拟南芥根生长缺陷突变体rei1的分离和鉴定

植物体根发育是一个复杂的过程,尽管对其研究颇多,但对其中的分子机制尚缺乏足够认识。以模式植物拟南芥（Arabidopsis thaliana）为研究材料,在T-DNA突变体库中分离到一个拟南芥根生长缺陷突变体rei1（root elongationinhi-bited1）。通过表型分析发现,rei1在生长发育方面与野生型存在明显的差异,突变体的根较野生型短,且角果较小,花出现部分的败育。对突变体进行显微结构分析,发现突变体的根在内部结构上表现为表皮及皮层细胞形态不规则,排列疏松且横向膨大。遗传学分析表明,rei1是单基因隐性突变且与一个T-DNA插入共分离,通过图位克隆的方法成功分离了缺失的候选基因。以上研究结果表明,REI1对植物的根发育具有非常重要的调节作用。     

拟南芥光形态突变体的筛选与基因鉴定

以哥伦比亚(Columbia)野生型拟南芥(Arabidopsis thaliana)为实验材料,用含有激活标记双元质粒pCB260的农杆菌浸花进行转化,构建拟南芥T-DNA插入突变体库.通过突变体的筛选和表型分析,获得了两株光形态突变体,子叶下胚轴伸长的光抑制效应减弱.通过TAIL-PCR(thermal asymmetric interlaced-PCR)技术,成功扩增出突变植株T-DNA插入位点侧翼序列,经NCBI序列比对,T-DNA分别插在CRY1第一和第三外显子部位.突变体的表型分析及PCR鉴定结果表明,T-DNA插入CRY1并影响到突变植株的光形态建成.     

拟南芥乙烯突变体在干旱胁迫下的形态学差异

为了揭示乙烯在植物与环境相互作用过程中的生物学功能,以拟南芥（Arabidopsis thaliana）的ein2-5、ein3-1、EIN3ox、EIL1ox 4种乙烯突变体与Col-0野生型为材料,对比研究它们在干旱胁迫条件下的生长和形态学变化。研究发现,干旱胁迫导致莲座叶直径、叶片面积、花序、水势等指标发生显著变化,同时不同突变体的形态适应特点呈现显著差异。这些结果表明,乙烯积极参与了植物形态塑造过程,与植物的抗旱性具有紧密相关性。     

ATMYB123和ATKOR1基因参与拟南芥根发育的调控

从拟南芥T-DNA插入突变体库中筛选到2个根发育相关基因ATMYB123和ATKOR1表达缺失的突变体atmyb123和atkor1,通过杂交构建这两个基因表达缺失的双突变体atmyb123/atkor1,以明确这两个基因在根发育中的作用。结果显示:(1)ATMYB123表达缺失突变体atmyb123植株地上部分发育减缓,种皮颜色变黄,而ATKOR1表达缺失突变体atkor1植株在这两方面与其野生型没有明显差异;两基因缺失均显著影响了拟南芥根的发育,根生长受到了严重抑制。(2)双突变体atmyb123/atkor1在植株形态和种皮颜色方面表现出单突变体AT-MYB123的特点,而其根长却介于两单突变体的中间。(3)进一步研究发现,培养基pH改变、NaCl处理、外源GA施用均没有改变突变体根生长趋势,显示这3种因素与两基因缺失突变引起的根发育抑制无关。研究表明,AT-MYB123和ATKOR1基因参与拟南芥根的发育调控,转录因子ATMYB123可能作为主调控因子参与ATKOR1对拟南芥根发育的调控。     

拟南芥维管组织的形态建成和分子调控

文章就近10年来拟南芥维管组织发育分化,形态建成、变异类型等,以及相关的调控机制的研究进展作简单介绍.     

