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subcellular localization of the S Locus F-box Protein AhSLF-S2 in Pollen and Pollen Tubes of Self-Incompatible Antirrhinum

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Journal of Integrative Plant Biology 2005年第1期47卷 76-83页

作者： Hong-YunWANG Yong-BiaoXUE InstituteofGeneticsandDevelopmentalBiology theChineseAcademyofSciencesBeijing100080China／／GraduateSchooloftheChineseAcademyofSciencesBeijing100039China InstituteofGeneticsandDevelopmentalBiology theChineseAcademyofSciencesBeijing100080China

The distribution of the S locus F-box (SLF) protein was examined by immunocytochemistry and Western blot techniques using an antibody against the C-terminal part of AhSLF-S2 in self-incompatible lines of Antirrhinum. Abundant gold particles were found where pollen tubes emerge in vitro. With the elongation of pollen tubes, binding sites for the antibody were found in the cytoplasm of the pollen tubes,including the peripheral part of the endoplasmic reticulum. After germination in vitro for 16 h, the product of AhSLF-S2 and possibly its allelic products could still be detectable, implying that the SLF protein has a role in the elongating process of pollen tubes. The present study provides evidence at the protein level that the SLF protein is present in pollen cytoplasm during pollen tube growth. These findings are discussed, as is their potential role in the self-incompatible response in Antirrhinum.

关键词： Antirrhinum pollen tube self-incompatibility S locus F-box protein subcellular localization

Activities, Quantitative Changes and subcellular localization of α-Amylase During Development of Apple Fruit

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Acta Botanica Sinica 2002年第1期44卷 34-41页

作者：王永章张大鹏中国农业大学果树分子发育实验室.植物生理学与生物化学国家重点实验室北京100094

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuberous roots, and fleshy fruit development. α_Amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was often shown extrachloroplastic in living cells. The present experiment showed that α_amylase activity was progressively increasing concomitantly with the decreasing starch concentrations during the development of apple ( Malus domestica Borkh cv. Starkrimson) fruit. The apparent amount of α_amylase assessed by Western blotting also increased during the fruit development, which is consistent with the seasonal changes in the enzyme activity. The enzyme subcellular_localization studies via immunogold electron_ microscopy technique showed that α_amylase visualized by gold particles was predominantly located in plastids, but the gold particles were scarcely found in other subcellular compartments. A high density of the enzyme was observed at the periphery of starch granules during the middle and late developmental stages. These data proved that the enzyme is compartmented in its functional sites in the living cells of the fruit. The predominantly plastid_distributed pattern of α_amylase in cells was shown unchanged throughout the fruit development. The density of gold particles (α_amylase) in plastids was increasing during the fruit development, which is consistent with the results of Western blotting. So it is considered that α_amylase is involved in starch hydrolysis in plastids of the fruit cells.

关键词： amylase subcellular localization apple fruit

Tissue-specific expression, developmentally and spatially regulated alternative splicing, and protein subcellular localization of OsLpa1 in rice

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2016年第2期17卷 100-109页

作者： Hai-ping LU Wei-qin PANG Wen-xu LI Yuan-yuan TAN Qing WANG Hai-jun ZHAO Qing-yao SHU State Key Laboratory of Rice Biology Institution of Crop Science Zhejiang University Hangzhou 310029 China Wuxi Hupper Bioseed Technology Academy Ltd. Wuxi 214000 China

The OsLpa1 gene（LOC_Os02g57400） was identified to be involved in phytic acid（PA） metabolism because its knockout and missense mutants reduce PA content in rice grain. However, little is known about the molecular characteristics of OsLpa1 in rice and of its homologues in other plants. In the present study, the spatial pattern of OsLpa1 expression was revealed using OsLpa1 promoter：：GUS transgenic plants（GUS： β-glucuronidase）; GUS histochemical assay showed that OsLpa1 was strongly expressed in stem, leaf, and root tissues, but in floral organ it is expressed mainly and strongly in filaments. In seeds, GUS staining was concentrated in the aleurone layers; a few blue spots were observed in the outer layers of embryo, but no staining was observed in the endosperm. Three OsLpa1 transcripts（OsLpa1.1, OsLpa1.2, OsLpa1.3） are produced due to alternative splicing; quantitative reversetranscriptase polymerase chain reaction（RT-PCR） analysis revealed that the abundance of OsLpa1.3 was negligible compared with OsLpa1.1 and OsLpa1.2 in all tissues. OsLpa1.2 is predominant in germinating seeds（about 5 times that of OsLpa1.1）, but its abundance decreases quickly with the development of seedlings and plants, whereas the abundance of OsLpa1.1 rises and falls, reaching its highest level in 45-d-old plants, with abundance greater than that of OsLpa1.2 in both leaves and roots. In seeds, the abundance of OsLpa1 continuously increases with seed growth, being 27.5 and 15 times greater in 28-DAF（day after flowering） seeds than in 7-DAF seeds for OsLpa1.1 and OsLpa1.2, respectively. Transient expression of chimeric genes with green fluorescence protein（GFP） in rice protoplasts demonstrated that all proteins encoded by the three OsLpa1 transcripts are localized to the chloroplast.

