Pineapple translation factor SUI1 and ribosomal protein L36 promoters drive constitutive transgene expression patterns in Arabidopsis thaliana

The availability of a variety of promoter sequences is necessary for the genetic engineering of plants, in basic research studies and for the development of transgenic crops. In this study, the promoter and 5′ untranslated regions of the evolutionally conserved protein translation factor SUI1 gene and ribosomal protein L36 gene were isolated from pineapple and sequenced. Each promoter was translationally fused to the GUS reporter gene and transformed into the heterologous plant system Arabidopsis thaliana. Both the pineapple SUI1 and L36 promoters drove GUS expression in all tissues of Arabidopsis at levels comparable to the CaMV35S promoter. Transient assays determined that the pineapple SUI1 promoter also drove GUS expression in a variety of climacteric and non-climacteric fruit species. Thus the pineapple SUI1 and L36 promoters demonstrate the potential for using translation factor and ribosomal protein genes as a source of promoter sequences that can drive constitutive transgene expression patterns.     

采后菠萝黑心病诱导因素的研究

本文叙述了外源赤霉素ＧＡ３、低温条件对采后菠萝果实黑心病发展的影响．说明外源赤霉素ＧＡ３和低温条件都是诱导菠萝黑心病的重要因子。菠萝黑心病是一种生理失调症。实验中,未发现化学杀菌剂对此种病症显现出防治效果,而采后贮前４０℃高温处理２４小时．则可以减少和延迟此病的发生,因此．后者是一种可以应用于商品菠萝果实黑心病防治的好方法。     

低温打蜡对贮藏菠萝黑心病控制的作用

研究了常温、低温、低温加打蜡 3种贮藏条件下菠萝果实硬度、可溶性固形物 (TSS)、可溶性糖醛酸、多聚半乳糖醛酸酶 (PG)和多酚氧化酶 (PPO)等因素的变化 ,及它们对黑心病发生及发展情况的影响。结果表明 ,低温加打蜡可以延缓果肉硬度、可溶性固形物和可溶性糖醛酸含量的下降 ,降低 PG和 PPO的活性 ,降低黑心病的发病率 ,较好的保持果实品质     

Increased dehydrin promoter activity caused by HvSPY is independent of the ABA response pathway

A barley SPINDLY protein, HvSPY, is a negative regulator of gibberellin (GA) action. It is also found to be a positive regulator of the promoter of a barley dehydrin (Dhn) gene which is abscisic acid (ABA) upregulated. To investigate whether HvSPY acts through the ABA signaling pathway to upregulate the Dhn promoter, functional characterization was carried out by co-bombardment experiments. These experiments used Dhn promoter-GUS reporter constructs and an effector construct to overexpress HvSPY protein in barley aleurone. ABA dose-response experiments with and without HvSPY overexpression showed that the induction by HvSPY occurred in addition to the ABA effect. Gibberellic acid (GA3) did not reduce the induction by ABA, but it had a small, although significant, effect on the ability of HvSPY to upregulate. The induction of promoter activity of Dhn by HvSPY required the intact protein, and a small deletion in the tetratricopeptide repeat (TPR) region reduced this ability significantly. When a promoter region containing an element for ABA responsiveness was mutagenized or deleted, the mutant promoters lost ABA responsiveness but remained responsive to HvSPY. In addition, HvSPY did not increase promoter activities of other ABA-upregulated genes. Taken together, these results indicate that HvSPY and ABA both regulate promoter activity of Dhn, and that HvSPY acts independently of the ABA signaling pathway.     

Gibberellin-responsive elements in the promoter of a barley high-pI alpha-amylase gene.