磷空间有效性对拟南芥根形态构型的影响

磷空间有效性显著影响拟南芥主、侧根生长。在均一的磷处理下,极度磷胁迫或过量供磷均会导致拟南芥主根变短和侧根密度降低。在分层的磷处理下,上层高磷下层低磷能明显促进主根伸长生长,提高侧根在高磷区域的密度,说明植物根系在下层低磷区感受到磷胁迫信号后,可促进上层高磷区侧根的形成和发育。     

拟南芥铵超敏感突变体amosd和vtc1对外源铵的响应

分析了不同外源铵浓度(0、1、5、10、20mmol.L-1)处理下,2个铵超敏感突变体amosd和vtc1对于外源铵处理的响应差异。结果表明,尽管amosd和vtc1都表现为对外源铵超敏感,但二者对外源铵处理浓度的敏感性上存在差异。随着外源铵浓度的增加,vtc1比amosd先表现出铵中毒症状,更高浓度(20mmol·L-1)铵处理时amosd受到的毒害程度表现更加严重,AMOSD遗传位点的缺失容易导致植物出现铵毒害死亡。其次,二者在遭受外源铵胁迫时表现的最敏感部位有所不同,主要的毒害特征上存在差异,amosd在铵胁迫下首先表现在叶片尤其是新叶的发育受阻,而vtc1则主要表现在根部尤其是主根的伸长受阻。通过分区供应实验证明,amosd主要对于地上部供铵处理超敏感,对根部供铵处理不表现超敏感特性;而vtc1则相反,对根部供铵处理超敏感,对地上部供铵不表现超敏感特性。由此可见,amosd和vtc1这2个铵超敏感突变体在拟南芥铵毒害范围和部位上存在较大差异,与vtc1有所不同,amosd是一个叶源铵超敏感型突变体。在农业机械化叶面喷施施肥日益增加和环境铵沉降日益严重的当下,叶源型铵超敏感突变体amosd的获得为揭示植物地上部铵毒害机制提供了一个理想的遗传材料,对系统全面认识植物铵毒害机制,提高作物耐铵性状具有十分重要的意义。     

低氮下拟南芥叶酰聚谷氨酸合成酶突变体atdfb根发育研究

利用叶酰聚谷氨酸合成酶功能缺失突变体atdfb解析叶酸在拟南芥根发育过程中的生物学功能。纯合T-DNA插入功能缺失突变体atdfb 在土壤培养条件下生长3周,与野生型表型无明显差异。在氮源充足的1/2MS培养基上,atdfb的主根显著短于野生型,互补植株的主根长度恢复到野生型水平,说明主根缩短的表型是由AtDFB基因功能缺失造成的。在1/2MS培养基生长11 d的突变体主根长度只有野生型的23%。在低氮条件下,突变体的生长发育几乎停滞,培养11 d的突变体主根长度只有野生型的4%;5-甲酰四氢叶酸(5-F-THF)可以恢复低氮条件下atdfb-3的表型,其主根长度、根毛长度及静止中心的细胞排列均得到恢复。进一步分析发现,低氮条件下培养少于3 d的atdfb-3补充充足的5-F-THF,3 d后能像野生型一样适应低氮环境。由此说明叶酸对拟南芥根部发育及对低氮环境的适应是必需的。     

拟南芥细胞死亡突变体mod1突变座位的精细物理图谱构建

拟南芥细胞死亡突变体mod1突变位点位于第2染色体分子标记IGS1和mi421之间. 以这一区域YAC重叠群中的YAC克隆末端DNA片段CIC9A3R, CIC11C7L, CIC2G5R及RFLP分子标记克隆CDs3为探针筛选TAMU BAC文库, 获得31个阳性BAC克隆. 用BAC克隆的末端DNA片段杂交所有阳性BAC克隆, 确立了由T6P5, T7M23, T12A21, T8L6及T18A18等克隆组成的MOD1基因所在区域的BAC重叠群. 同时在这一区域发展出11个CAPS分子标记和12个STS序标, 为MOD1基因的图位克隆与鉴定分析及这一区域的全序列测定奠定了基础.     

