Physiological Analysis of Two Arabidopsis thaliana Mutants in Response to CO2

[Objective] The 15urpose was to seek for the different phenotypes between wild type and Arabidopsis Mutants in response to CO2. [Method] The epidermis bioassays and seed germination test were carried out to analyze the physiological characteristics of two Arabidopsis mu- tants and their wild type. [Result] There existed distinct differences in stomata apertures, water loss and leaf temperature compared with wild type except for stomata density. In addition, seed germination test on the medium indicated that cdfl was insensitive to ABA, mannitol and NaCI, but cdsl performed contrary to cdil. [ Conclusion] There are some different physiological characteristics between wild type and mutants.

Abiotic Stresses and Phytohormones Regulate Expression of FAD2 Gene in Arabidopsis thaliana

Modification of unsaturated fatty acid （FA） levels has been found to accompany multiple abiotic stress acclimations in many plants. Delta 12 fatty acid desaturase （FAD2） plays a critical role in the synthesis of polyunsaturated FAs in plant cells by converting oleic acid （18：1） to linoleic acid （18：2）. To better understand the relationship between polyunsaturated FAs metabolism and stress adaptation, the expression of FAD2 gene and changes in the FA compositions under various abiotic stresses and phytohormone treatments in Arabidopsis thaliana was investigated in this study. A 1 423-bp promoter of the FAD2 gene was cloned and characterized from Arabidopsis. Several putative hormone- and stress- inducible cis-elements were identified in the cloned promoter, which include salt- and pathogen-inducible GT-1 motifs, low-temperature-responsive MYC element, dehydration-responsive MYB element, and GA signaling related WRKY71OS element. To investigate the fine regulation of FAD2 gene, a recombinant FAD2 promoter-GUS construct was introduced into Arabidopsis plants. Histochemical study showed that the promoter was ubiquitously active and responsive not only to exogenous phytohormones including ABA, 24-eBL, and SA but also to darkness, temperature, salt, and sucrose stresses in Arabidopsis seedlings. Consistent with the expression change, treatments with exogenous 24-eBL, ABA, SA, and NaCl resulted in reduction in polyunsaturated FAs in Arabidopsis seedlings. These findings suggest that the FAD2 gene with a wide variety of putative response elements in its promoter is responsive to multiple phytohormones and abiotic stresses and therefore may play an important role in stress responses of Arabidopsis during plant growth and seed development.

Cloning the Promoter of BcNA1 from Brassica napus and Fad2 Gene from Arabidopsis thaliana and Construction of the Plant Expression Vector

The upstream regulatory region of a seed specific gene was isolated from the genomic DNA of Brassica napus by PCR amplification. The cloned fragment contained 1755 nucleotides, and shared a sequence homology of 99.6% with the reported data. The coding region of oleic acid desaturase gene was then cloned from Arabidopsis thaliana. The sequencing analysis indicated that the sequence of the PCR product was just the same as reported before. In addition, the plant expression vector harboring the seed specific promoter and trans Fad2 gene was constructed.

Construction of Arabidopsis thaliana ugt84a2/ugt84a4 Double Mutants Mediated by CRISPR/Cas9

[Objectives]The CRISPR/Cas9(Clustered regulatory interspaced short palindromic repeat/Cas9)gene editing technology is the third generation of"genome fixed-point editing technology"following the"zinc finger endonuclease(ZFN)"and"transcription activator effector nuclease(TALEN)".Glucotransferase genes UGT84A2 and UGT84A4,can simultaneously convert hydroxycinnamate into 1-O-β-glucose esters as isozymes.The CRISPR/Cas9 technology was used to construct double mutants of Arabidopsis thaliana ugt84a2/ugt84a4.[Methods]A CRISPR/Cas9 double mutant expression vector was constructed using UGT84A2 and UGT84A4 as the target genes.The Agrobacterium-mediated dip dyeing method was used to transform wild-type A.thaliana,and the CRISPR/Cas9system was used to target and knock out A.thaliana UGT84A2 and UGT84A4 genes.[Results]The descendants of A.thaliana with the UGT84A2/UGT84A4 gene were sequenced and analyzed.Thirteen positively transformed plants obtained were analyzed according to the sequencing results,and the ugt84a2/ugt84a4 double mutants were screened.[Conclusions]This study provides a reference for the functional study of UGT84A2 and UGT84A4 isoenzyme genes in other species,as well as strong theoretical and method support for accelerating the development and utilization of UGT84A2/UGT84A4 functional gene resources.

