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1.
In a search for genes that are induced in citrus cell suspension in response to salt stress, a cDNA clone with high homology to cotton Lea5 gene was isolated. Data base analysis of the protein deduced from the nucleotide sequence indicates that, like in cotton, the protein from citrus contains regions with significant hydropathic character. The gene, designated C-Lea5, is expressed in citrus leaves as well as cell suspension. The steady-state level of C-Lea5 is increased in cell suspension that is grown in the presence of 0.2 M NaCl. This phenomenon is also observed in leaves of citrus plants irrigated with NaCl and in citrus seedlings which are exposed to drought and heat stress. We suggest that the osmotic stress resulted from elevated level of salt is responsible for the increase in the level of C-Lea5. 相似文献
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Zanek MC Reyes CA Cervera M Peña EJ Velázquez K Costa N Plata MI Grau O Peña L García ML 《Plant cell reports》2008,27(1):57-66
Citrus psorosis is a serious viral disease affecting citrus trees in many countries. Its causal agent is Citrus psorosis virus (CPsV), the type member of genus Ophiovirus. CPsV infects most important citrus varieties, including oranges, mandarins and grapefruits, as well as hybrids and citrus
relatives used as rootstocks. Certification programs have not been sufficient to control the disease and no sources of natural resistance have been found.
Pathogen-derived resistance (PDR) can provide an efficient alternative to control viral diseases in their hosts. For this
purpose, we have produced 21 independent lines of sweet orange expressing the coat protein gene of CPsV and five of them were
challenged with the homologous CPV 4 isolate. Two different viral loads were evaluated to challenge the transgenic plants,
but so far, no resistance or tolerance has been found in any line after 1 year of observations. In contrast, after inoculation
all lines showed characteristic symptoms of psorosis in the greenhouse. The transgenic lines expressed low and variable amounts
of the cp gene and no correlation was found between copy number and transgene expression. One line contained three copies of the cp gene, expressed low amounts of the mRNA and no coat protein. The ORF was cytosine methylated suggesting a PTGS mechanism,
although the transformant failed to protect against the viral load used. Possible causes for the failed protection against
the CPsV are discussed. 相似文献
5.
Expression of bacterial chitinase protein in tobacco leaves using two photosynthetic gene promoters 总被引:2,自引:0,他引:2
Jonathan D. G. Jones Caroline Dean David Gidoni David Gilbert Diane Bond-Nutter Rino Lee John Bedbrook Pamela Dunsmuir 《Molecular & general genetics : MGG》1988,212(3):536-542
Summary A bacterial chitinase gene from Serratia marcescens (chiA) was fused to (i) a promoter of the ribulose bisphosphate carboxylase small subunit (rbcS) gene and (ii) two different chlorophyll a/b binding protein (cab) gene promoters from petunia. The resulting constructions were introduced into Agrobacterium Ti plasmid-based plant cell transformation vectors and used to generate multiple independent transgenic tobacco plants. ChiA mRNA and protein levels were measured in these plants. On average, the rbcS/chiA fusion gave rise to threefold more chiA mRNA than either cab/chiA fusion. We investigated the influence of sequences around the translational initiation ATG codon on the level of ChiA protein. The rbcS/chiA and cab/chiA fusions in which the sequence in the vicinity of the translational initiation codon is ACC ATGGC gave rise to transformants with higher levels of ChiA protein than those carrying a cab/chiA fusion with the sequence CAT ATGCG in the same region. This difference in translational efficiency is consistent with previous findings on preferred sequences in this region of the mRNA. In those transformants showing the highest level of ChiA expression, ChiA protein accumulated to about 0.25% of total soluble leaf protein. These plants contained significantly higher chitinase enzymatic activity than control plants. 相似文献
6.
