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.

Construction of Plant Expression Vector of Heat Shock Factor Gene AtHsfA1a from Arabidopsis

[ Objective ] Heat shock factors （HSFs） are the major transcription factors of eukaryotic heat shock responses. This study aims to investigate the adversity stress tolerance functions of Arabidopsis heat shock factor AtHsfAla, which has important significance for in-depth understanding of adversity stress tolerance mechanisms of plants and further utilization of heat shock factor genes. [Method] Genomic DNA of Arabidopsis was extracted with CTAB method and purified to obtain Arabidopsis DNA samples for in vitro site-specific recombination cloning （ Gateway cloning） to construct plant expression vector of heat shock factor AtHs- fAla. Firstly, donor vector pDONR 201/AtHsfAla was constructed based on attB and attP site-specific recombination method （BP reaction）, to identify E. coli transformants harboring correct sequence of AtHsfAla by sequencing; secondly, plant expression vector pBTWG2/AttlsfAla overexpressing Arabidopsis heat shock factor AtHsfAla was constructed based on attL and attR site-specific recombination method （LR reaction）, to screen E. coli transformants harboring target plasmid. [ Result] Plant expression vector of Arabidopsis heat shock factor gene AtHsfAla was constructed successfully. [ Conclusion] This study not only provided experimental materials for acquiring transgenic plants overexpressing heat shock transcription factor AtHsfAla, but also laid the foundation for further investigation of the diversity of adversity stress tolerance functions reanlated by HSFs.

热激因子基因AtHSFa1a提高烟草的耐热性研究

克隆了拟南芥热激因子AtHSFa1a,并由热激启动子AtHSP70b控制,构建了双元表达载体pV-70bAtHSFa1a,并经农杆菌介导转化烟草,获得了转基因植株,并检测了其耐热性指标.结果显示:与对照相比,转基因烟草的电导率较低,丙二醛含量增幅较小,SOD酶活性较高,初步表明其对高温具有更高的耐受性.

热激因子AtHsfA1a突变对热胁迫中拟南芥抗坏血酸过氧化物酶的影响

【目的】探讨热激因子AtHsfA1a突变对热胁迫中拟南芥抗坏血酸过氧化物酶的影响。【方法】以T-DNA插入AtHsfA1a基因突变拟南芥及野生型植株为材料,采用分光光度法比较热胁迫下不同基因型植株的抗坏血酸过氧化物酶(APX,EC1.11.1.7)活性的变化,RT-PCR法研究AtHsfA1a对APX表达的影响,染色质免疫沉淀技术和凝胶阻滞电泳分析体内外研究AtHsfA1a与APX基因启动子区的结合情况。【结果】在热胁迫下AtHsfA1a基因突变拟南芥植株中的APX的活性低于野生型,且AtHsfA1a基因突变拟南芥植株中的APX的mRNA的相对量低于野生型;体内研究发现在热胁迫下AtHsfA1a基因突变拟南芥植株中未筛选出APX基因启动子区片段,而野生型筛选出APX基因启动子区片段,体外研究发现AtHsfA1a与APX基因启动子区的结合是直接的。【结论】热激因子AtHsfA1a突变对热胁迫中拟南芥APX的影响是从转录调控水平直接影响APX的表达。

拟南芥热激因子AtHsfA1a对细胞程序性死亡形态的影响

实验以热激因子At Hsf A1a不同基因型(野生型W和沉默型N)的拟南芥为材料,诱导其形成愈伤组织,将愈伤组织进行悬浮培养至单细胞,热激处理后,经过DAPI染色,在荧光显微镜下观察细胞涂片.结果表明,野生型拟南芥的单细胞均呈大小一致的完整圆形,且染色均匀,为正常的细胞形态.基因沉默型局部发生细胞质的浓缩,出现凋亡小体,说明拟南芥热激因子At Hsf A1a对细胞程序性死亡有一定的抑制作用.

ABA对拟南芥AtHsfA1a在冷胁迫响应中生理指标的影响

为从生理水平探讨低温下脱落酸(ABA)与拟南芥热激因子AtHsfA1a的关系,以拟南芥热激因子AtHsfA1a基因T-DNA插入突变型(MT)和野生型(WT)拟南芥为实验材料,研究外源ABA对AtHsfA1a不同基因型拟南芥在低温下的生理指标的影响.结果表明,低温可使幼苗中MDA和H2O2增加,AtHsfA1a基因突变型增加较明显,加入外源ABA后MDA和H2O2有所降低,AtHsfA1a基因突变型降低较少.而抗氧化酶变化趋势正好相反,突变型拟南芥的SOD、CAT、GR和APX活性在低温中比野生型升高小,ABA对冷胁迫中突变型拟南芥抗氧化酶SOD、CAT、GR和APX活性影响较小.说明外源ABA在AtHsfA1a提高抗冷性过程中有调控作用.