拟南芥中异源过表达CsLOX6和CsHPL2基因提高植株耐盐性和耐旱性

探究CsLOX6基因和CsHPL2基因对拟南芥(Arabidopsis thaliana)生长以及对干旱和盐胁迫抗性的影响,以野生型拟南芥(WT)、转基因型拟南芥(OE-CsLOX6、OE-CsHPL2)、突变体型拟南芥(TB-Atlox5)为材料,通过表型观察、转录组和代谢组学分析等方法评估4种拟南芥的耐旱性、耐盐性。结果表明,OE-CsLOX6的地上部鲜重和根长均显著高于其他基因型植株,地下部鲜重明显高于其他基因型植株;OE-CsHPL2的地上部鲜重、地下部鲜重和根长明显低于其他基因型植株。OE-CsLOX6、OE-CsHPL2和WT的代谢物含量存在较大差异,OE-CsLOX6和WT有64个差异代谢物,其中有6个上调,58个下调;OE-CsHPL2和WT有63个差异代谢物,其中有9个上调,54个下调。过表达CsLOX6基因和CsHPL2基因可以提高拟南芥对盐胁迫和干旱胁迫的抗性。在盐胁迫和干旱胁迫下,OE-CsLOX6、OE-CsHPL2的丙二醛含量要显著低于野生型植株,膜质过氧化程度低,SOD、POD和CAT活性要明显高于野生型,提高了植株的抗氧化能力,叶片长势明显优于野生型植株。

The Arabidopsis AS2 Gene Encoding a Predicted Leucine-zipper Protein Is Required for the Leaf Polarity Formation

The asymmetric leaves2 ( as2) is a classical Arabidopsis thaliana (L.) Heynh. mutant that shows leaf lobes and leaflet-like structures from the petioles of leaves. Genetic and molecular analyses have demonstrated that the AS2 function is required for repression of meristem-related homeobox genes in leaves. In this study, we describe phenotypic characterizations of new as2 alleles that are in the Landsberg erecta (Ler) genetic background. In addition to the as2 phenotypes reported previously, the new as2 mutants have some leaves with petiole growth underneath the leaf blade, showing a lotus-leaf structure. More severe rosettes leaves of the as2 mutants form a filament-like structure, reflecting a loss of the adaxial-abaxial polarity in leaves. Among as2 mutants analyzed in different genetic backgrounds, only those that are in the Ler genetic background resulted in a high frequency of the lotus-leaf structure. We have isolated the AS2 gene by map-based gene cloning. The predicted AS2 protein contains a leucine-zipper motif, and its N-terminus shares high levels of sequence similarity to those of a group of predicted proteins with no known biological functions. AS2 transcripts were detected in leaves, flowers and fruits, but absent in stems, consistent with the mutant phenotypes.

Extracellular and Intracellular Calcium both Involved in the Jasmonic Acid Induced Calcium Mobilization in Arabidopsis thaliana

The objective of this experiment is to evaluate the role of intracellular and extracellular Ca2＋ and calmodulin （CAM） in jasmonic acid （JA） signaling. The laser scanning microscopy was used to detect the changes of [Ca2＋]cyt of Arabidopsis thaliana leaf cells which pretreated with different types of calcium channel blocker. Moreover, the expression of VSP, one of JA response genes, was also investigated after pretreated with the above blocker and antagonist of CaM. The results showed that extracellular and intracellular calcium both involved in the JA-induced Ca2＋ mobilization, and then Ca2＋ exerted its functions through activating the CaM or CaM related proteins. The apoplast calcium influx and the calcium release from the calcium stores are both involved in the JA-induced calcium mobilization, then the JA-induced Ca2＋ transmited the JA signal through CaM or CaM related proteins, and regulated the JA responsive genes.

Ca^(2+)在茉莉酸甲酯诱导拟南芥气孔关闭中的信号转导作用

以拟南芥叶片下表皮为材料 ,分别用表皮生物分析法和激光扫描共聚焦显微镜成像技术 ,研究茉莉酸甲酯 (JA Me)促进气孔关闭过程中胞质Ca2 + 浓度的变化及其与气孔关闭的关系。结果表明 ,10 - 7到 10 - 3mol L的JA Me处理能促进拟南芥叶片的气孔关闭 ,其中 ,10 - 5mol L是最适浓度。用 10 - 5mol L的JA Me处理5min ,胞质Ca2 + 浓度从静息态的 10 5nmol L增加到 332 0nmol L ;质膜Ca2 + 通道阻断剂LaCl3和Ca2 + 螯合剂EGTA均能明显地降低JA Me对气孔关闭的促进作用。由此推测 ,胞质Ca2 + 可能是JA

基于水母发光蛋白的低温胁迫下拟南芥胞内Ca^(2+)信号转导研究

通过花序浸染法获得了转化有水母发光蛋白基因的拟南芥植株,利用该材料研究了低温胁迫下植物细胞内Ca2+浓度的变化.结果表明,低温刺激可引起细胞内Ca2+浓度升高,EGTA和LaCl3能抑制Ca2+浓度升高.