关键词： OsLpa1 Low phytic acid Expression pattern Alternative splicing subcellular localization

Influence of RNA interference on the mitochondrial subcellular localization of alpha-synuclein and on the formation of Lewy body-like inclusions in the cytoplasm of human embryonic kidney 293 cells induced by the overexpression of alphasynuclein

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Neural Regeneration Research 2012年第2期7卷 85-90页

作者： Tao Chen Xiaoping Liao Guoqiang Wen Yidong Deng Min Guo Zhigang Long Feng Ouyang Department of Neurology Hainan Provincial People＇s Hospital Haikou 570311 Hainan Province China

The specific and effective a-synuclein RNA interference （RNAi） plasmids, and the a-synuclein-pEGFP recombinant plasmids were co-transfected into human embryonic kidney 293 （HEK293） cells using the lipofectamine method. Using an inverted fluorescence microscope, a-synuclein proteins were observed to aggregate in the cytoplasm and nucleus. Wild-type a-synuclein proteins co-localized with mitochondria. Hematoxylin-eosin staining revealed round eosinophilic bodies （Lewy body-like inclusions） in the cytoplasm of some cells transfected with a-synuclein-pEGFP plasmid. However, the formation of Lewy body-like inclusions was not observed following transfection with the RNAi pSYN-1 plasmid. RNAi blocked Lewy body-like inclusions in the cytoplasm of HEK293 cells induced by wild-type a-synuclein overexpression, but RNAi did not affect the subcellular localization of wild-type a-synuclein in mitochondria.

关键词： RNA interference a-synuclein gene subcellular localization inclusion Parkinson'sdisease neural regeneration

Protein subcellular localization Prediction and Genomic Polymorphism Analysis of the SARS Coronavirus

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Tsinghua Science and Technology 2004年第4期9卷 384-390页

作者：季星来柳树群李岭孙之荣 Institute of Bioinformatics Department of Biological Sciences and BiotechnologyTsinghua University Beijing 100084 China Department of Medical Genetics China Medical University Shenyang 110001China

The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavi-rus (CoV). Several sequences of the complete genome of SARS-CoV have been determined. The subcellu-lar localization (SubLocation) of annotated open-reading frames of the SARS-CoV genome was predicted using a support vector machine. Several gene products were predicted to locate in the Golgi body and cell nucleus. The SubLocation information was combined with predicted transmembrane information to develop a model of the viral life cycle. The results show that this information can be used to predict the functions of genes and even the virus pathogenesis. In addition, the entire SARS viral genome sequences currently available in GenBank were compared to identify the sequence variations among different isolates. Some variations in the Hong Kong strains may be related to the special clinical manifestations and provide clues for understanding the relationship between gene functions and evolution. These variations reflect the evolu-tion of the SARS virus in human populations and may help development of a vaccine.

关键词： severe acute respiratory syndrome (SARS) subcellular localization viral life cycle genomic polymorphism

Expression, subcellular localization and protein-protein interaction of four isoforms of EcR/USP in the common cutworm

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Insect Science 2015年第1期22卷 95-105页

作者： Li-Xia Huang Yan-Jun Gong Jun Gu Bao-Juan Zeng Li-Hua Huang Qi-Li Feng Laboratory of Molecular and Developmental Entomology Guangdong Provincial Key Laboratory of Biotechnology for Plant DevelopmentSchool of Life Sciences South China Normal University Guangzhou China

