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Isolation and functional identification of ERF transcription factors in wheat

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Isolation and functional identification of ERF transcription...

Isolation and Characterization of GmSTY1,a Novel Gene Encoding a Dual-Specificity Protein Kinase in Soybean（ Glycme max L.）

2010中国作物学会学术年会

作者： zhao-shi xu,ming chen,lian-cheng li and you-zhi ma~* (National Key Facility of Crop Gene Resources and Genetic Improvement(NFCRI),Key Laboratory of Crop Genetics and Breeding,Ministry of Agriculture,Institute of Crop Science,Chinese Academy of Agriculture Sciences(CAAS),Beijing 100081,China)

Abiotic and biotic stresses,such as drought,high-salinity and low-temperature,as well as pathogen and pest attack,have adverse effects on plant growth and crop *** survive,plants have evolved defense mechanisms to perceive signals from their surroundings and to appropriately respond to the different stresses by modulating the expression of responsive *** factors function pivotally in signal transduction to activate or suppress defense gene expression,as well as in the regulation of interaction between different signaling *** than 1,500 genes encode transcription factors in

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Journal of Integrative Plant Biology 2006年第7期48卷 857-866页

作者： zhao-shi xu you-zhi ma Xian-Guo cheng li-Xia Cao lian-cheng li ming chen Key Laboratory of Crop Genetics and Breeding Ministry of Agriculture Institute of Crop Science the Chinese Academy of Agricultural Sciences Beijing 100081 China

Phosphorylation of protein klnases has profound effects on their activity and interaction with other proteins. Tyroslne phosphorylation was reported to be involved in various physiological processes in plants; however, no typical receptor tyrosine kinase has been isolated from plants thus far. Dual-specificity kinases are potentially responsible for the phosphorylation of both tyrosine and serine/threonine of target proteins. A cDNA clone encoding a putative dual-specificity protein kinase was isolated by screening the cDNA GAL4 activation domain （AD） fusion library of soybean （Glycine max L.）, and its entire length was obtained using 5＇-rapid ampUflcatlon of cDNA ends. The predicted polypeptide of 330 amino acid residues, designated as GmSTY1, contains all 11 conserved subdomains, which share common characteristics with both the serine/ threonine and tyroslne protein klnases reported thus far. In addition, three potential N-linked glycosylation sites （NXS/T）, as well as phosphorylation motifs （SXXXS/T）, were observed, suggesting that GmSTY1 may be post-translationally modified. Furthermore, a potential N-myristoylation motif （MGARCSK） was found, suggesting that the GmSTY1 protein could associate with membranes in vivo. Southern blotting analysis revealed a single-copy of GmSTY1 in the genome. Northern blotting analysis showed that this gene was upregulated by drought and salt treatment in a time-dependent manner; however, exogenous abscisic acid （ABA） could not significantly affect the mRNA accumulation of GmSTY1. Interestingly, the transcript of this gene was remarkably downregulated by cold treatment during the early stages of the response, but upregulated later. These results Indicate that the protein kinase was possibly regulated by abiotic stresses in an ABA-independent pathway.

关键词： abiotic stress dual-specificity Glycine max , serine/threonine/tyrosine protein kinase.

W55a Encodes a Novel Protein Kinase That Is Involved in Multiple Stress Responses

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Journal of Integrative Plant Biology 2009年第1期51卷 58-66页

作者： zhao-shi xu li liu zhi-Yong Ni Pei liu ming chen lian-cheng li Yao-Feng chen you-zhi ma National Key Facility of Crop Gene Resources and Genetic Improvement (NFCRI) Key Laboratory of Crop Genetics and Breeding Ministry of Agriculture Institute of Crop Science the Chinese Academy of Agricultural Sciences (CAAS) Beijing 100081 China Agronomy College Northwest Sci-Tech University of Agriculture and Forestry Yangling 712100 China

Protein kinases play crucial roles in response to external environment stress signals. A putative protein kinase, W55a, belonging to SNFl-related protein kinase 2 （SnRK2） subfamily, was isolated from a cDNA library of drought-treated wheat seedlings. The entire length of W55a was obtained using rapid amplification of 5＇ cDNA ends （5＇-RACE） and reverse transcription-polymerase chain reaction（RT-PCR）. It contains a 1 029-bp open reading frame （ORF） encoding 342 amino acids. The deduced amino acid sequence of W55a had eleven conserved catalytic subdomains and one Ser/Thr protein kinase active-site that characterize Ser/Thr protein kinases. Phylogenetic analysis showed that W55a was 90.38% homologous with rice SAPK1, a member of the SnRK2 family. Using nullisomic-tetrasomic and ditelocentric lines of Chinese Spring, W55a was located on chromosome 2BS. Expression pattern analysis revealed that W55a was upregulated by drought and salt, exogenous abscisic acid, salicylic acid, ethylene and methyl jasmonate, but was not responsive to cold stress. In addition, W55a transcripts were abundant in leaves, but not in roots or stems, under environmental stresses. Transgenic Arabidopsis plants overexpressing W55a exhibited higher tolerance to drought. Based on these findings, W55a encodes a novel dehydration-responsive protein kinase that is involved in multiple stress signal transductions.