Ca^2＋参与异三聚体G蛋白调控的拟南芥侧根生长发育过程

以拟南芥的野生型(ws)、异三聚体G蛋白α亚基基因GPA1缺失突变体(gpα1-1、gpα1-2)和超表达突变体(wGα、cGα)为材料,在含有不同质量浓度(0~0.2mg/L)NAA的培养基内,添加常用的钙通道抑制剂,对拟南芥侧根生长发育的形态进行了观测。结果表明:1)在不含Ca2+的培养基内,5种基因型侧根生长发育的差异显著性明显降低。2)在含有电压依赖型钙通道有机抑制剂(异搏定和地而流卓)的培养基内,5种基因型侧根的生长发育与未施加抑制剂的差异显著性一致。3)在含有不同浓度的3价无机离子的培养基内,5种基因型侧根的生长发育与未施加抑制剂的差异显著性不一致。初步表明了在NAA诱导的拟南芥侧根生长发育过程中Ca2+可能是G蛋白的下游信号。

Magnesium transporter AtMGT9 is essential for pollen development in Arabidopsis

Magnesium （Mg^2＋） is abundant in plant cells and plays a critical role in many physiological processes. A 10-member gene family AtMGT （also known as AtMRS2） was identified in Arabidopsis, which belongs to a eukaryote subset of the CorA superfamily, functioning as Mg^2＋ transporters. Some family members （AtMGT1 and AtMGT10） function as high-affinity Mg^2＋ transporter and could complement bacterial mutant or yeast mutant lacking Mg^2＋ transport capability. Here we report an AtMGT family member, AtMGT9, that functions as a low-affinity Mg^2＋ transporter, and is essential for pollen development. The functional complementation assay in Salmonella mutant strain MM281 showed that AtMGT9 is capable of mediating Mg^2＋ uptake in the sub-millimolar range of Mg^2＋. The AtMGT9 gene was expressed most strongly in mature anthers and was also detectable in vascular tissues of the leaves, and in young roots. Disruption of AtMGT9 gene expression resulted in abortion of half of the mature pollen grains in heterozygous mutant ＋/mgt9, and no homozygous mutant plant was obtained in the progeny of selfed ＋/mgt9 plants. Transgenic plants expressing AtMGT9 in these heterozygous plants can recover the pollen phenotype to the wild type. In addition, At- MGT9 RNAi transgenic plants also showed similar abortive pollen phenotype to mutant ＋/mgt9. Together, our results demonstrate that AtMGT9 functions as a low-affinity Mg^2＋ transporter that plays a crucial role in male gametophyte development and male fertility.

Primary evidence for involvement of IP_(3) in heat-shock signal transduction in Arabidopsis

The role of inositol 1,4,5-trisphosphate （IP3） in transducing heat-shock （HS） signals was examined in Arabidopsis. The whole-plant IP3 level increased within 1 min of HS at 37℃. After 3 min of HS, the IP3 level reached a maximum 2.5 fold increase. Using the transgenic Arabidopsis plants that have AtHsp 18.2 promoter-β-glucuronidase （GUS） fusion gene, it was found that the level of GUS activity was up-regulated by the addition of caged IP3 at both non-HS and HS temperatures and was down-regulated by the phospholipase C （PLC） inhibitors {1-[6-（（ 1713-3-Methoxyestra-1,3,5（10）-trien- 7-yl）amino）hexyl]-2,5-pyrrolidinedione } （U-73122）. The intracellular-free calcium ion concentration （[Ca^2＋]i） increased during HS at 37℃ in suspension-cultured Arabidopsis cells expressing apoaequorin. Treatment with U-73122 prevented the increase of [Ca^2＋]i to some extent. Above results provided primary evidence for the possible involvement of IP3 in HS signal transduction in higher plants.

