Plant growth and crop productivity are severely affected by abiotic stress on a global scale.WD40 repeat-containing proteins play a significant role in the development and environmental adaptation of eukaryotes.In thi...Plant growth and crop productivity are severely affected by abiotic stress on a global scale.WD40 repeat-containing proteins play a significant role in the development and environmental adaptation of eukaryotes.In this study,OsABT,a stress response gene,was cloned from rice(Oryza sativa L.cv.Nipponbare).OsABT encodes a protein containing seven WD40 domains.Expression analysis revealed that the OsABT gene was first up-regulated and then down-regulated following treatment with abscisic acid(ABA)and NaCl,but was down-regulated when treated with PEG8000.Subcellular localization results showed that OsABT was located in the cytoplasm and nucleus of Arabidopsis roots.OsABT transgenic Arabidopsis showed significantly increased tolerance to ABA and salt stress during plant seedling development.However,the transgenic lines were more sensitive to drought stress.Moreover,OsABT can interact with OsABI2,a component of ABA signaling pathway.These results showed that OsABT plays a positive regulatory role in response to salt stress and a negative role in response to drought stress in Arabidopsis.展开更多
植物免疫调控基因RTP5(Resistance to Phytophthora 5)编码一个含WD40结构域的蛋白,该基因负调控植物对疫霉的抗性。为获得沉默马铃薯中RTP5同源基因StRTP5a和StRTP5b的遗传材料,以便进一步探究StRTP5a和StRTP5b基因的生物学功能。以四...植物免疫调控基因RTP5(Resistance to Phytophthora 5)编码一个含WD40结构域的蛋白,该基因负调控植物对疫霉的抗性。为获得沉默马铃薯中RTP5同源基因StRTP5a和StRTP5b的遗传材料,以便进一步探究StRTP5a和StRTP5b基因的生物学功能。以四倍体马铃薯品种‘Désirée’的cDNA为模板,扩增到靶向StRTP5a和StRTP5b基因的DNA片段,通过Gateway技术将目的片段重组至植物表达载体pHellsgate12,构建同步靶向StRTP5a和StRTP5b基因的目的表达载体pHells12-StRTP5a/b i,然后利用根癌农杆菌介导的遗传转化方法将目的表达载体转化至‘Désirée’中。经PCR和qPCR分析后,获得8株2个目的基因同时沉默的马铃薯株系,转化株系中StRTP5a和StRTP5b基因表达量同时降幅最高达70%以上。展开更多
In this study, we isolated a WD40-repeat gene from Artemisia annua glandular trichomes. This gene shows 69.97% sequence similarity to Arabidopsis TTG1 at aminoacid level. Sub-cellular localization study shows that AaW...In this study, we isolated a WD40-repeat gene from Artemisia annua glandular trichomes. This gene shows 69.97% sequence similarity to Arabidopsis TTG1 at aminoacid level. Sub-cellular localization study shows that AaWD40 protein diffuses in both cell nucleus and cytosol. The correct nuclear localization of AaWD40 was observed when co-expressed with AabHLH, a putative A. thaliana AtTTG1 homologue cloned from Artemisia annua glandular trichomes. When AaWD40 gene was ectopically over expressed in Arabidopsis transparent testa glabrous1-1 (ttg1-1) mutants of A. thaliana, PAs production in seeds was restored, and the trichomeless phenotypes of mutant were rescued. Real-time PCR analysis results revealed that ETC1, CPC, TTG2 and BAN (the downstream targets of AtTTG1 depend on regulatory complex), which regulate the epidermal differentiation and anthocyanin biosynthesis were differentially expressed as a result of AaWD40 over expression. Furthermore, the CLV1, CLV2, CLV3 and WUS, which are required to maintain the stem-cell niche of Arabidopsis shoot apex, were also modulated by AaWD40 and Arabidopsis TTG1. The transcriptions of AP2/ERF, bHLH, MYB, WRKY and NACs family proteins, which are mostly involved in defense, stress response and development regulation, were remarkably modulated by AaWD40 over expression. We hypothesize that WD40 repeat proteins act as a crucial factor in regulating a wide variety of cellular functions in A. thaliana.展开更多