关键词： chromosomal location induction kinetics SnRK2 stress response transgenic Arabidopsis wheat.

Isolation and Expression Analysis of a Novel Abiotic Stress-Induced Gene W89 from Wheat

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Agricultural Sciences in China 2007年第4期6卷 391-398页

作者： ZHANG Rui-yue xu zhao-shi li lian-cheng chen ming ma you-zhi National Key Facility of Crop Gene Resources and Genetic Improvement Key Laboratory of Crop Genetics and Breeding Ministry of Agriculture/Institute of Crop Science Chinese Academy of Agricultural Sciences Beijing 100081 P.R.China

Water stress and cold stress are important factors restricting plant growth. However, there is little knowledge on the function of stress-responsive genes in plants. Therefore, it is necessary to clone some important genes to study the mechanism of plant adaptation to abiotic stress for improvement of plant resistance. A putative water stress-induced gene, W89, was cloned from the eDNA library of drought-treated wheat seedlings by phage hybridization in situ, and its entire length was obtained using 5＇-rapid amplification of eDNA ends （RACE） and reverse transcription-polymerase chain reaction （RT-PCR）. The full-length eDNA of W89 consists of 2 392 bp and contains a 1 896 bp open reading frame （ORF） encoding a 631 amino acid protein. Southern blot analysis indicated that W89 was a single-copy gene. RT-PCR analysis revealed that the expression of W89 was upregulated by drought, cold, and abscisic acid （ABA）. Amino acid sequence analysis discovered that W89 had a conserved region of DUF248 （pfam03141）, which contained a methyltransferase domain with a sterile alpha motif （SAM）-binding motif. Phylogenetic analysis showed that W89 was 66% identical to Oryza sativa dehydration-responsive protein （BAD67956）. It was supposed that W89 was a novel dehydration-responsive protein encoding gene. On the basis of the functions of methyltransferase and the SAM-binding motif, the SAM-binding motif of W89 was supposed to be connected with other proteins or transcription factors to transduce stress signals and finally regulate the expression of stress-responsive genes on the early stage of drought stress.

关键词： wheat water stress cold gene cloning

Isolation and Functional Analysis of the bZIP Transcription Factor Gene TaABP1 from a Chinese Wheat Landrace

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Journal of Integrative Agriculture 2012年第10期11卷 1580-1591页

作者： CAO Xin-you chen ming xu zhao-shi chen Yao-feng li lian-cheng YU Yue-hua liU Yang-na mayou-zhi National Key Facility for Crop Genetic Resources and Genetic Improvement Key Laboratory of Crop Genetics and BreedingMinistry of Agriculture/Institute of Crop Sciences Chinese Academy of Agricultural Sciences Crop Research Institute Shandong Academy of Agricultural Sciences College of Agronomy Northwest A&F University

In plants, basic leucine zipper （bZIP） transcription factors play important roles in regulatory processes, including stress response, pathogenic defense and light response as well as organ and tissue differentiation. Chinese wheat landrace Pingyaoxiaobaimai （PYXBM）, an original parent of drought tolerant wheat varieties grown in northern China, is significantly tolerant to abiotic stresses such as drought, cold and nutrient deficiencies. In order to isolate key stress-responsive genes and then improve stress tolerances of conventional varieties, a bZIP transcription factor gene was isolated from a cDNA library of drought-treated PYXBM using the in situ plaque hybridization method, and was designated as Triticum aestivum L. abscisic acid （ABA）-responsive element binding protein 1 （TaABP1）. It encodes 372 amino acids, and contains three conserved domains （C1-C3） in the N terminal and a bZIP domain in the C terminal which is a typical protein structure for the group member of bZIP family. Transcriptional activation analysis showed that TaABP1 activated the expression of downstream reporter genes in yeast without ABA application. TaABP1 protein fused with green fluorescent protein （GFP） demonstrated that the localization of TaABP 1 protein is in the nucleus. Expression pattern assays indicated that TaABP1 was strongly induced by ABA, high salt, low temperature and drought, and its expression was stronger in stems and leaves than in the roots of wheat. Furthermore, overexpression of TaABP1 in tobacco showed significant improvement of drought tolerance. Data suggested that TaABP1 may be a good candidate gene for improving stress tolerance of wheat by genetic transformation and elucidation of the role of this gene will be useful for understanding the mechanism underlying drought tolerance of Chinese wheat landrace PYXBM.