LecRKIII.2基因调控拟南芥对非生物胁迫和外源激素的响应

在拟南芥Arabidopsis thaliana中,凝集素类受体激酶(lectin receptor-like kinases,LecRKs)包含86个成员,在植物对生物及非生物胁迫反应中起关键作用。AtLecRKIII.2作为LecRKs家族的一员,其具体功能未见报道。本文通过实时定量PCR和三引物法鉴别AtLecRKIII.2基因的过表达植株及纯合缺失突变体。启动子顺式作用元件分析发现,AtLecRKIII.2基因有多种与调控胁迫、激素等反应相关的功能元件。组织表达模式分析表明,AtLecRKIII.2基因于果荚中具有较高的转录水平,茎中的转录水平次之。逆境及激素响应模式分析表明,AtLecRKIII.2基因对外源生长素(3-indoleacetic acid,IAA)、油菜素内酯(brassinolide,BL)、赤霉素(gibberellins,GAs)、PEG8000、高温和低温均具有不同程度的响应。萌发率及根长实验结果表明,NaCl或Mannitol处理后,LecRKIII.2-OE的种子萌发率高于野生型,而lecrkiii.2的种子萌发率低于野生型。用生长素处理后,LecRKIII.2-OE根的伸长被显著抑制,且其体内与生长素应答和运输相关基因的转录水平显著变化。上述研究结果表明,AtLecRKIII.2基因正调控拟南芥对盐胁迫、渗透胁迫的耐受性和涉及根的生长发育。研究结果为进一步深入研究AtLecRKIII.2基因在植物非生物胁迫及激素信号转导中的生理功能提供参考数据。

拟南芥MGT6调控高镁条件下Ca^(2+)/Mg^(2+)动态平衡的机制研究

镁是植物生长发育所必需的营养元素之一,在一系列生理生化过程起着重要的作用。高镁胁迫影响植物正常生长发育,但到目前为止有关植物响应高镁胁迫的机制报道较少。镁转运蛋白6(magnesium transporter 6,MGT6)是植物中第一个被报道定位于细胞质膜,并在低镁条件下介导植物根部吸收镁离子的蛋白质。本研究发现,MGT6的突变体和RNA干扰植株在高镁条件下出现叶片变黄、地上部分变小的表型,该表型能被一定浓度的钙离子拮抗。这暗示MGT6可能通过调控钙镁离子动态平衡来响应高镁胁迫。

拟南芥2-甲基-6-叶绿基-1，4-苯醌甲基转移酶启动子的分离及表达特性分析

维生素E是一类人体必需的脂溶性抗氧化剂,具有重要的生理功能。2-甲基-6-叶绿基-1,4-苯醌甲基转移酶(MPBQMT)是天然维生素E合成途径中的关键酶之一,催化MPBQ甲基化,生成DMPBQ。从拟南芥分离了MPBQ MT基因1018bp的启动子序列,构建了含该启动子和GUS报告基因的植物表达载体,通过农杆菌介导转化拟南芥,获得了转基因植株。GUS组织化学染色结果表明,在MPBQ MT启动子驱动下,报告基因GUS在拟南芥的茎、叶、花萼、雄蕊、种荚均有表达,且在茎、叶、种荚中表达量较高,而在根、花瓣和种子中则没有观察到GUS基因的表达,表明MPBQ MT基因可能仅在拟南芥幼嫩茎、叶、种荚等绿色组织中特异性高表达。

拟南芥突变体lac8-2和lac8-3的鉴定

本课题组已通过基因工程方法证实,拟南芥At5g01040的编码蛋白Lac8具有漆酶活性.本研究通过PCR扩增技术,成功筛选出Lac8基因的T-DNA插入突变体lac8-2和lac8-3的纯合子.RT-PCR分析表明,突变体中没有Lac8基因的表达.因此,lac8-2和lac8-3均可作为Lac8基因的功能缺失突变体,这为进一步研究Lac8基因在拟南芥生长发育中的功能奠定了材料基础.

