Microarray-based Screening of Target Genes Regulated by Heat Shock Factor AtHsfA1a in Arabidopsis thaliana

[ Objective] This study aimed to screen target genes regulated by heat shock factor AtHsfAla in Arabidopsis thaliana. [ Method] Using AtHsfAla-in- serted mutant athsfala （SALK-068042） and wild-type A. thaliana seedlings as experimental materials, target genes regulated by heat shock factor AtHsfAla were screened by microarray assay. Differentially expressed genes were screened by multiple method. Specific functions of differentially expressed genes were analyzed by gene ontology （GO） analysis. Signal transduction pathways, in which differentia｜ly expressed genes were involved, were analyzed by pathway analysis. Gene-gene interaction network was constructed by Signal-Net. [ Result] A total of 3 672 differentially expressed genes were screened out. Up-regulated differentially expressed genes were involved in 198 functions and 7 signal transduction pathways; down-regulated differentially expressed genes were involved in 94 functions and 10 signal transduction pathways. In the signal transduction network, it was found that cwlNV4 and HXK3 had relatively high ability of mediation; AT1 G14240 and cwlNV4 ex- hibited the most interactions with other genes, which were located in key positions throughout the gene-gene interaction network. [ Conclusion] Heat shock factor AtHsfAla regulates a large number of target genes in A. thaliana.

Subcellular Localization of APX1 Protein in Arabidopsis thaliana

To predict the subcellular location of ascorbate peroxidase 1 （ APX1 ） protein in Arabidopsis thaliana, a plasmid expressing APXl-enbance green fluores- cent protein （EGFP） fusion protein was constructed, transformed into Agrobacterium tumefaciens GV3101, and transiently expressed in tobacco leaves. The localization of APX1 protein in the cells was determined by detecting the distribution of the fusion protein in tobacco epidermal cells. The results showed APX1 protein was mainly distributed in the cytoplasm of the plant.

Development of a New Cold-Tolerant Maize(Zea mays L.)Germplasm Using the ICE1 Gene from Arabidopsis thaliana

To develop cold-tolerant maize germplasms and identify the activation of INDUCER OF CRT/DRE-BINDING FACTOR EXPRESSION(ICE1)expression in response to cold stress,RT-PCR was used to amplify the complete open reading frame sequence of the ICE1 gene and construct the plant expression vector pCAMBIA3301-ICE1-Bar.Immature maize embryos and calli were transformed with the recombinant vector using Agrobacterium tumefaciens-mediated transformations.From the regenerated plantlets,three T1 lines were screened and identi-fied by PCR.A Southern blot analysis showed that a single copy of the ICE1 gene was integrated into the maize(Zea mays L.)genomes of the three T1 generations.Under low temperature-stress conditions(4°C),the relative conductivity levels decreased by 27.51%–31.44%,the proline concentrations increased by 12.50%–17.50%,the malondialdehyde concentrations decreased by 16.78%–18.37%,and the peroxidase activities increased by 19.60%–22.89%in the T1 lines compared with those of the control.A real-time quantitative PCR analysis showed that the ICE1 gene was ectopically expressed in the roots,stems,and leaves of the T1 lines.ICE1 positively regulates the expression of the CBF genes in response to cold stress.Thus,this study showed the successful transformation of maize with the ICE1 gene,resulting in the generation of a new maize germplasm that had increased tolerance to cold stress.

Regulation of AtbZIP1 Gene Expression by T-DNA Insertion in Arabidopsis thaliana

bZIP transcription factor family is one of the largest groups of the plant transcription factor families and plays an important role in plant growth and adaption to the abiotic stresses. In this study, two AtbZIP1 mutant Arabidopsis （bzipl） were used with T-DNA inserted into two different sites, designated as SALK-556773 and SALK-660942, in order to identify different effects on AtbZIP1 gene expression by different T-DNA insertion sites. PCR and RT-PCR results revealed that T-DNA insertion in CDS region could effectively inhibit AtbZIP1 gene expression, while T-DNA insertion in 3＇-UTR couldn＇t. The phenotype analysis further confirmed the differences and showed that T-DNA insertion in CDS region decreased plants＇ drought resistance, while in 3＇-UTR couldn＇t. The phenotype assays also suggested that AtbZIP1 held pivotal roles in plant response to drought stress.