Gholamreza Salehi Jouzani Irina V. Goldenkova Eleonora S. Piruzian 《Plant Cell, Tissue and Organ Culture》2008,92(3):321-325
The full-modified Bacillus thuringiensis cry3a (cry3aM) gene was designed and synthesized for effective expression in plants. A plant expression vector pC29RBCS-leader-cry3aM–licBM2 was constructed for potato transformation. In this vector, the cry3aM sequence was fused in reading frame with a new reporter gene (licBM2) and a leader sequence for the rbcs gene. The reporter gene encoded thermostable lichenase and the leader sequence encoded a signal peptide for transporting
protein product to chloroplasts. The vector contained the light-inducible promoter for rbcs gene isolated from Arabidopsis thaliana. Transgenic plants were obtained by Agrobacterium mediated transformation using microtuber explants. Transgenic plantlets were selected by kanamycin resistance and confirmed
as transgenic by PCR with specific primers, evaluation of lichenase activity, and bioassay of Colorado potato beetle neonate
larvae. Promoter activity assays under light induction (kinetic analysis) using lichenase activity and bioassay both showed
high and stable expression of hybrid genes in transgenic plantlets. Furthermore, the presence of lichenase as a reporter protein
in the composition of hybrid protein was shown to facilitate selection and analysis of the expression level of hybrid genes
in transgenic plants. 相似文献
7.
Guo W Duan Y Olivares-Fuster O Wu Z Arias CR Burns JK Grosser JW 《Plant cell reports》2005,24(8):482-486
Valencia orange [Citrus sinensis (L.) Osbeck] is the leading commercial citrus species in the world for processed juice products; however, the presence of thermostable pectin methylesterase (TSPME) reduces its juice quality. A long-term strategy of this work is to eliminate or greatly reduce TSPME activity in Valencia orange. Previous work resulted in the isolation of a putative TSPME gene, CsPME4, associated with a thermostable protein fraction of Valencia orange juice. To begin research designed to overexpress CsPME4 to verify the thermostability of the protein product and/or to downregulate the gene, a sense gene cassette containing a gene-specific sequence from a putative TSPME cDNA and the enhanced green fluorescent protein (GFP) as a selectable marker was constructed (M2.1). In the work reported here, M2.1 plasmid DNA was transformed (polyethylene glycol-mediated) into protoplasts isolated from an embryogenic suspension culture of Valencia somaclone line B6-68, in an effort to obtain transgenic Valencia lines. A vigorous transformed line was identified via GFP expression, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. One transgenic proembryo expressing GFP was recovered and multiple shoots were regenerated. The recovery of multiple transgenic plants was expedited by in vitro grafting. Polymerase chain reaction analysis revealed the presence of the PME gene in transgenic plants, and subsequent Southern blot analysis confirmed the presence of the eGFP gene. These transgenic plants show normal growth and minor morphological variation. The thermostability of PME in these plants will be assessed after flowering and fruit set. This is the first successful transfer of a target fruit-quality gene by protoplast transformation with recovery of transgenic plants in citrus. This method of transformation has the advantage over Agrobacterium-mediated transformation in that it requires no antibiotic-resistance genes. 相似文献
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Jing Fan Xue Gao Ying-Wu Yang Wei Deng Zheng-Guo Li 《Plant Molecular Biology Reporter》2007,25(3-4):145-153
A cDNA subtraction library had been constructed to identify differentially expressed genes in peel pitting of citrus fruit.
Based on the sequence of a cDNA fragment homologous to NAC gene family, the full-length cDNA of 1,203 nucleotides was cloned from “navel” orange by rapid amplification of cDNA ends.