尿黑酸对拟南芥酪氨酸降解缺陷突变体sscd1的影响

为了了解尿黑酸对拟南芥酪氨酸降解途径的影响,探讨植物中酪氨酸降解途径在长日照下是否被抑制,本研究以拟南芥野生型Col-0和酪氨酸降解途径缺陷突变体sscd1、hgo为实验材料,通过在培养基添加尿黑酸处理,分析长、短日照下尿黑酸对幼苗生长的影响,同时采用q RT-PCR分析尿黑酸对酪氨酸降解途径关键基因HGO、MAAI和SSCD1表达的影响。结果发现:在短日照下,尿黑酸处理能够加速酪氨酸降解途径缺陷突变体sscd1的死亡;在长日照下,尿黑酸也能导致sscd1突变体的死亡;尿黑酸处理上调拟南芥野生型、酪氨酸降解缺陷突变体sscd1中MAAI和HGO的表达,其中MAAI表达在sscd1突变体中的上调幅度更大。以上结果说明,外源尿黑酸处理确实可以激活植物体内酪氨酸降解途径,而且植物中酪氨酸降解途径在长日照下不会被抑制。此外,研究结果进一步证明酪氨酸降解途径中间代谢产物的积累可导致sscd1突变体的死亡。     

Resistance and biomass in Arabidopsis: a new model for Salicylic Acid perception

Salicylic acid (SA) is an essential hormone for plant defence and development. SA perception is usually measured by counting the number of pathogens that grow in planta upon an exogenous application of the hormone. A biological SA perception model based on plant fresh weight reduction caused by disease resistance in Arabidopsis thaliana is proposed. This effect is more noticeable when a chemical analogue of SA is used, like Benzothiadiazole (BTH). By spraying BTH several times, a substantial difference in plant biomass is observed when compared with the mock treatment. Such difference is dose‐dependent and does not require pathogen inoculation. The model is robust and allows for the comparison of different Arabidopsis ecotypes, recombinant inbreed lines, and mutants. Our results show that two mutants, non‐expresser of pathogenesis‐related genes 1 (npr1) and auxin resistant 3 (axr3), fail to lose biomass when BTH is applied to them. Further experiments show that axr3 responds to SA and BTH in terms of defence induction. NPR1‐related genotypes also confirm the pivotal role of NPR1 in SA perception, and suggest an active program of depletion of resources in the infected tissues.     

Salicylic acid inhibits jasmonic acid-induced resistance of Arabidopsis thaliana to Spodoptera exigua

The role of salicylic acid (SA) in plant responses to pathogens has been well documented, but its direct and indirect effects on plant responses to insects are not so well understood. We examined the effects of SA, alone and in combination with jasmonic acid (JA), on the performance of the generalist herbivore, Spodoptera exigua, in wild-type and mutant Arabidopsis thaliana genotypes that varied genetically in their ability to mount SA- and JA-mediated defence responses. In one experiment, growth of S. exigua larvae was highest on the Wassilewskija wild-type, intermediate on the Columbia wild-type and the JA-deficient fad mutant, and lowest on the nim1-1 and jar1-mutants, which are defective in the SA and JA pathways, respectively. Activity of guaiacol peroxidase, polyphenoloxidase, n-acetylglucosaminidase, and trypsin inhibitor varied by genotype but did not correlate with insect performance. SA treatment increased growth of S. exigua larvae by approximately 35% over all genotypes, but had no discernable effect on activities of the four defence proteins. In a second experiment, growth of S. exigua was highest across treatments on the cep1 mutant, a constitutive expressor of high SA levels and systemic acquired resistance, and lowest on the fad mutant, which is JA-deficient. JA treatment generally increased activity of all four defence proteins, increased total glucosinolate levels and reduced insect growth by approximately 25% over all genotypes. SA generally inhibited expression of JA-induced resistance to S. exigua when both hormones were applied simultaneously. Across genotypes and treatments, larval mass was negatively correlated with the activity of trypsin inhibitor and polyphenoloxidase and with total glucosinolate levels, and insect damage was negatively correlated with the activity of polyphenoloxidase. SA had little effect on the induction of defence protein activity by JA. However, SA attenuated the induction of glucosinolates by JA and therefore may explain better the interactive effects of SA and JA on insect performance. This study illustrates that direct and indirect cross-effects of SA on resistance to S. exigua can occur in A. thaliana. Effects of SA may be mediated through effects on plant defence chemistry or other aspects of the suitability of foliage for insect feeding and growth.     