Determining the transcriptional regulation pattern of PgTIP1 in transgenic Arabidopsis thaliana by constructing gene

The seed size, seed mass, and growth rate of transgenic Arabidopsis plants containing PgTIP1, a ginseng tonoplast aquaporin gene, are significantly higher than those of wild-type Arabidopsis plants. Whole genome expression and bioinformatics analysis, including analysis of co-expression networks and transcription factors (Tfscan), were used to determine the key genes that are activated after the expression of PgTIP1 and the transcription factors that play important roles in the regulation of the genes controlling growth of Arabidopsis thaliana seeds by using transgenic Arabidopsis plants containing PgTIP1. Differential gene analysis showed that transformation of exogenous PgTIP1 to Arabidopsis induced endogenous gene expression changes. Analysis of gene co-expression networks revealed 2 genes, PIP1 (plasma membrane aquaporin 1 gene) and RD26 (responsive to desiccation 26 gene;a NAC transcription factor), that were localized in the core of the networks. Analysis of the transcriptional regulation network of transgenic Arabidopsis plants containing PgTIP1 showed that PIP1 and RD26 were regulated via DNA binding with a finger domain on transcription factor 2 (Dof2). In this study, we demonstrated that Dof2 induces up-regulation of PIP1 and RD26 after transformation with PgTIP1. The results of this study provide a new means for conducting research into and controlling growth of Arabidopsis thaliana seeds.

Arabidopsis RAV1 is down-regulated by brassinosteroid and may act as a negative regulator during plant development

RAV1 is a novel DNA-binding protein with two distinct DNA-binding domains unique in higher plants,but its role in plant growth and development remains unknown. Using cDNA array,we found that transcription of RAV1 is downregulated by epibrassinolide (epiBL) in Arabidopsis suspension cells. RNA gel blot analysis revealed that epiBL-regulated RAV1 transcription involves neither protein phosphorylation/dephosphorylation nor newly synthesized protein,and does not require the functional BRI1,suggesting that this regulation might be through a new BR signaling pathway.Overexpressing RAV1 in Arabidopsis results in a retardation of lateral root and rosette leaf development,and the underexpression causes an earlier flowering phenotype,implying that RAV1 may function as a negative regulatory component of growth and development.

Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development

Pollen germination on the surface of compatible stigmatic tissues is an essential step for plant fertilization. Here we report that the Arabidopsis mutant bcll is male sterile as a result of the failure ofpollen germination. We show that the bcll mutant allele cannot be transmitted by male gametophytes and no homozygous bcll mutants were obtained. Analysis of pollen developmental stages indicates that the bcll mutation affects pollen germination but not pollen maturation. Molecular analysis demonstrates that the failure of pollen germination was caused by the disruption of AtBECLIN 1. AtBECLIN 1 is expressed predominantly in mature pollen and encodes a protein with significant homology to Beclin1/Atg6/Vps30 required for the processes of autophagy and vacuolar protein sorting （VPS） in yeast. We also show that AtBECLIN 1 is required for normal plant development, and that genes related to autophagy, VPS and the glycosylphosphatidylinositol anchor system, were affected by the deficiency of AtBECLIN 1.

Characterization of a novel developmentally retarded mutant (drm1) associated with the autonomous flowering pathway in Arabidopsis

A developmentally retarded mutant (drm1) was identified from ethyl methanesulfonate (EMS)-mutagenized M2 seedsin Columbia (Col-0) genetic background. The drm1 flowers 109 d after sowing, with a whole life cycle of about 160 d.It also shows a pleiotropic phenotype, e.g., slow germination and lower germination rate, lower growth rate, curlingleaves and abnormal floral organs. The drm1 mutation was a single recessive nuclear mutation, which was mapped tothe bottom of chromosome 5 and located within a region of 20-30 kb around MXK3.1. There have been no mutantswith similar phenotypes reported in the literature, suggesting that DRM1 is a novel flowering promoting locus. Thefindings that the drm1 flowered lately under all photoperiod conditions and its late flowering phenotype was significantlyrestored by vernalization treatment suggest that the drm1 is a typical late flowering mutant and most likely associatedwith the autonomous flowering pathway. The conclusion was further confirmed by the revelation that the transcriptlevel of FLC was constantly upregulated in the drm1 at all the developmental phases examined, except for a very earlystage. Moreover, the transcript levels of two other important repressors, EMF and TFL1, were also upregulated in thedrm1, implying that the two repressors, along with FLC, seems to act in parallel pathways in the drm1 to regulateflowering as well as other aspects of floral development in a negatively additive way. This helps to explain why the drm1exhibits a much more severe late-flowering phenotype than most late-flowering mutants reported. It also implies that theDRM1 might act upstream of these repressors.