It was designated as CsNAC, encoding a protein of 305 amino acids. The calculated molecular weight of the CsNAC protein was 35.2 kDa, and theoretical
isoelectric point was 6.72. Sequence comparison showed that the CsNAC protein had a strikingly conserved region at the N terminus,
which is considered as the characteristic of the NAC protein family. CsNAC protein was orthologous to Arabidopsis
thaliana ATAF1. Phylogenetic analysis confirmed CsNAC belonged to the ATAF subfamily, which plays an important role in response to
stress stimuli. RNA gel blot analysis showed that the expression of CsNAC gene was rapidly and strongly induced by stresses such as wounding and no oxygen. Low temperature (4°C) and exposure to ethylene
also increased the expression level of CsNAC gene. However, its expression was suppressed by high temperature (40°C) but not affected by low oxygen (3%). Our results
may provide the basis for future research of NAC-like gene’s role in stress-induced citrus peel pitting.
Sequence data of CsNAC from this article have been deposited at GenBank under accession number EF596736. 相似文献
9.
Systemic induction of a Phytolacca insularis antiviral protein gene by mechanical wounding, jasmonic acid, and abscisic acid 总被引:5,自引:0,他引:5
We have isolated a gene encoding a ribosome-inactivating protein (RIP) from Phytolacca insularis, designated as P. insularis antiviral protein 2 (PIP2). The PIP2 gene contained an open reading frame encoding a polypeptide of 315 amino acids. The deduced amino acid sequence of PIP2 was similar to those of other RIPs from Phytolacca plants. Recombinant PIP2 was expressed in Escherichia coli and was used to investigate its biological activities. Recombinant PIP2 inhibited protein synthesis in rabbit reticulocyte lysate by inactivating ribosomes through N-glycosidase activity. It also exhibited antiviral activity against tobacco mosaic virus (TMV). Expression of the PIP2 gene was developmentally regulated in leaves and roots of P. insularis. Furthermore, expression of the PIP2 gene was induced in leaves by mechanical wounding. The wound induction of the PIP2 gene was systemic. Expression of the PIP2 gene also increased in leaves in a systemic manner after treatment with jasmonic acid (JA) and abscisic acid (ABA), but not with salicylic acid (SA). These results imply that plants have employed the systemic synthesis of the defensive proteins to protect themselves more efficiently from infecting viruses. 相似文献
10.
Cold acclimation in plants is a polygenic phenomenon involving increased expression of several genes. The gene products participate either directly or indirectly towards increasing cold tolerance. Evidence of proteins having a direct effect on cold tolerance is emerging but limited. With isolated protoplasts from warm-grown kale (Brassica oleracea) as a model system, we tested protein fractions from winter bittersweet nightshade, Solanum dulcamara, stems for the presence of proteins that have a cryoprotective effect. Purification of one such fraction resulted in isolation of a 25 kDa protein. N-terminal Edman degradation amino acid sequence analysis showed that it has high homology to osmotin and osmotin-like proteins. When added to warm-grown protoplasts, it increased the cryosurvival of frozen-thawed protoplasts by 24% over untreated or BSA-treated controls at –8 °C. A cDNA library which was made in November from stems and leaves of S. dulcamara was successfully screened for the corresponding cDNA clone. The deduced amino acid sequence indicated that the protein consists of 206 amino acid residues including a N-terminal signal sequence and a putative C-terminal propeptide. The mature protein, without the N-terminal signal sequence, was expressed in Escherichia coli. The partially purified protein in the supernatant fraction of the culture medium had cryoprotective activity. 相似文献
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A tobacco cDNA clone encoding a GATA-1 zinc finger protein homologous to regulators of nitrogen metabolism in fungi 总被引:5,自引:0,他引:5