Ecdysone receptor （EcR） and ultraspiracle （USP） form heterodimers to mediate ecdysteroid signaling during molting and metamorphosis. Various EcR/USP heterodimers have been reported. However, it is unclear what kind of EcR/USP combination is adopted by lepidopteran insects during the larval-pupal metamorphosis and whether the EcR/USP heterodimer varies among different tissues. To address these questions, two isoforms of each EcR and USP were cloned from the common cutworm, their messenger RNA expression patterns were examined by real-time quantitative polymerase chain reaction in different tissues during the larval-pupal metamorphosis and in the midgut in response to hormonal induction. Furthermore, their subcellular localization and protein-protein interaction were explored by transient expression and far-western blotting, respectively. All the four genes were significantly up-regulated in prepuae and/or pupae. The expression profiles of EcRB1 and USP1 were nearly identical to each other in the epidermis, fat body and midgut, and a similar situation also applied to EcRA and USP2. The three genes responded to 20-hydroxyecdysone （20E） induction except for USP2, and USP1 could be up-regulated by both 20E and juvenile hormone. The four proteins mainly localized in the nucleus and the nuclear localization was promoted by 20E. The protein-protein interaction between each EcR and USP was found in vitro. These results suggest that two types of EcR/USP heterodimer （EeRA/USP2 and EcRB 1/USP1） may exist simultaneously in the common cutworm, and the latter should play more important roles during the larval-pupal metamorphosis. In addition, the types of EcR/USP heterodimer do not vary in the tissues which undergo histolysis and regeneration during metamorphosis.

关键词： ecdysone receptor protein interaction subcellular localization ultraspiracle

Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

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World Journal of Gastroenterology 2006年第47期12卷 7649-7653页

作者： Li-Cheng Dai Di-Yong Xu Xing Yao Li-Shan Min Ning Zhao Bo-Ying Xu Zheng-Ping Xu Yong-Liang Lu Huzhou Key Laboratory of Molecular Medicine Huzhou Central Hospital Huzhou 313000 Zhejiang Province China Department of Research Center for Environmental Genomics Zhejiang University School of Medicine Hangzhou 310031 Zhejiang Province China Huzhou Teachers College Huzhou 313000 Zhejiang Province China

AIM： To construct an expression plasmid encoding human wild-type midkine （MK） and enhanced green fluorescence protein （EGFP） fusion protein （MK-EGFP）, and to analyze the subcellular localization of MK in different cardnoma cell lines. METHODS： Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells, respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected. RESULTS： Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines. CONCLUSION： This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

关键词： Midkine subcellular localization Laser scanning confocal microscopy

Expression and subcellular localization of cysteine- and glycine-rich protein-2 in rat undifferentiated olfactory stem cells

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Neural Regeneration Research 2008年第5期3卷 558-560页

作者： Xue Gao Zengyu Cao Jun Chen Shunli Liu Dingjun Zha Feng Wang Yang Chen Li Qiao Lianjun Lu Jianhua Qiu Department of Otorhinolaryngology Head and Neck Surgery Xijing Hospital the Fourth Mifitary Medical University Xi＇an 710032 ShaanxiProvince China

BACKGROUND： As a member of the LIM protein family Ⅱ, cysteine- and glycine-rich protein-2 （CRP2） has been demonstrated to play a role in the regulation of growth and differentiation of eukaryotic cells. Our previous study has demonstrated that CRP2 can be detected in the embryonic rat inner ear but not in the adult rat inner ear. However, at present, the expression of LIM protein family H members in stem or precursor cells has not been described. OBJECTIVE： To determine the expression and sub-cellular localization of CRP2 in olfactory stem cells. DESIGN, TIME AND SETTING： An experiment with repeated measures was performed in the Laboratory of Otorhinolaryngology, Head and Neck Surgery, Xijing Hospital, the Fourth Military Medical University from February 2008 to April 2008. MATERIALS： Olfactory stem cells, and rabbit-anti-CRP2 polyclonal antibody were prepared and kept in our laboratory. METHODS： Reverse transcription polymerase chain reaction and Western blot analysis were used to detect expression of CRP2 in olfactory stem cells. Immunocytochemistry was also used to localize CRP2 in olfactory stem cells. MAIN OUTCOME MEASURES： The expression and sub-cellular localization of CRP2 in rat olfactory stem cells. RESULTS： CRP2 expression was found in olfactory stem cells, and CRP2 was distributed in both the nucleus and the cytoplasm. CONCLUSION： Confirmation of the expression and distribution of CRP2 in olfactory stem cells.

关键词： cysteine- and glycine-rich protein-2 LIM domain subcellular localization olfactory stem cells

An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts

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Horticultural Plant Journal 2023年第3期9卷 425-436页

作者： Wenhui Yang Jiaqin Ren Wanrong Liu Dan Liu Kaidong Xie Fei Zhang Pengwei Wang Wenwu Guo Xiaomeng Wu National Key Laboratory for Germplasm Innovation&Utilization of Horticultural Crops College of Horticulture and Forestry SciencesHuazhong Agricultural UniversityWuhanHubei 430070China Hubei Hongshan Laboratory WuhanHubei 430070China

Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol(PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNAgRNA(inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

关键词： Citrus Callus protoplast Transient transfection subcellular localization Genome editing