Deletion analysis has previously shown that the major gibberellic acid (GA)- and abscisic acid (ABA)-responsive elements in the promoter of a high-pI alpha-amylase gene of barley are located downstream of -174 (Jacobsen and Close, 1991). We have used transient expression assays in barley aleurone protoplasts to identify sequences between -174 and +53 that confer GA and ABA responsiveness on expression of a beta-glucuronidase reporter gene. Using alpha-amylase promoter fragments and synthetic oligonucleotides fused to minimal promoters, we have shown that the hormone-responsive region is located between -174 and -108. A single copy of this region fused to a minimal alpha-amylase promoter (-41) conferred both GA- and ABA-responsive expression on the reporter gene comparable to the positive control, Am(-174)IGN. Multiple copies of this region were able to activate even greater levels of expression. Site-directed mutagenesis was used to determine the functional importance of the conserved motifs (-169pyrimidine box, -143TAACAAA box, and -124TATCCAC box) and nonconserved intervening sequences within the region between -174 and -108. Our results showed that both the TAACAAA and TATCCAC boxes play an important role in GA-regulated expression. We propose that the TAACAAA box is a gibberellin response element, that the TATCCAC box acts cooperatively with the TAACAAA box to give a high level of GA-regulated expression, and that together these motifs form important components of a gibberellin response complex in high-pI alpha-amylase genes. The TAACAAA box also appears to be the site of action of ABA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fusion genetic analysis of gibberellin signaling mutants

A fusion genetic strategy was used to identify gibberellin (GA) signaling mutants in transgenic Arabidopsis expressing the beta-glucuronidase (GUS) and firefly luciferase (LUC) reporter genes under control of the GA-responsive GASA1 promoter. Initial analyses determined the spatial and temporal patterns of reporter expression, and showed that reporter induction by GA was antagonized by ABA. gamma-Irradiated M2 progeny with altered reporter activities were identified by LUC bioimaging followed by GUS assays and northern hybridization of the endogenous GASA1 mRNA. Genetic analysis showed that three mutants, which overexpressed both reporters and endogenous GASA1, were caused by recessive (goe1 and goe2, for GASA over-expressed) and semi-dominant (goe3) mutations at different loci. These mutants are altered in their sensitivity to GA and the GA biosynthetic inhibitor paclobutrazol, and in the expression of several GA signaling related genes.     

Evaluation of heterologous promoters in transgenic Populus tremula × P. alba plants

The pattern and expression level of β-glucuronidase (gus) reporter gene regulated by six heterologous promoters were studied in transgenic Populus tremula × P. alba plants obtained by Agrobacterium-mediated transformation. Binary vector constructs used contained the following promoter sequences: the CaMV35S from cauliflower mosaic virus; its duplicated version fused to the enhancer sequence from alfalfa mosaic virus; CsVMV from cassava vein mosaic virus; ubiquitin 3 from Arabidopsis thaliana (UBQ3); S-adenosyl-L-methionine synthetase (Sam-s) from soybean; and the rolA from Agrobacterium rhizogenes. Histochemical staining of root, stem and leaf tissues showed phloem and xylem-specific gus expression under rolA promoter, and constitutive expression with the other putative constitutive promoters. Quantitative GUS expression of 10 – 15 independently transformed in vitro grown plants, containing each promoter, was determined by fluorimetric GUS assays. The UBQ3-gus fusion induced the highest average expression level, although an extensive variation in expression levels was observed between independent transgenic lines for all the constructs tested.     

低温与GA_3诱导菠萝黑心病的生理机制

菠萝黑心病是PPO催化氧化酚类物质形成褐色产物所致。低温或GA_3处理提高了PPO活性及其底物——儿茶酚、绿原酸和咖啡酸的含量,也导致了PAL活性增加;低温还使乙烯释放率增大。这些变化均有利于黑心病的发生和发展。     