In higher plants, the expression of the nitrate assimilation pathway is highly regulated. Although the molecular mechanisms involved in this regulation are currently being elucidated, very little is known about the trans-acting factors that allow expression of the nitrate and nitrite reductase genes which code for the first enzymes in the pathway. In the fungus Neurospora crassa, nit-2, the major nitrogen regulatory gene, activates the expression of unlinked structural genes that specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein containing a single zinc finger motif defined by the C-X2-CX17-C-X2-C sequence. This DNA-binding domain recognizes the promoter region of N. crassa nitrogen-related genes and fragments derived from the tomato nia gene promoter. The observed specificity of the binding suggests the existence of a NIT2-like homolog in higher plants. PCR and cross-hybridization techniques were used to isolate, respectively, a partial cDNA from Nicotiana plumbaginifolia and a full-length cDNA from Nicotiana tabacum. These clones encode a NIT2-like protein (named NTL1 for nit-2-like), characterized by a single zinc finger domain, defined by the C-X2-C-X18-C-X2-C amino acids, and associated with a basic region. The amino acid sequence of NTL1 is 60% homologous to the NIT2 sequence in the zinc finger domain. The Ntl1 gene is present as a unique copy in the diploid N. plumbaginifolia species. The characteristics of Ntl1 gene expression are compatible with those of a regulator of the nitrate assimilation pathway, namely weak nitrate inducibility and regulation by light. 相似文献
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Molecular cloning and expression of the hot pepper ERabp1 gene encoding auxin-binding protein 总被引:3,自引:0,他引:3
Soon-Yong Choi 《Plant molecular biology》1996,32(5):995-997
The 22 kDa auxin-binding proteins in higher plants have received considerable attention as candidates for an auxin receptor. A cDNA clone Ca-ERabp1 of hot pepper (Capsicum annum) was isolated using the oligonucleotides as PCR primers. The cDNA codes for a polypeptide related to the major 22 kDa auxin-binding protein from maize and Arabidopsis ERabp1. The deduced amino acid sequence contains an endoplasmic reticulum retention signal, the KDEL sequence located at the C-terminal end, and has two possible auxin-binding sites, HRHSCE and YDDWSVPHTA conserved sequences. Northern hybridization analysis revealed that the Ca-ERabp1 gene is differentially expressed in total RNA isolated from different organs of a pepper plant, showing the highest level of expression in fruits but barely detectable in leaves and roots. 相似文献
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Summary Exposure of normal, tall rice (Oryza sativa) seedlings to 5-azacytidine, a powerful inhibitor of DNA methylation in vivo, induced both demethylation of genomic DNA and dwarf plants. Genes that had been affected by treatment were identified by differential screening of a cDNA library, and a ras-related gene, rgp1, was subsequently isolated. The cDNA of rgp1 was found to encode a deduced protein sequence of 226 amino acids with a relative molecular mass of 24850, which was most closely related to the ras-related ypt3 protein of fission yeast, Shizosaccharomyces pombe. The rgp1 protein, expressed in transformed Escherichia coli, clearly showed GTP-binding activity. During seedling growth, rgp1 expression was first observed 14 days after germination, reaching a maximum level between 28 and 42 days, and gradually decreased thereafter until 63 days when it attained the same level of expression as in 14-day-old seedlings. Expression of rgp1 was found to be markedly reduced throughout the growth period of both 5-azacytidine-induced dwarf plants and their progenies, relative to levels in untreated tall control plants. These results suggest that expression of rgp1 may be influenced, either directly or indirectly, by DNA methylation, and that the rgp1 protein may play an important role in plant growth and development. 相似文献
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Yu Jingjuan Peng Peng Zhang Xiujun Zhao Qian Zhy Dengyun Sun Xuehui Liu Junqi Ao Guangming 《Molecular breeding : new strategies in plant improvement》2004,14(1):1-7