拟南芥水杨酸受体表达与在体转录水平的研究

目的:从拟南芥叶中克隆水杨酸结合蛋白(SA binding protein 2,SABP2,也称水杨酸受体)基因sabp2进行异源表达并测定其活性.方法:从拟南芥叶RNA中通过反转录PCR扩增sabp2,将PCR产物克隆至载体pMD - 19T simple中,经测序验证后,再基于pET28a构建重组表达载体,转化至大肠杆菌BL21( DE3)并表达,检测重组蛋白的活性.另一方面,对sabp2在拟南芥中转录水平进行了研究.结果:PCR获得792bp的sabp2基因,并成功构建异源表达载体pET28a - sabp2.优化结果表明,在0.4mmol/L IPTG诱导下20℃培养8h,表达产物活性较强,具天然SABP2的特征性酯酶活性.该基因在拟南芥叶中转录模式呈SA应激性和组织特异性.结论:sabp2成功表达,不仅为筛选SA受体拮抗剂提供新的原核体系,而且为探讨SA与SABP2相互作用在植物防御过程中时空变化奠定基础.     

Comparative proteomic analysis of Arabidopsis thaliana roots between wild type and its salt-tolerant mutant

Two-dimensional electrophoresis (2-DE) showed the variation expression of Arabidopsis thaliana root proteins between wild type and its salt-tolerant mutant obtained from cobalt-60 γ ray radiation. Forty-six differential root protein spots were reproducibly presented on 2-DE maps, and 29 spots were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MS). Fifteen protein spots corresponding to 10 proteins, and 14 protein spots corresponding to 9 proteins were constitutively up-regulated and down-regulated in the salt-tolerant mutant root. Bioinformatic analysis indicated that those differential proteins might be involved in the regulation of redox homeostasis, nucleotide metabolism, signal transduction, stress response and defense, carbohydrate metabolism, and cell wall metabolism. Peroxidase 22 might be a versatile enzyme and might play dual roles in both cell wall metabolism and regulation of redox homeostasis. Our work provides not only new insights into salt-responsive proteins in root, but also the potential salt-tolerant targets for further dissection of molecular mechanism adapted by plants during salt stress.     

Salicylic acid prevents Trichoderma harzianum from entering the vascular system of roots

Trichoderma is a soil‐borne fungal genus that includes species with a significant impact on agriculture and industrial processes. Some Trichoderma strains exert beneficial effects in plants through root colonization, although little is known about how this interaction takes place. To better understand this process, the root colonization of wild‐type Arabidopsis and the salicylic acid (SA)‐impaired mutant sid2 by a green fluorescent protein (GFP)‐marked Trichoderma harzianum strain was followed under confocal microscopy. Trichoderma harzianum GFP22 was able to penetrate the vascular tissue of the sid2 mutant because of the absence of callose deposition in the cell wall of root cells. In addition, a higher colonization of sid2 roots by GFP22 compared with that in Arabidopsis wild‐type roots was detected by real‐time polymerase chain reaction. These results, together with differences in the expression levels of plant defence genes in the roots of both interactions, support a key role for SA in Trichoderma early root colonization stages. We observed that, without the support of SA, plants were unable to prevent the arrival of the fungus in the vascular system and its spread into aerial parts, leading to later collapse.     

