破坏光敏色素A改变生长素反应因子8的表达谱

拟南芥生长素反应因子8（auxin response factor8,ARF8）受光诱导表达,涉及光信号转导。为阐明光信号通过光受体传递给ARF8的过程和机理,首先利用半定量RT-PCR分析发现,PhyA的突变促进幼苗中ARF8的表达,而PhyB、Cry1和Cry2的突变对ARF8的表达没有明显影响。进而,利用GUS染色以及半定量和定量RT-PCR方法,系统分析PhyA基因的突变对ARF8基因表达的影响。结果表明,在黑暗、白光和远红光条件下,PhyA突变均明显提高拟南芥（Arabidopsis thaliana）幼苗子叶和叶片中ARF8的表达水平,并且明显降低其在幼苗下胚轴、茎尖和根尖中的表达水平。上述结果说明,PhyA基因的突变组成型地改变了ARF8基因的表达谱。然而,ARF8的突变并未明显改变PhyA的表达。说明在远红光信号通路中,ARF8位于PhyA的下游。     

拟南芥光敏色素A反义RNA载体的构建及转化

光敏色素A是植物远红光信号的关键受体,在植物光信号转导途径中起重要作用。一些具有重要功能的基因(如生长素反应因子8:auxin response factor 8,ARF8)受到PhyA调控。本实验拟通过构建PhyA基因反义株系,研究PhyA调控ARF8等重要功能基因表达的机理。以培养7 d的拟南芥幼苗为材料提取RNA,利用RT-PCR技术扩增出PhyA的全长CDS,将其反向插入表达载体pMD1得到反义表达载体pMD1-PhyA CDSR,并通过农杆菌介导转化拟南芥,经卡那霉素抗性平板筛选和PCR鉴定得到13个PhyA转基因株系。pMD1-PhyA CDSR及其转基因株系的获得为研究PhyA调控ARF8等基因表达的机理奠定了材料基础。     

拟南芥GH3.9基因的过表达及其表型分析

为研究GH3.9基因在植物生长发育过程中的作用,利用RT-PCR成功克隆到GH3.9基因,该基因全长为1 750bp。通过构建pEGAD-GH3.9过表达载体转化拟南芥,获得过表达GH3.9基因纯系转基因株系GH3.9ox-3和GH3.9ox-7。对拟南芥野生型(WT)和转基因株系(GH3.9ox-3和GH3.9ox-7)幼苗用不同光强和光质进行处理,结果显示:在蓝光、红光、远红光等不同光照强度下培养,过表达株系幼苗下胚轴的生长均明显受到抑制,且较野生型明显;采用不同光周期处理拟南芥幼苗,过表达幼苗下胚轴的伸长明显低于野生型;对成年植株表型进行观察,发现过表达株系植株矮小、雄蕊变短、果荚短小。研究表明:GH3.9基因参与了拟南芥生长发育调控,过表达GH3.9基因对拟南芥生长有抑制作用。     

拟南芥F-box基因At3g16740的表达分析

拟南芥At3g16740基因为F-box基因家族成员,其功能尚不清楚.通过连续和瞬时光照处理分析,发现蓝光、红光和远红光都诱导At3g16740基因的表达,其中远红光的诱导作用最明显.蓝光受体cry1、cry2,红光受体phyB或远红光受体phyA突变导致At3g16740基因表达的光诱导作用减弱或者消失,表明该基因为光信号通路相关基因.通过实时荧光定量PCR分析At3g16740基因在拟南芥不同组织器官中的表达,发现其在拟南芥根、茎、叶、花和果荚中都有表达,花和果荚中的表达量最高,推测该基因可能参与植物花和/或果荚的发育.酵母双杂交分析发现,At3g16740蛋白通过F-box结构域与拟南芥ASK(arabidopsis-SKP1-like)家族成员ASK1、ASK2和ASK11相互作用,表明At3g16740是SCF(Skp、Cullin、F-box)复合物的成员.     