AIM: To investigate role of putative mitogen-activated protein kinase activator with WD40 repeats (MAWD)/ MAWD binding protein (MAWBP) in gastric cancer (GC). METHODS: MAWBP and MAWD mRNA expression level was examined...AIM: To investigate role of putative mitogen-activated protein kinase activator with WD40 repeats (MAWD)/ MAWD binding protein (MAWBP) in gastric cancer (GC). METHODS: MAWBP and MAWD mRNA expression level was examined by real-time reverse transcriptasepolymerase chain reaction and semi-quantitative polymerase chain reaction in six GC cell lines. Western blotting was used to examine the protein expression levels. We developed GC cells that stably overexpressed MAWBP and MAWD, and downregulated expression by RNA interference assay. Proliferation and migration of these GC cells were analyzed by 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), soft agar, tumorigenicity, migration and transwell assays. The effect of expression of MAWBP and MAWD on transforming growth factor (TGF)-β1-induced epithelialmesenchymal transition (EMT) was examined by transfection of MAWBP and MAWD into GC cells. We detected the levels of EMT markers E-cadherin, N-cadherin and Snail in GC cells overexpressing MAWBP and MAWD by Western blotting. The effect of MAWBP and MAWD on TGF-β signal was detected by analysis of phosphorylation level and nuclear translocation of Smad3 using Western blotting and immunofluorescence. RESULTS: Among the GC cell lines, expression of endogenous MAWBP and MAWD was lowest in SGC7901 cells and highest in BGC823 cells. MAWBP and MAWD were stably overexpressed in SGC7901 cells and knocked down in BGC823 cells. MAWBP and MAWD inhibited GC cell proliferation in vitro and in vivo . MTT assay showed that overexpression of MAWBP and MAWD suppressed growth of SGC7901 cells (P < 0.001), while knockdown of these genes promoted growth of BGC823 cells (P < 0.001). Soft agar colony formation experiments showed that overexpression of MAWBP and MAWD alone or together reduced colony formation compared with vector group in SGC7901 (86.25±8.43, 12.75±4.49, 30±6.41 vs 336.75±22.55, P < 0.001), and knocked-down MAWBP and MAWD demonstrated opposite effects (131.25±16.54, 88.75±11.12, 341.75±22.23 vs 30.25±8.07, P < 0.001). Tumorigenicity experiments revealed that overexpressed MAWBP and MAWD inhibited GC cell proliferation in vivo (P < 0.001). MAWBP and MAWD also inhibited GC cell invasion. Transwell assay showed that the number of traverse cells of MAWBP, MAWD and coexpression group were more than that in vector group (84±16.57, 98.33±9.8, 29±16.39 vs 298±11.86, P < 0.001). Coexpression of MAWBP and MAWD significantly decreased the cells traversing the matrix membrane. Conversely, knocked-down MAWBP and MAWD correspondingly promoted invasion of GC cells (100.67±14.57, 72.66±8.51, 330.67±20.55 vs 27±11.53, P < 0.001). More importantly, coexpression of MAWBP and MAWD promoted EMT. Cells that coexpressed MAWBP and MAWD displayed a pebble-like shape and tight cell-cell adhesion, while vector cells showed a classical mesenchymal phenotype. Western blotting showed that expression of E-cadherin was increased, and expression of N-cadherin and Snail was decreased when cells coexpressed MAWBP and MAWD and were treated with TGF-β1. Nuclear translocation of p-Smad3 was reduced by attenuating its phosphorylation. CONCLUSION: Coexpression of MAWBP and MAWD inhibited EMT, and EMT-aided malignant cell progression was suppressed.展开更多
基金supported by the Major Program of the Zhejiang Province for Food Crop Breeding(Grant No.2016C02050-6)the Key Program of Hangzhou Agricultural Scientific Research(Grant No.20191203B08)。
文摘Plant growth and crop productivity are severely affected by abiotic stress on a global scale.WD40 repeat-containing proteins play a significant role in the development and environmental adaptation of eukaryotes.In this study,OsABT,a stress response gene,was cloned from rice(Oryza sativa L.cv.Nipponbare).OsABT encodes a protein containing seven WD40 domains.Expression analysis revealed that the OsABT gene was first up-regulated and then down-regulated following treatment with abscisic acid(ABA)and NaCl,but was down-regulated when treated with PEG8000.Subcellular localization results showed that OsABT was located in the cytoplasm and nucleus of Arabidopsis roots.OsABT transgenic Arabidopsis showed significantly increased tolerance to ABA and salt stress during plant seedling development.However,the transgenic lines were more sensitive to drought stress.Moreover,OsABT can interact with OsABI2,a component of ABA signaling pathway.These results showed that OsABT plays a positive regulatory role in response to salt stress and a negative role in response to drought stress in Arabidopsis.