The immune components ENHANCED DISEASE SUSCEPTIBILITY 1 and PHYTOALEXIN DEFICIENT 4 are required for cell death caused by overaccumulation of ceramides in Arabidopsis

Sphingolipids have key functions in plant membrane structure and signaling. Perturbations of plant sphingolipid metabolism often induce cell death and salicylic acid (SA) accumulation; SA accumulation, in turn, promotes sphingolipid metabolism and further cell death. However, the underlying molecular mechanisms remain unclear. Here, we show that the Arabidopsis thaliana lipase-like protein ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and its partner PHYTOALEXIN DEFICIENT 4 (PAD4) participate in sphingolipid metabolism and associated cell death. The accelerated cell death 5 (acd5) mutants accumulate ceramides due to a defect in ceramide kinase and show spontaneous cell death. Loss of function of EDS1, PAD4 or SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) in the acd5 background suppressed the acd5 cell death phenotype and prevented ceramide accumulation. Treatment with the SA analogue benzothiadiazole partially restored sphingolipid accumulation in the acd5 pad4 and acd5 eds1 double mutants, showing that the inhibitory effect of the pad4-1 and eds1-2 mutations on acd5-conferred sphingolipid accumulation partly depends on SA. Moreover, the pad4-1 and eds1-2 mutations substantially rescued the susceptibility of the acd5 mutant to Botrytis cinerea. Consistent with this, B. cinerea-induced ceramide accumulation requires PAD4 or EDS1. Finally, examination of plants overexpressing the ceramide synthase gene LAG1 HOMOLOGUE2 suggested that EDS1, PAD4 and SA are involved in long-chain ceramide metabolism and ceramide-associated cell death. Collectively, our observations reveal that EDS1 and PAD4 mediate ceramide (especially long-chain ceramide) metabolism and associated cell death, by SA-dependent and SA-independent pathways.     

Novel software for analysis of root gravitropism: comparative response patterns of Arabidopsis wild-type and axr1 seedlings

In an earlier study (Evans, Ishikawa & Estelle 1994, Planta 194, 215-222) we used a video digitizer system to compare the kinetics of auxin action on root elongation in wild-type seedlings and seedlings of auxin response mutants of Arabidopsis thaliana (L.) Heynh. We have since modified the system software to allow determination of elongation on opposite sides of vertical or gravistimulated roots and to allow continuous measurement of the angle of orientation of sequential subsections of the root during the response. We used this technology to compare the patterns of differential growth that generate curvature in roots of the Columbia ecotype and in the mutants axr1-3, axr1-12 and axr2, which show reduced gravitropic responsiveness and reduced sensitivity to inhibition by auxin. The pattern of differential growth during gravitropism differed in roots of wild-type and axr1 seedlings. In wild-type roots, initial curvature resulted from differential inhibition of elongation in the distal elongation zone (DEZ). This was followed by an acceleration of elongation along the top side of the DEZ. In roots of axr1-3, curvature resulted from differential stimulation of elongation whereas in roots of axr1-12 the response was variable. Roots of axr2 did not exhibit gravitropic curvature. The observation that the pattern of differential growth causing curvature is dramatically altered by a change in sensitivity to auxin is consistent with the classical Cholodny-Went theory of gravitropism which maintains that differential growth patterns induced by gravistimulation are mediated primarily by gravi-induced shifts in auxin distribution. The new technology introduced with this report allows automated determination of stimulus response patterns in the small but experimentally popular roots of Arabidopsis.     

拟南芥草酸不敏感突变体的筛选与分析

草酸是多种真菌的致病因子.在含 1.2 mmol/L 草酸和 10 μmol/L 雌二醇的 MS 缺钙培养基上,从大约含 6000个独立株系的拟南芥化学诱导突变体库中筛选草酸不敏感的突变体.初筛获得的可能的草酸不敏感突变体单株收种后,进一步复筛获得 5 株较抗草酸的突变体 D33、D74、D154、D282 和 D630.对它们的 TAIL-PCR 的第三步产物回收、测序、比对的结果表明:D33 的 T-DNA 插入位点位于 At2g39720(Zinc finger)and At2g39730 (Rubisco activase) 之间,D74、D154、D282 和 D630 都插在 At5g10450 (14-3-3 protein GF14 lambda) 的第一个内含子上.突变体后继的遗传分析与分子分析正在进行中.