The Arabidopsis homologs of CCR4-associated factor 1 show mRNA deadenylation activity and play a role in plant defence responses

Messenger RNA （mRNA） turnover in eukaryotic cells begins with shortening of the poly （A） tail at the 3＇ end, a process called deadenylation. In yeast, the deadenylation reaction is predominantly mediated by CCR4 and CCR4- associated factor 1 （CAF1）, two components of the well-characterised protein complex named CCR4-NOT. We report here that AtCAF1a and AtCAF1b, putative Arabidopsis homologs of the yeast CAF1 gene, partially complement the growth defect of the yeast call mutant in the presence of caffeine or at high temperatures. The expression of At-CAF1a and AtCAFlb is induced by multiple stress-related hormones and stimuli. Both AtCAF1a and AtCAFlb show deadenylation activity in vitro and point mutations in the predicted active sites disrupt this activity. T-DNA insertion mutants disrupting the expression of AtCAF1a and/or AtCAF1b are defective in deadenylation of stress-related mRNAs, indicating that the two AtCAF1 proteins are involved in regulated mRNA deadenylation in vivo. Interestingly, the single and double mutants of AtCAF1a and AtCAFlb show reduced expression of pathogenesis-related （PR） genes PR1 and PR2 and are more susceptible to Pseudomonas syringae pv tomato DC3000 （Pst DC3000） infection, whereas transgenic plants over-expressing AtCAFla show elevated expression of PR1 and PR2 and increased resis-tance to the same pathogen. Our results suggest roles of the AtCAF1 proteins in regulated mRNA deadenylation and defence responses to pathogen infections.

Transcriptome-wide effect of Salix SmSPR1 in etiolated seedling of Arabidopsis

Microtubules and their regulatory proteins are involved in the regulation of plant cell morphology.SPIRAL1(SPR1),a plant-specific microtubule-binding protein,is critical in regulating the anisotropic growth of plant cells.Our previous study showed that overexpressed S alix SmS PR1 genes in Arabidopsis thaliana caused right-handed spiral elongation in etiolated seedlings,but there were no morphological differences between wild-type and transgenic seedlings under varied light conditions.We then studied the transcriptional regulation patterns in transgenic plants engineered with the S mSPR1 gene.Transcriptomic results showed that a large number of differentially expressed genes were involved in plant light signal reception,chlorophyll synthesis and photosystem structure.Eleven gene families with 42 photosynthesis-related genes and 6 light-responsive genes were involved in regulation of cell morphology.Our results showed that these genes in the SmSPR1-ox line were particularly down-regulated under dark conditions.In addition,33 TFs showed differences between S mSPR1-ox and wild-type lines.Taken together,the transcriptome analysis provides new insight into investigating the molecular mechanisms of light-induced cell morphological changes mediated by the microtubule binding protein SPR1.

Expression of the C-Terminal Domain of Mammalian TET3 DNA Dioxygenase in Arabidopsis thaliana Induces Heritable Methylation Changes at rDNA Loci

In plants, demethylation of 5-methylcytosine (5 mC) residues is controlled by DNA glycosylases, while in mammals it requires oxidation of 5 mC by TET proteins, a group of Fe(II)/2-oxoglutaratedependent dioxygenases. We analysed the effects of expressing the C-terminal catalytic domain of the human TET3 gene (TET3c) in Arabidopsis thaliana, using an rDNA region as a methylation reporter. In TET3c transformants, epialleles with hypomethylation or hypermethylation patterns can be induced, which is each stably retained in progeny lines even after removal of the TET3c transgene. In TET3c transformants, 5-hydroxymethylcytosine (5 hmC) marks are detected, indicative of the oxidative activity of the transgenic enzyme. 5-formylcytosine (5 fC) is only detectable in TET3c transformants with a DNA glycosylase mutant background suggesting further oxidation of 5 hmC residues to 5 fC by TET3c, and efficient recognition and removal of 5 fC by plant glycosylases. The results suggest that TET3c can be employed to induce heritable locus-specific changes in DNA methylation, and that accumulation of 5 hmC can be used as a marker for TET3c target regions.

Isolation and Characterization of an Arabidopsis Bushy and Dwarf Mutant

Apical dominance is a phenomenon that the growth of axillary meristems is inhibited by the primary shoot or inflorescence. Recent researches have begun to reveal the molecular mechanisms of apical dominance by isolating and identifying mutants with altered apical dominance. Here we report isolation of a bushy and dwarf 1 (bud1) mutant from Arabidopsis thaliana L. through a T-DNA tagging approach. The phenotypes of bul1 plants include loss of apical dominance, reduced plant size and dwarfism, suggesting that the bud1 mutant may be involved in auxin metabolism, transport or signalling. Using a reporter gene driven by an auxin-responsive promoter, we found that the expression pattern of auxin response element was altered in bud1. The auxin sensitivity and transport assay indicates that these two processes are normal in bud1. These results suggest that the bud1 phenotypes may result from an alteration in auxin metabolism. Genetic analysis demonstrates that bud1 is a semidominant mutant and cosegregates with a T-DNA insertion, which indicates that BUD1 gene could be cloned by iPCR approach.