胁迫相关基因CIPK14在PHYA介导抑制拟南芥远红光黄化苗转绿过程中的作用

本文对CBL互作蛋白激酶, CIPK14参与拟南芥光敏色素A介导的, 远红光黄化苗转绿抑制调控进行了研究. 结果发现, 拟南芥光敏色素A功能缺失突变体(phyA)远红光黄化苗(4天)转入白光处理后, 仅0.5 h叶片迅速转绿; 相同条件下, CIPK14基因插入失活突变体(cipk14)远红光黄化苗, 经过15 h白光处理之后叶片才开始转绿; 野生型(Col-4)远红光黄化苗转绿时间介于突变体phyA与cipk14之间. 基因表达分析表明, 上述不同基因型拟南芥远红光黄化苗转绿的快慢, 与原叶绿素酸酯还原酶基因表达量存在正相关性. 结合研究发现——CIPK14基因受到远红光调节, 并且表达具有时钟节律性认为, Ca²⁺调节蛋白CIPK14,可能在PhyA信号传导途径的上游分支介入PhyA介导的远红光黄化苗转绿抑制调控.     

GA2ox8基因过量表达诱导蓝光下拟南芥光形态建成

检测不同光照强度蓝光下,过量表达GA2ox8基因的转基因植株光形态建成表型的结果表明,突变体比各自的母本的下胚轴短,茎尖角度和子叶张开度较大,花青素和叶绿素的含量较高,并且其差异与GA2ox8基因的表达量呈正相关.RT-PCR检测光调节基因表达水平的结果显示,暗培养条件下其突变体幼苗中的水平比各自的母本高,蓝光下35S::GFP-GA2ox8-1.35S::GFP-GA2ox8-8与母本co1-4之间差异不明显,但scc7-D中的水平比母本crylcry2高.这似乎说明,GA2ox8基因过量表达可诱导蓝光下拟南芥幼苗光形态建成.     

红光下拟南芥光敏素调节基因的蛋白质鉴定与mRNA分析

光敏素是植物体内一种重要的光受体家族,它们可介导植物对外界红光和远红光的应答,在植物的生理发育过程中发挥重要作用.基因芯片分析结果表明,PHYA和PHYB在转导红光信号至光应答基因的过程中起关键作用.为了获得与PHYA,PHYB相关的光应答基因,本研究采用双向电泳技术比较分析了在持续红光下生长7天的拟南芥光敏素双突变体phyAphyB和野生型col-4幼苗的全蛋白图谱.采用基质辅助激光解吸飞行时间串联质谱（MALDI-TOF-TOF）进行肽质谱指纹图谱分析,成功鉴定到了32个差异蛋白点.选取其中的10个蛋白点,对应于10个不同的基因,进行了RT-PCR分析,并用Q-PCR对其中的2个基因表达情况进行了验证.结果表明,光敏素可能从mRNA水平或蛋白质水平来调节基因的表达.蛋白质组学分析为寻找光敏素依赖基因提供了新的途径。     

拟南芥QUA1基因在光信号途径中的表达与功能分析

光信号在植物生长发育过程中具有非常重要的作用。不同的光信号通过调节植物下游基因的表达,进而影响细胞分化、结构和功能的改变,以及组织和器官的形成,参与植物光形态建成。QUA1 (QUASIMODO1)是拟南芥糖基转移酶家族中的一个成员,参与植物细胞壁中果胶的合成。本文以拟南芥qua1-1/cry1以及qua1-1/phyB双突变体为材料,对QUA1基因在光信号途径中的功能进行了分析。结果显示,qua1-1突变体在暗、蓝光、红光以及远红外光培养条件下下胚轴的伸长均受到抑制,QUA1基因的表达同样受到光信号的调节,而且突变体中多种光信号调节基因的表达也受到了影响。通过对qua1-1突变体下胚轴的观察发现,突变体下胚轴表皮细胞长度明显变短。与cry1以及phyB突变体相比,qua1-1/cry1和qua1-1/phyB双突变体下胚轴长度明显变短,而且双突变体中光信号调节基因的表达也有明显变化,表明QUA1可能参与了CRY1以及PHYB介导的蓝光及红光信号传导。以上结果表明QUA1影响了下胚轴细胞的伸长以及光信号调节基因的表达,并参与调控多种光信号传导途径。     

拟南芥CKL3基因表达对非生物胁迫的响应

以拟南芥为材料,采用PCR和RT-PCR技术在DNA和RNA水平上鉴定出了与CKL3基因对应的T-DNA插入纯合突变体,并对其表型变化进行了观察.半定量RT-PCR检测CKL3基因在拟南芥不同器官和非生物胁迫响应中表达的结果表明,CKL3基因在根、花、叶中表达较高,在茎、叶柄中表达较弱;盐胁迫下CKL3基因表达下降,蓝光下CKL3基因表达升高,但热激和红光对此基因表达量的影响不大.     

