ZTL的相互作用很可能在红光和蓝光信号转导途径中发挥作用,且PRRs基因极有可能参与了红光和蓝光对CO的调控.     

转CkLEA4基因拟南芥种子萌发期的抗逆性分析

该研究在实验室前期研究的基础上,将受脱水、盐胁迫和ABA诱导的柠条锦鸡儿CkLEA4基因转入野生型拟南芥,并利用实时荧光定量PCR从8株纯合体中筛选出3个表达量不同的株系,比较野生型和转CkLEA4基因过表达拟南芥种子在不同胁迫处理下的萌发率,以探讨CkLEA4基因在植物抵抗逆境胁迫中的功能。结果发现:(1)在不同浓度NaCl、甘露醇及ABA处理下,转CkLEA4基因过表达拟南芥种子的萌发率均高于野生型,随着NaCl、甘露醇及ABA浓度增加,各株系萌发率均降低,但野生型的萌发率下降幅度均高于3个过表达株系,并且在200mmol/L NaCl和400mmol/L甘露醇处理下,过表达株系子叶绿化率均显著高于野生型。(2)在低浓度ABA处理下,CkLEA4过表达植株子叶的绿化率也高于野生型。研究表明,柠条锦鸡儿CkLEA4基因提高了拟南芥种子萌发阶段对盐、ABA及渗透胁迫的耐受性。     

拟南芥类受体蛋白激酶基因CRK45对外源钙离子的响应

以拟南芥野生型和类受体蛋白激酶基因CRK45的T-DNA插入突变体crk45为材料,采用差异基因表达筛选技术检测ABA处理后野生型和crk45中基因表达的差异。结果显示:(1)crk45突变体中有1个基因的表达比野生型高约4倍。(2)NCBI数据库检索表明,该基因编码的蛋白具有EF手型结构,蛋白序列全长为130个氨基酸,是典型的Ca2+结合蛋白,故命名为CRK45抑制的钙离子结合蛋白(CICBP)。(3)Northern blotting分析结果显示,ABA处理后crk45突变体中CICBP的表达明显升高,证明CICBP基因的确受ABA诱导,且其表达受CRK45的抑制。(4)外源75mmol/L的Ca2+处理后,crk45突变体的萌发率(30.8%)显著高于野生型(17.16%),说明在Ca2+介导下CRK45的功能是抑制种子萌发。(5)qRT-PCR检测显示,野生型中CRK45的表达受Ca2+诱导明显升高,而crk45突变体中的表达一直保持很低,说明crk45突变体是一个基因敲除突变体;Ca2+处理后crk45突变体中CICBP基因表达上调,而野生型中CICBP的表达反而降低,说明Ca2+处理下CRK45抑制CICBP基因的表达。研究表明,ABA或Ca2+处理后,CRK45通过负调控CICBP基因的表达,从而抑制拟南芥种子萌发。     

DELLA蛋白缺失对拟南芥干旱胁迫耐受性的影响

为探索DELLA蛋白缺失对拟南芥耐旱能力的影响,对拟南芥野生型Ler和DELLA蛋白缺失突变体della进行干旱处理,测定存活率、萌发率、离体叶片的失水率、脯氨酸、可溶性糖和丙二醛含量,并对发挥植物细胞脱水保护功能的胚胎晚期丰富蛋白编码基因LEA和ABA应答基因LOX3、COR15b、COR413的表达量进行了检测。结果表明:(1)干旱21d后复水,della突变体的存活率明显高于野生型Ler;(2)della突变体在含甘露醇的固体培养基上的萌发率显著高于Ler;(3)della突变体离体叶片的失水速率明显低于Ler;(4)干旱胁迫后,della突变体脯氨酸、可溶性糖和丙二醛含量的积累低于Ler;(5)干旱胁迫后,della突变体的LEA基因上调表达程度高于Ler,而ABA应答基因上调表达程度低于Ler。研究表明,DELLA蛋白的缺失有助于提高植物抗旱能力。     

沉默VHA-c4基因对拟南芥根长和种子萌发的影响

为了研究植物液泡H+-ATPase c亚基VHA-c4基因在拟南芥响应非生物胁迫中的作用,本研究构建了拟南芥VHA-c4基因沉默载体,利用农杆菌介导,将线性化的沉默载体dsDNA片段整合在拟南芥基因组上,筛选获得了能够不同程度特异沉默VHA-c4基因的7个转基因株系c4-1～c4-7,并对VHA-c4基因沉默效果最好的株系c4-2进行NaCl、ABA、6%葡萄糖胁迫处理。结果显示:在特定浓度梯度的NaCl处理后,沉默株系c4-2的主根相对伸长量和种子萌发率都被明显抑制,并且被抑制效果远远高于野生型株系。然而,在特定浓度梯度的ABA和葡糖糖处理后,沉默株系c4-2的主根相对伸长量和种子的萌发率虽然也都有一定程度的抑制,但是其被抑制效果远远低于野生型拟南芥。结果显示沉默了VHA-c4基因后,显著降低了拟南芥对NaCl的耐受性,影响了其的生长发育,然而对ABA和葡糖糖的抑制作用却表现为很不敏感,这可能是因为H~+-ATPase在盐胁迫信号通路和ABA信号通路中起着不同的(正负)调控功能。     