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.

Identification of T-DNA Inserted Mutant Gene Transcription by Using Real-time PCR in Arabidopsis

AtERF4 （ethylene response factor） is a negative regulator in jasmonic acid mediated signal transduction pathway and ethylene mediated signal transduction pathway of Arabidopsis. It could respond to abscisic acid （ABA） and ethylene stimulus ATSYR1 gene encodes a syntaxin localizing at the plasma membrane in Arabidopsis, which can be induced by abiotic stress. To identify mutation lines for gene functional analysis, real-time PCR was employed to detect the expression level of AtERF4 and ATSYR1 in homozygous T-DNA insertion mutant line, respectively. Real-time PCR is a powerful tool which can be used to detect steady-state mRNA levels specifically, sensitively and reproducibly. Comparing to other forms of quantitative RT-PCR, the amount of amplified products can be detected by real-time PCR instantly and thus is a preferable alternative. In this study, RNA with T-DNA inserting into exon could be detected in AtERF4 knock-out mutation line. The results indicated that AtERF4 had been trucked in transcription level. On the other hand, T-DNA inserting into the promoter of gene ATSYR1 had no effect on reducing the expression level ofATSYR1 gene. Further molecular and phenotype studies now are ongoing to clarify the potential consequences of AtERF4 and ATSYR1 deficiency in Arabidopsis

灯盏花叶片数调控基因磷酸蛋白酶1的克隆与功能分析

【目的】探究灯盏花叶片数相关基因磷酸蛋白酶1(EbPP1)的功能,为揭示灯盏花叶片数目和开花时间分子机理奠定基础。【方法】从灯盏花叶片克隆EbPP1基因,将其构建至植物过表达载体RP101。用蘸花法侵染刚抽薹拟南芥植株进行异源表达,通过筛选获得若干株T_(1)、T_(2)、T_(3)代拟南芥阳性植株。最后统计T_(3)代阳性植株叶片数目和开花时间,通过实时荧光定量(RT-qPCR)检测EbPP1表达量,并测定拟南芥植株内源激素含量。【结果】成功克隆全长EbPP1基因,并构建该基因的重组过表达载体RP101-GFP-EbPP1。在拟南芥中实现稳定的遗传转化,于T_(3)代中获得24株阳性EbPP1转基因植株,并对T_(3)代阳性植株进行实时荧光定量分析,RT-qPCR结果显示转基因拟南芥中EbPP1基因过量表达,其表达量从野生型的1.034增加至136.330;转基因拟南芥在表型上呈现出叶片数增多、开花时间延迟的特征;通过LC-MS定量测定植株内源激素含量显示,T_(3)代转基因拟南芥中吲哚-3-甲酸(ICA)和赤霉素24(GA24)等多种激素的含量明显高于野生型,其中生长素前体物质L-色氨酸(TRP)尤为显著,达到野生型的3倍。【结论】本研究成功在拟南芥植株中异源表达EbPP1基因,与野生型相比,转基因拟南芥中叶片数增多,EbPP1基因表达量增高,内源激素含量升高。表明EbPP1可能通过调控植物激素水平,进而影响灯盏花叶片数目和开花时间。本研究为进一步解析灯盏花叶片数目发育相关机制和开花分子机理奠定基础,也为选育高品质灯盏花新品种提供一定遗传基础。

过表达VHA-c1基因对拟南芥根长及ABA与糖响应的影响

为初步探讨液泡H+-ATPase c亚基基因(VHA-c1)在植物生长发育及信号转导中的作用,该实验构建了拟南芥VHA-c1的过表达载体,转化并获得转基因拟南芥纯合株系,通过半定量RT-PCR技术分析了VHA-c1的表达量,然后对其进行暗培养、ABA和糖处理。结果显示:(1)该实验获得7个T2代转基因纯系,其mRNA表达水平均高于野生型,表明过表达载体的构建是有效的。(2)黑暗条件下,6个拟南芥VHA-c1过表达纯合株系的根长变短。(3)光照条件下,4个转基因株系主根伸长和子叶的展开以及7个转基因株系的种子萌发对ABA的抑制不敏感。(4)分别有5个和4个转基因株系的种子萌发对葡萄糖和蔗糖的抑制不敏感。推测VHA-c1可能参与了ABA和糖介导的信号转导途径,并可能影响了拟南芥根细胞的扩展。