GA20x8基因过量表达诱导蓝光下拟南芥光形态建成

检测不同光照强度蓝光下,过量表达GA20x8基因的转基因植株光形态建成表型的结果表明,突变体比各自的母本的下胚轴短,茎尖角度和子叶张开度较大,花青素和叶绿素的含量较高,并且其差异与GA20x8基因的表达量呈正相关。RT—PCR检测光调节基因表达水平的结果显示,暗培养条件下其突变体幼苗中的水平比各自的母本高,蓝光下35S：：GFP—GA20x8-1,35S：：GFP—GA20x8—8与母本col-4之间差异不明显,但scc7-D中的水平比母本crylcry2高。这似乎说明,GA20x8基因过量表达可诱导蓝光下拟南芥幼苗光形态建成。     

Genetic identification of FIN2, a far red light-specific signaling component of Arabidopsis thaliana

Phytochrome A (PhyA) mediates most, if not all various plant responses to far-red (FR) light. Here, we report a novel genetic mutation that impairs a variety of responses in the PhyA-signaling pathway of Arabidopsis thaliana . The mutation was isolated by screening seedlings that show reduced sensitivity to continuous far-red (FRc) light irradiation, but not to continuous red (Rc) light irradiation. The mutation named fin2–1 is not allelic to a PHYA mutation. Furthermore, immunoblot analysis indicated that the amount of the phytochrome A apoprotein in the fin2–1 mutant was comparable to that in wild type. Seedling of the fin2–1 mutant showed defects in hypocotyl growth inhibition and apical hook and cotyledon opening in FRc light but not in Rc light. The results showed that the mutation occurred in a downstream signaling component potentially specific to PhyA. Other PhyAmediated responses such as FR-preconditioned blocking of greening, anthocyanin accumulation, reduction of gravitropic response, and expression of the CAB and CHS genes were impaired by the fin2–1 mutation: the degree of the mutant effect on the responses was variable. However, FR light-mediated seed germination and photoperiodic flowering responses were not affected significantly in the mutant. These results showed that FIN2 defines an upstream branch point in the PhyA signaling pathway.     

拟南芥Atkin8a和Atkin8b基因表达特性

以拟南芥动蛋白(kinesin)kin-8家族的AtKin8a和AtKin8b这两个动蛋白基因作为研究对象,以组成型表达的肌动蛋白基因(Actin2)作为对照,利用半定量RT-PCR的方法,分析其在拟南芥各器官中的表达状况。结果表明:AtKin8a和AtKin8b基因主要在花器官中特异表达;随后克隆AtKin8a和AtKin8b基因启动子区域并与GUS基因融合,转基因植株花器官GUS染色表明:AtKin8a和AtKin8b基因的表达主要分别在胚珠、花药部位。由此推测它们可能分别在胚珠、花药发育过程中发挥作用。     

Functional and biochemical analysis of the N-terminal domain of phytochrome A

Phytochrome A (phyA) is a versatile plant photoreceptor that mediates responses to brief light exposures (very low fluence responses, VLFR) as well as to prolonged irradiation (high irradiance responses, HIR). We identified the phyA-303 mutant allele of Arabidopsis thaliana bearing an R384K substitution in the GAF subdomain of the N-terminal half of phyA. phyA-303 showed reduced phyA spectral activity, almost normal VLFR, and severely impaired HIR. Recombinant N-terminal half oat of PHYA bearing the phyA-303 mutation showed poor incorporation of chromophore in vitro, despite the predicted relatively long distance (>13 A) between the mutation and the closest ring of the chromophore. Fusion proteins bearing the N-terminal domain of oat phyA, beta-glucuronidase, green fluorescent protein, and a nuclear localization signal showed physiological activity in darkness and mediated VLFR but not HIR. At equal protein levels, the phyA-303 mutation caused slightly less activity than the fusions containing the wild-type sequence. Taken together, these studies highlight the role of the N-terminal domain of phyA in signaling and of distant residues of the GAF subdomain in the regulation of phytochrome bilin-lyase activity.     