拟南芥钾转运突变体atkup12鉴定及非生物胁迫的敏感性检测

[目的]鉴定获得拟南芥HAK/KUP/KT高亲和钾离子转运突变体atkup12,通过检测种萌期atkup12突变体在低钾、盐及氧化胁迫下的生长指标以初步明确拟南芥AtKUP12基因是否参与植物对非生物胁迫的响应。[方法]以拟南芥atkup12突变体基因组和总RNA反转后的cDNA为模板,通过PCR扩增确定AtKUP12基因T-DNA插入失活的纯合突变体。将野生型和atkup12突变体点种于0.5μmol/L低钾、不同NaCl浓度和1μmol/L甲基紫精的胁迫培养基,测定并比较突变体与野生型拟南芥在根长、种子萌发率及子叶绿化率间的差异。[结果]利用双引物法,结合反转录PCR,在DNA及RNA水平鉴定获得了AtKUP12基因的纯合突变体。低钾胁迫下,atkup12突变体种苗的根长比较野生型拟南芥短40%左右,较野生型受到了显著抑制;不同NaCl浓度胁迫下,atkup12突变体种子的萌发率较野生型显著降低;1μmol/L甲基紫精显著的抑制了突变体种苗的子叶绿化率。[结论]成功鉴定获得了AtKUP12基因T-DNA插入失活的纯合突变体,且AtKUP12基因的缺失会增加植物对盐、低钾及氧化等非生物胁迫的敏感性。     

拟南芥干旱突变体远红外成像技术的筛选和特性鉴定

利用化学诱变剂甲基磺酸乙酯(EMS)对模式植物拟南芥(Arabidopsis thaliana)进行化学诱变获得突变体筛选群体。在干旱胁迫下,以叶片的温度差异为筛选指标,利用远红外成像技术进行突变体的筛选,获得了对干旱不敏感突变体dri1(drought-insensitive 1)和敏感突变体drs1(drought-sensitive 1)。实验结果表明dri1和drs1为单基因隐性突变,气孔密度同野生型无差异,而叶片温度、气孔开度和叶片失水率则有明显改变。在MS培养基上的种子萌发实验表明在ABA、甘露醇和NaCl胁迫下dri1萌发率要比野生型高,而drs1则比野生型低。对突变基因的研究有待进一步进行。     

拟南芥高迁移率族蛋白B族基因表达模式分析

为了解高迁移率族蛋白B族(HMGB)基因在拟南芥中的表达模式及作用方式,该研究克隆了拟南芥中5个编码HMGB的基因:AtHMGB1、AtHMGB2、AtHMGB3、AtHMGB4、AtHMGB5,并运用荧光实时定量PCR方法检测野生型拟南芥中以上5种基因在不同器官中的表达及在外源植物激素(ABA、2,4-D)处理前后的表达差异,选取AtHMGB2、AtHMGB4和AtHMGB5分别转化拟南芥并筛选出超表达株系,随即检测ABA诱导下超表达AtHMGB的转基因拟南芥的表型。研究证实:在野生型拟南芥中AtHMGB2在拟南芥各个器官中的表达量远高于其它家族成员,AtHMGB4和AtHMGB5在花、果荚和根中的表达略高于茎和叶;在ABA处理前后AtHMGB家族成员的表达水平有显著差异,其中AtHMGB2的表达被ABA显著负调控;ABA诱导下超表达AtHMGB2的转基因拟南芥与野生型相比出现萌发及生长迟缓现象,但超表达AtHMGB4与AtHMGB5的转基因拟南芥在ABA诱导下的种子萌发和幼苗生长与野生型相比差异不大。研究发现,AtHMGB家族成员在转录水平上响应ABA的方式各有不同,对理解AtHMGB家族成员的生物学功能提供了新的基础。     

拟南芥耐旱、耐高盐突变体sdt1的获得

在有20000个独立转化株系的拟南芥(Arabidopsis thaliana)激发标签突变体库中,筛选到1 株耐旱、耐盐突变体sdt1(high-salinity and drought tolerant 1)。实验结果表明在连续26d不浇水的干旱胁迫条件下,sdt1可保持正常生长而野生型全部萎蔫死亡。此外,在外施150 mmol/L NaCl水溶液的高盐胁迫条件下．sdt1可正常生长,而野生型全部死亡。在0．5xMS培养基上进行的发芽实验表明sdt1对内源和外施ABA的敏感性均低于野生型,为一个ABA不敏感突变体。对叶片失水率的测定结果表明,在干旱胁迫下sdt1的失水率显著小于野生型。Southern杂交结合sdt1的遗传分析表明该突变体为紧密相邻的2个T-DNA串联插入,对突变的功能基因有待进一步分子鉴定。     