文摘植物免疫调控基因RTP5(Resistance to Phytophthora 5)编码一个含WD40结构域的蛋白,该基因负调控植物对疫霉的抗性。为获得沉默马铃薯中RTP5同源基因StRTP5a和StRTP5b的遗传材料,以便进一步探究StRTP5a和StRTP5b基因的生物学功能。以四倍体马铃薯品种‘Désirée’的cDNA为模板,扩增到靶向StRTP5a和StRTP5b基因的DNA片段,通过Gateway技术将目的片段重组至植物表达载体pHellsgate12,构建同步靶向StRTP5a和StRTP5b基因的目的表达载体pHells12-StRTP5a/b i,然后利用根癌农杆菌介导的遗传转化方法将目的表达载体转化至‘Désirée’中。经PCR和qPCR分析后,获得8株2个目的基因同时沉默的马铃薯株系,转化株系中StRTP5a和StRTP5b基因表达量同时降幅最高达70%以上。
文摘In this study, we isolated a WD40-repeat gene from Artemisia annua glandular trichomes. This gene shows 69.97% sequence similarity to Arabidopsis TTG1 at aminoacid level. Sub-cellular localization study shows that AaWD40 protein diffuses in both cell nucleus and cytosol. The correct nuclear localization of AaWD40 was observed when co-expressed with AabHLH, a putative A. thaliana AtTTG1 homologue cloned from Artemisia annua glandular trichomes. When AaWD40 gene was ectopically over expressed in Arabidopsis transparent testa glabrous1-1 (ttg1-1) mutants of A. thaliana, PAs production in seeds was restored, and the trichomeless phenotypes of mutant were rescued. Real-time PCR analysis results revealed that ETC1, CPC, TTG2 and BAN (the downstream targets of AtTTG1 depend on regulatory complex), which regulate the epidermal differentiation and anthocyanin biosynthesis were differentially expressed as a result of AaWD40 over expression. Furthermore, the CLV1, CLV2, CLV3 and WUS, which are required to maintain the stem-cell niche of Arabidopsis shoot apex, were also modulated by AaWD40 and Arabidopsis TTG1. The transcriptions of AP2/ERF, bHLH, MYB, WRKY and NACs family proteins, which are mostly involved in defense, stress response and development regulation, were remarkably modulated by AaWD40 over expression. We hypothesize that WD40 repeat proteins act as a crucial factor in regulating a wide variety of cellular functions in A. thaliana.
基金Supported by The National Bio-Tech 863 Program, No.2006AA02A402National Natural Science Foundation of China, No. 30901717
文摘AIM: To investigate role of putative mitogen-activated protein kinase activator with WD40 repeats (MAWD)/ MAWD binding protein (MAWBP) in gastric cancer (GC). METHODS: MAWBP and MAWD mRNA expression level was examined by real-time reverse transcriptasepolymerase chain reaction and semi-quantitative polymerase chain reaction in six GC cell lines. Western blotting was used to examine the protein expression levels. We developed GC cells that stably overexpressed MAWBP and MAWD, and downregulated expression by RNA interference assay. Proliferation and migration of these GC cells were analyzed by 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), soft agar, tumorigenicity, migration and transwell assays. The effect of expression of MAWBP and MAWD on transforming growth factor (TGF)-β1-induced epithelialmesenchymal transition (EMT) was examined by transfection of MAWBP and MAWD into GC cells. We detected the levels of EMT markers E-cadherin, N-cadherin and Snail in GC cells overexpressing MAWBP and MAWD by Western blotting. The effect of MAWBP and MAWD on TGF-β signal was detected by analysis of phosphorylation level and nuclear translocation of Smad3 using Western blotting and immunofluorescence. RESULTS: Among the GC cell lines, expression of endogenous MAWBP and MAWD was lowest in SGC7901 cells and highest in BGC823 cells. MAWBP and MAWD were stably overexpressed in SGC7901 cells and knocked down in BGC823 cells. MAWBP and MAWD inhibited GC cell proliferation in vitro and in vivo . MTT assay showed that overexpression of MAWBP and MAWD suppressed growth of SGC7901 cells (P < 0.001), while knockdown of these genes promoted growth of BGC823 cells (P < 0.001). Soft agar colony formation experiments showed that overexpression of MAWBP and MAWD alone or together reduced colony formation compared with vector group in SGC7901 (86.25±8.43, 12.75±4.49, 30±6.41 vs 336.75±22.55, P < 0.001), and knocked-down MAWBP and MAWD demonstrated opposite effects (131.25±16.54, 88.75±11.12, 341.75±22.23 vs 30.25±8.07, P < 0.001). Tumorigenicity experiments revealed that overexpressed MAWBP and MAWD inhibited GC cell proliferation in vivo (P < 0.001). MAWBP and MAWD also inhibited GC cell invasion. Transwell assay showed that the number of traverse cells of MAWBP, MAWD and coexpression group were more than that in vector group (84±16.57, 98.33±9.8, 29±16.39 vs 298±11.86, P < 0.001). Coexpression of MAWBP and MAWD significantly decreased the cells traversing the matrix membrane. Conversely, knocked-down MAWBP and MAWD correspondingly promoted invasion of GC cells (100.67±14.57, 72.66±8.51, 330.67±20.55 vs 27±11.53, P < 0.001). More importantly, coexpression of MAWBP and MAWD promoted EMT. Cells that coexpressed MAWBP and MAWD displayed a pebble-like shape and tight cell-cell adhesion, while vector cells showed a classical mesenchymal phenotype. Western blotting showed that expression of E-cadherin was increased, and expression of N-cadherin and Snail was decreased when cells coexpressed MAWBP and MAWD and were treated with TGF-β1. Nuclear translocation of p-Smad3 was reduced by attenuating its phosphorylation. CONCLUSION: Coexpression of MAWBP and MAWD inhibited EMT, and EMT-aided malignant cell progression was suppressed.