关键词： wheat expression pattern stress tolerance sub-cellular localization transcriptional activation

The NAC-like transcription factor Si NAC110 in foxtail millet(Setaria italica L.) confers tolerance to drought and high salt stress through an ABA independent signaling pathway

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Journal of Integrative Agriculture 2017年第3期16卷 559-571页

作者： XIE li-na chen ming MIN Dong-hong FENG Lu xu zhao-shi ZHOU Yong-bin xu Dong-bei li lian-cheng ma you-zhi ZHANG Xiao-hong College of Agronomy North west A &F University Yangling 712100 P.R. China National Key Facility for Crop Gene Resources and Genetic Improvement Ministry of Agriculture/Institute of Crop Sciences Chinese Academy of Agricultural Sciences Beijing 100081 P.R. China College of Sciences Northwest A&F University Yangling 712100 P.R. China

Foxtail millet（Setaria italica（L.）***）is a naturally stress tolerant *** to other gramineous crops,it has relatively stronger drought and lower nutrition stress tolerance *** date,the scope of functional genomics research in foxtail millet（*** L.）has been quite ***（NAM,ATAF1/2 and CUC2）-like transcription factors are known to be involved in various biological processes,including abiotic stress *** our previous foxtail millet（*** L.）RNA seq analysis,we found that the expression of a NAC-like transcription factor,SiNAC110,could be induced by drought stress;additionally,other references have reported that SiNAC110 expression could be induced by abiotic ***,we here selected SiNAC110 for further characterization and functional ***,the predicted SiNAC110 protein encoded indicated SiNAC110 has a conserved NAM（no apical meristem）domain between the 11–139 amino acid *** analysis then indicated that SiNAC110 belongs to subfamily III of the NAC gene *** localization analysis revealed that the SiNAC110-GFP fusion protein was localized to the nucleus in Arabidopsis *** expression profiling analysis indicated that expression of SiNAC110 was induced by dehydration,high salinity and other abiotic *** functional analysis using SiNAC110 overexpressed Arabidopsis plants indicated that,under drought and high salt stress conditions,the seed germination rate,root length,root surface area,fresh weight,and dry weight of the SiNAC110 overexpressed lines were significantly higher than the wild type（WT）,suggesting that the SiNAC110 overexpressed lines had enhanced tolerance to drought and high salt ***,overexpression of SiN AC110 did not affect the sensitivity of SiNAC110 overexpressed lines to abscisic acid（ABA）*** analysis of genes involved in proline synthesis,Na＋/K＋transport,drought responses,and aqueous transport proteins were higher in th

关键词： foxtail millet (Setaria italica (L.) NAC-like transcription factor drought stress high salt stress ABA-independent pathway

Analysis of Specific Binding and Subcellular Localization of Wheat ERF Transcription Factor W17

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Agricultural Sciences in China 2008年第6期7卷 647-655页

作者： zhao Yun-xiang liU Pei xu zhao-shi chen ming li lian-cheng chen Yao-feng XIONG Xiang-jin ma you-zhi Institute of Crop Science Chinese Academy of Agricultural Sciences/The National Key Facility for Crop Gene Resources and Genetic Improvement （NFCR1）/Key Laboratory of Crop Genetics and Breeding Ministry of Agriculture Beijing 100081 P.R.China Agronomy College Northwest A ＆ F University Yangling 712100 P.R.China

The study aims to detect the subcellular localization of ERF （ethylene-responsive element binding factor） transcription factor W17 protein, the interaction between W 17 and cis-acting regulatory elements GCC-box and DRE in vitro, the binding and transactivating ability in vivo, and the role of W17 in higher plant stress-signal pathway. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS 1000/He. Transformed cells were incubated for 24 h at 22℃ in the dark and green fluorescence was monitored under a confocal microscope. The gene W17 was fused N-terminus of GST （glutathione-S-transferase） in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 （DE3）. IPTG （0.5 mmol L-1） was added to induce the expression of recombinant GST/W17 for 3 h. The fused proteins were purified by GST purification columns, and then subjected to gel retardation assay with a 32p-labeled GCC or DRE sequence. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. The GFP fused with W17 protein was localized in the nuclei; SDS-PAGE assay demonstrated that the fused protein GST/W17 could be induced and purified with molecular weight at around 42.2 kD under the induction of IPTG. Purified fused protein was able to specifically bind to both the wild-type GCC-box and DRE element, but had no interaction with either the mutant DRE or GCC-box; W17 protein can bind to GCC-box and transactive downstream GUS gene in vivo. W17 can localize into the nuclei, and it may be involved not only in biotic stresses controlled by GCC-box, but also in abiotic stresses （e.g., salt-） induced signaling pathway.