拟南芥ABA敏感突变体asm1的分离与鉴定

以拟南芥(Arabidopsis thaliana)为研究材料,从T-DNA突变体库中筛选分离得到1株脱落酸(ABA)敏感突变体asm1(ABA sensitive mutant 1,asm1),在含有ABA的培养基中,与野生型相比,asm1突变体的根伸长明显受到抑制,且其种子萌发结果显示asm1对ABA同样表现出敏感特性。在生长发育方面,asm1突变体抽苔时间提前,植株矮化,并且荚果长度明显小于野生型。利用远红外成像系统分析发现,在干旱胁迫下asm1突变体叶面温度高于野生型;失水率分析显示突变体失水率降低以及水分散失减少。遗传学分析表明,asm1是单基因隐性突变且与一个T-DNA插入共分离;通过图位克隆成功获得候选基因ASM1。RT-PCR结果显示,在突变体中ASM1的表达受到抑制,并且能够调控多种ABA信号通路和胁迫应答基因的表达水平。研究结果表明,ASM1可能参与调控ABA信号转导并应答干旱胁迫。     

Genetic linkages between circadian clock-associated components and phytochrome-dependent red light signal transduction in Arabidopsis thaliana

The current best candidates for Arabidopsis thaliana clock components are CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and its homolog LHY (LATE ELONGATED HYPOCOTYL). In addition, five members of a small family, PSEUDO-RESPONSE REGULATORS (including PRR1, PRR3, PRR5, PRR7 and PRR9), are believed to be another type of clock component. The originally described member of PRRs is TOC1 (or PRR1) (TIMING OF CAB EXPRESSION 1). Interestingly, seedlings of A. thaliana carrying a certain lesion (i.e. loss-of-function or misexpression) of a given clock-associated gene commonly display a characteristic phenotype of light response during early photomorphogenesis. For instance, cca1 lhy double mutant seedlings show a shorter hypocotyl length than the wild type under a given fluence rate of red light (i.e. hypersensitivity to red light). In contrast, both toc1 single and prr7 prr5 double mutant seedlings with longer hypocotyls are hyposensitive under the same conditions. These phenotypes are indicative of linkage between the circadian clock and red light signal transduction mechanisms. Here this issue was addressed by conducting combinatorial genetic and epistasis analyses with a large number of mutants and transgenic lines carrying lesions in clock-associated genes, including a cca1 lhy toc1 triple mutant and a cca1 lhy prr7 prr5 quadruple mutant. Taking these results together, we propose a genetic model for clock-associated red light signaling, in which CCA1 and LHY function upstream of TOC1 (PRR1) in a negative manner, in turn, TOC1 (PRR1) serves as a positive regulator. PRR7 and PRR5 also act as positive regulators, but independently from TOC1 (PRR1). It is further suggested that these signaling pathways are coordinately integrated into the phytochrome-mediated red light signal transduction pathway, in which PIF3 (PHYTOCHROME-INTERACTING FACTOR 3) functions as a negative regulator immediately downstream of phyB.     

Ectopic expression of ABSCISIC ACID 2/GLUCOSE INSENSITIVE 1 in Arabidopsis promotes seed dormancy and stress tolerance

Abscisic acid (ABA) is an important phytohormone that plays a critical role in seed development, dormancy, and stress tolerance. 9-cis-Epoxycarotenoid dioxygenase is the key enzyme controlling ABA biosynthesis and stress tolerance. In this study, we investigated the effect of ectopic expression of another ABA biosynthesis gene, ABA2 (or GLUCOSE INSENSITIVE 1 [GIN1]) encoding a short-chain dehydrogenase/reductase in Arabidopsis (Arabidopsis thaliana). We show that ABA2-overexpressing transgenic plants with elevated ABA levels exhibited seed germination delay and more tolerance to salinity than wild type when grown on agar plates and/or in soil. However, the germination delay was abolished in transgenic plants showing ABA levels over 2-fold higher than that of wild type grown on 250 mm NaCl. The data suggest that there are distinct mechanisms underlying ABA-mediated inhibition of seed germination under diverse stress. The ABA-deficient mutant aba2, with a shorter primary root, can be restored to normal root growth by exogenous application of ABA, whereas transgenic plants overexpressing ABA2 showed normal root growth. The data reflect that the basal levels of ABA are essential for maintaining normal primary root elongation. Furthermore, analysis of ABA2 promoter activity with ABA2::beta-glucuronidase transgenic plants revealed that the promoter activity was enhanced by multiple prolonged stresses, such as drought, salinity, cold, and flooding, but not by short-term stress treatments. Coincidently, prolonged drought stress treatment led to the up-regulation of ABA biosynthetic and sugar-related genes. Thus, the data support ABA2 as a late expression gene that might have a fine-tuning function in mediating ABA biosynthesis through primary metabolic changes in response to stress.     