报春PF sHSP 17-1基因转化拟南芥及耐热鉴定

根据灰岩皱叶报春PF sHSP 17-1热激蛋白基因序列,采用PCR法从差减文库中克隆得到该基因,并构建pCAMBIA1301-PF sHSP 17-1植物表达载体,通过根癌农杆菌介导的花器浸染法转化拟南芥,经潮霉素筛选、PCR和RT-PCR法鉴定后,对转基因植株和野生型对照进行高温胁迫,分析其耐热性差异。结果显示,在40℃、42℃、44℃处理下,转基因拟南芥的相对电导率和丙二醛含量升高的幅度均显著低于野生型对照,而脯氨酸含量的积累明显高于对照,可溶性蛋白的含量在热胁迫下也比对照稳定,仅有轻微下降。试验证明,PF sHSP 17-1基因对植物的耐热性有重要作用。

拟南芥P5CS1基因转化羽衣甘蓝增强耐盐性分析

【目的】分析转拟南芥△1-吡咯啉-5-羧酸合成酶(P5CS1)基因羽衣甘蓝的耐盐性,为获得较强的耐盐性羽衣甘蓝品种及其抗逆育种提供理论依据。【方法】将拟南芥P5CS1基因(AtP5CS1)经农杆菌介导转入羽衣甘蓝植物中,在盐胁迫下,分别检测转基因植株与野生型植株的AtP5CS1 mRNA表达量、幼苗脯氨酸含量、株系根系性状、整株干质量和鲜质量、叶片相对水含量、叶片电导率和整株存活率。【结果】在150 mmol/L NaCl胁迫下,转基因植株的P5CS1基因mRNA可正常表达,与对照相比,转基因株系Y1、Y2的主根和最长侧根长度较长,侧根数目较多,整株干质量和鲜质量较重;而且相对水含量显著高于对照植株(P<0.05,下同),脯氨酸含量及存活率均极显著高于对照植株(P<0.01),叶片相对电导率显著低于对照植株。【结论】转AtP5CS1基因植株的耐盐表型优于对照,即AtP5CS1基因在羽衣甘蓝中的表达明显改善了转基因植株的耐盐性。

光敏启动子控制的酵母Hem1基因在烟草中表达

拟南芥hemA1基因启动子是一种光响应型启动子,它控制着植物体内5-氨基乙酰丙酸(ALA)昼夜节律型合成.将该启动子与酿酒酵母Hem1基因构建的二价载体转入烟草中,获得转基因植株.以转二价基因的烟草T0代种子为材料,用不同浓度卡那霉素(Km)溶液浸种,结果显示,种子发芽率和子叶绿化率随着Km浓度升高而降低.将1 000 mg·L-1Km溶液中长出的162株抗性植株移植至盆钵中培养,GUS检测出的阳性比率为92%.用特异引物PCR法检测Km抗性-GUS阳性植株,发现含有拟南芥HemA1基因启动子的比率为92.5%,含有酿酒酵母Hem1基因的比率为88%,含有二价重组基因的比率为84.2%.RT-PCR检测表明,Hem1基因在黑暗中的表达量明显低于光照下,证明光敏启动子有效地控制了结构基因Hem1的表达.生理指标分析表明,与野生型相比,转基因植株ALA合成速率、叶绿素含量明显增加,叶绿素b/a比值提高.野生型植株基部叶片SPAD值显著降低,而转基因植株基部叶片SPAD值保持较高水平.以上结果说明,外源二价基因已经成功整合到烟草基因组中,并且能够在光照条件下过量表达,合成过量ALA.

拟南芥肌醇-1-磷酸合酶类蛋白基因的克隆表达

以野生型拟南芥总RNA为模板,逆转录PCR反应获得拟南芥肌醇-1-磷酸合酶类蛋白基因cDNA(AtMIPS1),开放读码框为1533 bp,编码510个氨基酸.与已报道物种MIPS基因氨基酸序列比较分析表明,AtMIPS1与植物MIPS基因的氨基酸同源性和相似性较高达86%～90%与95%～96%,并含有MIPS基因的保守区域'334SYNHLGNNDG'.将该cDNA序列不改变阅读框架克隆到pET28a(+)原核表达载体上,SDS-PAGE结果表明:在0.12g/L IPTG诱导2 h的条件下得到最佳的蛋白表达效果,AtMIPS1的成功表达为其功能研究打下基础.