The sb401 gene from potato (Solanum berthaultii) encoding a pollen-specific protein with high lysine content was successfully integrated into the genome of maize plants and its expression was correlated with increased levels of lysine and total protein content in maize seeds. A plasmid vector containing the sb401 gene under the control of a maize seed-specific expression storage protein promoter (P19z) was constructed and introduced into maize calli using microprojectile bombardment. The integration of the sb401 gene into the maize genome was confirmed by Southern blot analysis and its expression was confirmed by Western blot analysis. Quantification of lysine and protein content in R1 maize seeds showed that, compared to the non-transgenic maize control, the lysine content increased by 16.1% to 54.8%, and total protein content increased by 11.6% to 39.0%. There was no visible morphological change in vegetative parts and seeds of the transgenic maize plants. Lysine and protein analysis of the transgenic maize grains showed that the levels of lysine and total protein remained high for six continuous generations, indicating that the elevated lysine and total protein levels were heritable. These results indicate that the sb401 gene could be successfully employed in breeding programmes aimed at improving the nutritional value of maize. 相似文献
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Plant glutathione peroxidases 总被引:22,自引:0,他引:22
Yuval Eshdat Doron Holland Zehava Faltin Gozal Ben-Hayyim 《Physiologia plantarum》1997,100(2):234-240
Oxidative stress in plants causes the induction of several enzymes, including superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2). The first two are responsible for converting superoxide to H2 O2 and its subsequent reduction to H2 O, and the third is involved in recycling of ascorbate. Glutathione peroxidases (GPXs, EC 1.11.1.9) are a family of key enzymes involved in scavenging oxyradicals in animals. Only recently, indications for the existence of this enzyme in plants were reported. Genes with significant sequence homology to one member of the animal GPX family, namely phospholipid hydroperoxide glutathione peroxidase (PHGPX), were isolated from several plants. Cit-SAP, the protein product encoded by the citrus csa gene, which is induced by salt-stress, is so far the only plant PHGPX that has been isolated and characterized. This protein differs from the animal PHGPX in its rate of enzymatic activity and in containing a Cys instead of selenocysteine (Sec) as its presumed catalytic residue. The physiological role of Cit-SAP and its homologs in other plants is not yet known. 相似文献
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Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as −26°C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants. 相似文献
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为探讨龙眼(Dimocarpus longan)体胚CDC48基因的表达方式,采用RT-PCR和RACE方法,从龙眼胚性愈伤组织中克隆得到1条长度为2620 bp、含有完整开放阅读框的DlCDC48基因cDNA序列(GenBank登录号:EU606206)和长度为2418 bp的DNA序列(GenBank登录号:FJ590953)。DlCDC48编码1个含有805个氨基酸的蛋白质。DlCDC48基因不含内含子。生物信息学分析表明:DlCDC48蛋白为不具跨膜结构域的亲水性胞质蛋白,不具有信号肽,定位在细胞核;与其他植物的CDC48有较高的同源性。将DlCDC48基因构建成原核表达载体,经IPTG诱导表达了1个分子量约为89 kD的蛋白。利用实时荧光定量PCR(qPCR)技术,DlCDC48在龙眼体胚发育过程中的各个阶段均有表达,其中球形胚时的表达量最低,胚性紧实球形结构阶段的表达量最高。这为进一步研究CDC48基因在植物体胚发生中的作用奠定基础。 相似文献
19.
Madhumita?Joshi Chen?Niu Geraldine?Fleming Sulekha?Hazra Ye?Chu C.?Joseph?Nairn Hongyu?Yang Peggy?Ozias-Akins
Summary The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a
most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced
GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient
expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third
construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The
brightest and most dense fluorescent signals observed during transient expression were from p524EGFP. 1 and EYFP. Optimized
bombardment conditions consisted of 0.6 μm diameter gold particles, 12410 kPa bombardment pressure, 95 kPa vacuum pressure,
and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence
microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since
quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance
gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected
either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny. 相似文献
20.
Caspase-resistant VirD2 protein provides enhanced gene delivery and expression in plants 总被引:1,自引:0,他引:1
Reavy B Bagirova S Chichkova NV Fedoseeva SV Kim SH Vartapetian AB Taliansky ME 《Plant cell reports》2007,26(8):1215-1219
Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein
has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis
of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These
results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to
combat Agrobacterium infection.
Brian Reavy and Svetlana Bagirova contributed equally to this work. 相似文献