Transgenic Bt cotton driven by the green tissue-specific promoter shows strong toxicity to lepidopteran pests and lower Bt toxin accumulation in seeds

A promoter of the PNZIP(Pharbitis nil leucine zipper)gene(1.459 kb)was cloned from Pharbitis nil and fused to the GUS(b-glucuronidase)and Bacillus thuringiensis endotoxin(Cry9C)genes.Several transgenic PNZIP::GUS and PNZIP::Cry9C cotton lines were developed by Agrobacterium-mediated transformation.Strong GUS staining was detected in the green tissues of the transgenic PNZIP::GUS cotton plants.In contrast,GUS staining in the reproductive structures such as petals,anther,and immature seeds of PNZIP::GUS cotton was very faint.Two transgenic PNZIP::Cry9C lines and one transgenic cauliflower mosaic virus(Ca MV)35S::Cry9C line were selected for enzyme-linked immunosorbent assay(ELISA)and insect bioassays.Expression of the Cry9C protein in the 35S::Cry9C line maintained a high level in most tissues ranging from24.6 to 45.5μg g~(-1) fresh weight.In green tissues such as the leaves,boll rinds,and bracts of the PNZIP::Cry9C line,the Cry9C protein accumulated up to 50.2,39.7,and 48.3μg g~(-1) fresh weight respectively.In contrast,seeds of the PNZIP::Cry9C line(PZ1.3)accumulated only 0.26μg g~(-1) fresh weight of the Cry9C protein,which was 100 times lower than that recorded for the seeds of the Ca MV 35S::Cry9C line.The insect bioassay showed that the transgenic PNZIP::Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm.The PNZIP promoter could effectively drive Bt toxin expression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds.These features should allay public concerns about the safety of transgenic foods.We propose the future utility of PNZIP as an economical,environmentally friendly promoter in cotton biotechnology.     

Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses

Phytochrome A signaling shows two photobiologically discrete outputs: so-called very-low-fluence responses (VLFR) and high-irradiance responses (HIR). By modifying previous screening protocols, we isolated two Arabidopsis mutants retaining VLFR and lacking HIR. Phytochrome A negatively or positively regulates phytochrome B signaling, depending on light conditions. These mutants retained the negative but lacked the positive regulation. Both mutants carry the novel phyA-302 allele, in which Glu-777 (a residue conserved in angiosperm phytochromes) changed to Lys in the PAS2 motif of the C-terminal domain. The phyA-302 mutants showed a 50% reduction in phytochrome A levels in darkness, but this difference was compensated for by greater stability under continuous far-red light. phyA-302:green fluorescent protein fusion proteins showed normal translocation from the cytosol to the nucleus under continuous far-red light but failed to produce nuclear spots, suggesting that nuclear speckles could be involved in HIR signaling and phytochrome A degradation. We propose that the PAS2 domain of phytochrome A is necessary to initiate signaling in HIR but not in VLFR, likely via interaction with a specific partner.     

Mechanism of gene expression of Arabidopsis glutathione S-transferase, AtGST1, and AtGST11 in response to aluminum stress

The gene expression of two Al-induced Arabidopsis glutathione S-transferase genes, AtGST1 and AtGST11, was analyzed to investigate the mechanism underlying the response to Al stress. An approximately 1-kb DNA fragment of the 5'-upstream region of each gene was fused to a beta-glucuronidase (GUS) reporter gene (pAtGST1::GUS and pAtGST11::GUS) and introduced into Arabidopsis ecotype Landsberg erecta. The constructed transgenic lines showed a time-dependent gene expression to a different degree in the root and/or leaf by Al stress. The pAtGST1::GUS gene was induced after a short Al treatment (maximum expression after a 2-h exposure), while the pAtGST11::GUS gene was induced by a longer Al treatment (approximately 8 h for maximum expression). Since the gene expression was observed in the leaf when only the root was exposed to Al stress, a signaling system between the root and shoot was suggested in Al stress. A GUS staining experiment using an adult transgenic line carrying the pAtGST11::GUS gene supported this suggestion. Furthermore, Al treatment simultaneously with various Ca depleted conditions in root region enhanced the gene expression of the pAtGST11::GUS in the shoot region. This result suggested that the degree of Al toxicity in the root reflects the gene response of pAtGST11::GUS in the shoot via the deduced signaling system. Both transgenic lines also showed an increase of GUS activity after cold stress, heat stress, metal toxicity, and oxidative damages, suggesting a common induction mechanism in response to the tested stresses including Al stress.