关键词： ERF/AP2 domain ERF DRE element GCC-box subcellular localization

The G-Protein β Subunit AGB1 Promotes Hypocotyl Elongation through Inhibiting Transcription Activation Function of BBX21 in Arabidopsis

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Molecular Plant 2017年第9期10卷 1206-1223页

作者： Dong-bei xu Institute of Botany Jiangsu Province and Chinese Academy of Sciences No.1 Qianhu Houcun Zhongshanmen Wai Nanjing Jiangsu Province 210014 PR China Beijing Engineering Research Center for Hybrid Wheat Beijing Academy of Agriculture and Forestry Sciences Beijing 100097 China College of Agronomy Northwest A&F University Yangling Shaanxi 712100 China

Hypocotyl development in Arabidopsis thaliana is regulated by light and endogenous hormonal cues, mak- ing it an ideal model to study the interplay between light and endogenous growth regulators. BBX21, a B-box （BBX）-Iike zinc-finger transcription factor, integrates light and abscisic acid signals to regulate hypocotyl elongation in Arabidopsis. Heterotrimeric G-proteins are pivotal regulators of plant development. The short hypocotyl phenotype of the G-protein I^-subunit （AGB1） mutant （agbl-2） has been previously identified, but the precise role of AGB1 in hypocotyl elongation remains enigmatic. Here, we show that AGB1 directly interacts with BBX21, and the short hypocotyl phenotype of agbl-2 is partially suppressed in agb1-2bbx21-1 double mutant. BBX21 functions in the downstream of AGB1 and overexpression of BBX21 in agbl-2 causes a more pronounced reduction in hypocotyl length, indicating that AGB1 plays an oppositional role in relation to BBX21 during hypocotyl development. Furthermore, we demonstrate that the C-terminal region of BBX21 is important for both its intracellular localization and its transcriptional activation activity that is inhibited by interaction with AGB1. ChiP assays showed that BBX21 specifically associates with its own promoter and with those of BBX22, HY5, and GA2oxl. which is not altered in agbl-2. These data suggest that the AGB1-BBX21 interaction only affects the transcrip- tional activation activity of BBX21 but has no effect on its DNA binding ability. Taken together, our data demonstrate that AGB1 positively promotes hypocotyl elongation through repressing BBX21 activity.

关键词： Arabidopsis BBX21 G-protein 13 subunit hypocotyl elongation transcriptional activation

G-protein β subunit AGB1 positively regulates salt stress tolerance in Arabidopsis

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Journal of Integrative Agriculture 2015年第2期14卷 314-325页

作者： ma Ya-nan chen ming xu Dong-bei FANG Guang-ning WANG Er-hui GAO shi-qing xu zhao-shi li lian-cheng ZHANG Xiao-hong MIN Dong-hong ma you-zhi College of Agronomy Northwest A&F University/State Key Laboratory of Arid Region Crop Adversity Biology Institute of Crop Sciences Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement/Key Laboratory of Biology and Genetic Improvement of Triticeae CropsMinistry of Agriculture Beijing Hybrid Wheat Engineering and Technology Research Center

The heterotrimeric GTP-binding proteins（G-proteins） in eukaryotes consisted of α, β and γ subunits and are important in molecular signaling by interacting with G-protein-coupled receptors（GPCR）, on which to transduce signaling into the cytoplast through appropriate downstream effectors. However, downstream effectors regulated by the G-proteins in plants are currently not well defined. In this study, the transcripts of AGB1, a G protein β subunit gene in Arabidopsis were found to be down-regulated by cold and heat, but up-regulated by high salt stress treatment. AGB1 mutant（agb1-2） was more sensitive to high salt stress than wild-type（WT）. Compared with WT, the cotyledon greening rates, fresh weight, root length, seedling germination rates and survival rates decreased more rapidly in agb1-2 along with increasing concentrations of Na Cl in normal（MS） medium. Physiological characteristic analysis showed that compared to WT, the contents of chlorophyll, relative proline accumulation and peroxidase（POD） were reduced, whereas the malonaldehyde（MDA） content and concentration ratio of Na＋/K＋ were increased in agb1-2 under salt stress condition. Further studies on the expression of several stress inducible genes associated with above physiological processes were investigated, and the results revealed that the expressions of genes related to proline biosynthesis, oxidative stress response, Na＋ homeostasis, stress- and ABAresponses were lower in agb1-2 than in WT, suggesting that those genes are possible downstream genes of AGB1 and that their changed expression plays an important role in determining phenotypic and physiologic traits in agb1-2. Taken together, these findings indicate that AGB1 positively regulates salt tolerance in Arabidopsis through its modulation of genes transcription related to proline biosynthesis, oxidative stress, ion homeostasis, stress- and ABA-responses.

关键词： Arabidopsis heterotrimeric G-protein β subunit physiological processes salt stress tolerance