菲油果LEAFY基因的表达模式及启动子克隆

LEAFY(简称LFY)是植物花分生组织特征基因,在植物由营养生长向生殖生长转变过程中起着重要作用,是启动开花的枢纽。菲油果是一种新兴的果树资源,为研究菲油果LFY基因(FsLFY)的表达调控规律,本研究通过实时荧光定量PCR技术研究了FsLFY基因的时空表达模式,并通过染色体步移技术克隆了该基因的启动子序列。荧光定量PCR结果表明,FsLFY基因在菲油果花蕾不同发育阶段以及其他组织器官中均有表达。在花蕾中,小蕾期最高,中蕾期最低;组织器官中,营养枝茎段最高,花瓣最低。FsLFY基因启动子序列长度为2436 bp(GenBank登录号:KF766536),运用PLACE、PlantCARE等在线软件对其序列进行顺式作用元件分析,结果显示该序列不仅含有CAAT-box、TATA-box等核心启动子元件,而且还具有响应水分、光、赤霉素(GA)以及其他功能未知的顺式调控元件,表明FsLFY基因的表达受多种外界环境条件的调控。本研究为阐明菲油果的开花机理,以及通过分子育种手段使菲油果早花早果奠定了理论基础。     

Mechanism of the Increase of Polyphenol Oxidase Activity in Pineapple Fruit Induced by GA3

No increase in polyphenol oxidase (PPO) activity was found by addition of GA3 solution in the enzyme extraction. But acid phosphatase activity was found to be increased in pineapple fruit (Armnas comosus (L.) Merr. ) treated with GA3, which was mainly caused by the significant increase in inorganic phosphate (Pi) content. Phospholipase D activities decreased gradually during the period of fruit ripening but GA3 treatment slowed down these processes. On the other hand, the increase of PPO activities in fruit treated with GA3 was enhanced by the addition of ABA treatment.     

水稻水孔蛋白RWC3的分布及其受GA和蔗糖的调控

水扎蛋白能介导水分的跨膜运输,在植物的生长发育过程中起重要作用.对RWC3启动子-GUS转基因水稻的组织化学染色表明,水稻(Oryza sativa L.)水孔蛋白RWC3可能在包括营养和生殖器官在内的各部位中广泛表达.同是发现,赤霉素(GA)能提高转基因植物的愈伤组织、悬俘细胞和叶片中的GUS活必性,而GA合成的抑制ancymidol降低了GUS活性.进一步研究发现,蔗糖能抑制GA对GUS活性的提高,说明GA和蔗糖在对RWC3表达调控的信号传递过程中可能存在着相互作用.     

A novel aspartic acid protease gene from pineapple fruit (Ananas comosus): Cloning,characterization and relation to postharvest chilling stress resistance

A full-length cDNA encoding a putative aspartic acid protease (AcAP1) was isolated for the first time from the flesh of pineapple (Ananas comosus) fruit. The deduced sequence of AcAP1 showed all the common features of a typical plant aspartic protease phytepsin precursor. Analysis of AcAP1 gene expression under postharvest chilling treatment in two pineapple varieties differing in their resistance to blackheart development revealed opposite trends. The resistant variety showed an up-regulation of AcAP1 precursor gene expression whereas the susceptible showed a down-regulation in response to postharvest chilling treatment. The same trend was observed regarding specific AP enzyme activity in both varieties. Taken together our results support the involvement of AcAP1 in postharvest chilling stress resistance in pineapple fruits.     

Distribution of Water Channel Protein RWC3 and Its Regulation by GA and Sucrose in Rice （Oryza sativa）

Water channel proteins facilitate water flux across cell membranes and play important roles in plant growth and development. By GUS histochemical assay in RWC3 promoter-GUS transgenic rice (Oryza sativa L. cv. Shenxiangjin 4), one of the members of water channel proteins in rice, RWC3, was found to distribute widely in variety of organs, from vegetative and reproductive organs. Further studies showed that gibberellin (GA) enhanced the GUS activity in the transgenic calli, suspension cells and leaves, whereas ancymidol (anc), an inhibitor of GA synthesis, reduced the GUS activity. Sucrose was found to inhibit the effects induced by addition of GA, suggesting a possible cross-talk between GA and sucrose signaling on regulation of the RWC3 gene expression.