拟南芥AtNCED2基因启动子区域序列克隆及其活性分析

目的:克隆拟南芥AtNCED2基因启动子区域序列,并分析其组织器官特异性及对外界刺激的响应。方法:通过PCR从拟南芥基因组中克隆AtNCED2基因5’侧翼2295bp启动子区域序列(AtNCED2p),并进行生物信息学分析。构建AtNCED2p驱动GUS的植物双元表达载体pAtNCED2p::GUS,通过根癌农杆菌介导法将其转化野生型拟南芥,检测转基因植株中GUS表达的组织器官特异性。结果:该启动子序列中存在TATA-box、CAAT-box、根器官特异性元件、ABA响应元件、低温响应元件、昼夜节律响应元件等顺式作用元件。GUS活性主要集中在转基因拟南芥根尖及侧根发生部位。外源ABA处理的转基因植株根中GUS活性为174.8nmol4-MU min-1mg-1蛋白,明显高于对照值91.7nmol 4-MU min-1mg-1蛋白。结论:AtNCED2基因可能在根的生长和发育中起作用,且外源ABA处理增强其在根中的表达。

The salt-activated CBF1/CBF2/CBF3-GALS1 module fine-tunes galactan-induced salt hypersensitivity in Arabidopsis

Plant growth and development are significantly hampered in saline environments,limiting agricultural productivity.Thus,it is crucial to unravel the mechanism underlying plant responses to salt stress.β-1,4-Galactan(galactan),which forms the side chains of pectic rhamnogalacturonan I,enhances plant sensitivity to high-salt stress.Galactan is synthesized by GALACTAN SYNTHASE1(GALS1).We previously showed that Na Cl relieves the direct suppression of GALS1 transcription by the transcription factors BPC1 and BPC2 to induce the excess accumulation of galactan in Arabidopsis(Arabidopsis thaliana).However,how plants adapt to this unfavorable environment remains unclear.Here,we determined that the transcription factors CBF1,CBF2,and CBF3 directly interact with the GALS1 promoter and repress its expression,leading to reduced galactan accumulation and enhanced salt tolerance.Salt stress enhances the binding of CBF1/CBF2/CBF3 to the GALS1 promoter by inducing CBF1/CBF2/CBF3 transcription and accumulation.Genetic analysis suggested that CBF1/CBF2/CBF3 function upstream of GALS1 to modulate salt-induced galactan biosynthesis and the salt response.CBF1/CBF2/CBF3 and BPC1/BPC2 function in parallel to regulate GALS1 expression,thereby modulating the salt response.Our results reveal a mechanism in which salt-activated CBF1/CBF2/CBF3 inhibit BPC1/BPC2-regulated GALS1 expression to alleviate galactan-induced salt hypersensitivity,providing an activation/deactivation fine-tune mechanism for dynamic regulation of GALS1 expression under salt stress in Arabidopsis.

SnoRNP is essential for thermospermine-mediated development in Arabidopsis thaliana

Polyamines have been discovered for hundreds of years and once considered as a class of phytohormones.Polyamines play critical roles in a range of developmental processes.However,the molecular mechanisms of polyamine signaling pathways remain poorly understood.Here,we measured the contents of main types of polyamines,and found that endogenous level of thermospermine(T-Spm)in Arabidopsis thaliana is comparable to those of classic phytohormones and is significantly lower than those of putrescine(Put),spermidine(Spd),and spermine(Spm).We further found a nodule-like structure around the junction area connecting the shoot and root of the T-Spm biosynthetic mutant acl5 and obtained more than 50 suppressors of acl5 nodule structure(san)through suppressor screening.An in-depth study of two san suppressors revealed that NAP57 and NOP56,core components of box H/ACA and C/D snoRNPs,were essential for T-Spm-mediated nodule-like structure formation and plant height.Furthermore,analyses of rRNA modifications showed that the overall levels of pseudouridylation and 2′-O-methylation were compromised in san1 and san2 respectively.Taken together,these results establish a strong genetic relationship between rRNA modification and T-Spm-mediated growth and development,which was previously undiscovered in all organisms.

桑树病程相关蛋白基因MuPR1-2的克隆及功能分析

病程相关蛋白(pathogenesis-related proteins,PRs)是植物体内受病原物或其他环境因子胁迫而诱导表达的一类蛋白质,在植物抗病反应过程中起着非常重要的作用。在桑树叶片中扩增得到MuPR1-2基因(Gen Bank登录号:KC453994),该基因编码区全长609 bp,编码蛋白质含有202个氨基酸,具有一个典型的信号肽和PR蛋白结构域以及PR1蛋白所共有的α-β-α夹心结构。将Mu PR1-2与绿色荧光蛋白基因gfp融合转入拟南芥,通过对转基因拟南芥根尖的显微结构观察,证明MuPR1-2蛋白定位于细胞壁或分泌到细胞间隙。转入MuPR1-2基因的拟南芥对丁香假单胞菌番茄致病变种(Pst DC3000)和灰霉菌具有较强的抵抗能力,表明Mu PR1-2在植物防御功能中具有重要作用。