The regulator of G-protein signaling proteins involved in sugar and abscisic acid signaling in Arabidopsis seed germination

The regulator of G-protein signaling (RGS) proteins, recently identified in Arabidopsis (Arabidopsis thaliana; named as AtRGS1), has a predicted seven-transmembrane structure as well as an RGS box with GTPase-accelerating activity and thus desensitizes the G-protein-mediated signaling. The roles of AtRGS1 proteins in Arabidopsis seed germination and their possible interactions with sugars and abscisic acid (ABA) were investigated in this study. Using seeds that carry a null mutation in the genes encoding RGS protein (AtRGS1) and the alpha-subunit (AtGPA1) of the G protein in Arabidopsis (named rgs1-2 and gpa1-3, respectively), our genetic evidence proved the involvement of the AtRGS1 protein in the modulation of seed germination. In contrast to wild-type Columbia-0 and gpa1-3, stratification was found not to be required and the after-ripening process had no effect on the rgs1-2 seed germination. In addition, rgs1-2 seed germination was insensitive to glucose (Glc) and sucrose. The insensitivities of rgs1-2 to Glc and sucrose were not due to a possible osmotic stress because the germination of rgs1-2 mutant seeds showed the same response as those of gpa1-3 mutants and wild type when treated with the same concentrations of mannitol and sorbitol. The gpa1-3 seed germination was hypersensitive while rgs1-2 was less sensitive to exogenous ABA. The different responses to ABA largely diminished and the inhibitory effects on seed germination by exogenous ABA and Glc were markedly alleviated when endogenous ABA biosynthesis was inhibited. Hypersensitive responses of seed germination to both Glc and ABA were also observed in the overexpressor of AtRGS1. Analysis of the active endogenous ABA levels and the expression of NCED3 and ABA2 genes showed that Glc significantly stimulated the ABA biosynthesis and increased the expression of NCED3 and ABA2 genes in germinating Columbia seeds, but not in rgs1-2 mutant seeds. These data suggest that AtRGS1 proteins are involved in the regulation of seed germination. The hyposensitivity of rgs1-2 mutant seed germination to Glc might be the result of the impairment of ABA biosynthesis during seed germination.     

PSEUDO-RESPONSE REGULATOR 7 and 9 are partially redundant genes essential for the temperature responsiveness of the Arabidopsis circadian clock

Environmental time cues, such as photocycles (light/dark) and thermocycles (warm/cold), synchronize (entrain) endogenous biological clocks to local time. Although much is known about entrainment of the Arabidopsis thaliana clock to photocycles, the determinants of thermoperception and entrainment to thermocycles are not known. The Arabidopsis PSEUDO-RESPONSE REGULATOR (PRR) genes, including the clock component TIMING OF CAB EXPRESSION 1/PRR1, are related to bacterial, fungal, and plant response regulators but lack the conserved Asp that is normally phosphorylated by an upstream sensory kinase. Here, we show that two PRR family members, PRR7 and PRR9, are partially redundant; single prr7-3 or prr9-1 mutants exhibit modest period lengthening, but the prr7-3 prr9-1 double mutant shows dramatic and more than additive period lengthening in the light and becomes arrhythmic in constant darkness. The prr7-3 prr9-1 mutant fails both to maintain an oscillation after entrainment to thermocycles and to reset its clock in response to cold pulses and thus represents an important mutant strongly affected in temperature entrainment in higher plants. We conclude that PRR7 and PRR9 are critical components of a temperature-sensitive circadian system. PRR7 and PRR9 could function in temperature and light input pathways or they could represent elements of an oscillator necessary for the clock to respond to temperature signals.     

Maternal Effects Govern Variable Dominance of Two Abscisic Acid Response Mutations in Arabidopsis thaliana

Three abscisic acid (ABA)-controlled responses (seed dormancy, inhibition of germination by applied ABA, and stomatal closure) were compared in wild-type versus homo- and heterozygotes of two Arabidopsis thaliana ABA-insensitive mutants, abi1 and abi2. We found that sensitivity of seeds to applied ABA is partially maternally controlled but that seed dormancy is determined by the embryonic genotype. The effects of the abi1 and abi2 mutations on ABA sensitivity of seed germination ranged from recessive to nearly fully dominant, depending on the parental source of the mutant allele. This maternal effect disappeared during vegetative growth. Stomatal regulation in heterozygotes showed substantial variability, but the average water loss was intermediate between that of homozygous mutants and wild type.