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Purification, Biological Activities, and molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb （Amaryllidaceae）

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Journal of Integrative Plant Biology 2006年第2期48卷 223-231页

作者： Chuan-Fang Wu Jian Li Jie An Li-Qing Chang Fang Chen Jin-Ku Bao College of Life Sciences Sichuan University Chengdu 610064 China

A novel mannose-bindlng aggiutinln was purified from bulbs of Zephyranthes candida Herb by extraction, precipitation with 80% （NH4）2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel flitration on Sephscryl S-100. The purified Z. candida agglutlnln （ZCA） migrated as a single band of 12 kDa on sodium dodecyi suifate-poiyecryiamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the iectln, as datermlned by gel filtration chromatography, was 48 kDa. The results Indicated that ZCA was composed of four Identical subunlts of 12 kDa each （homotetramerlc nature）. The ZCA agglutlhated rabbit erythrocytes, Escherichla coil and Saccharomyces cerevislae ceils at concentrations of 0.95, 1.90, and 31.30 μg/mL, respectively. Bloassays Indicated that ZCA has a significant effect on wheat aphid survival. Mortality after 7 d was 〉 90% at 0.26%. A degenerate primer was designed In accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3＇- and 5＇-rapid amplification of cDNA ends. The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA Includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa. The result show that the zca gene encodes a protein precursor with a signal peptlde, a mature protein, and a C-terminal cleavage amino acids sequence. molecular modeling of ZCA Indicated that Its three-dimensional atructure strongly resembies that of the snowdrop aggiutinin. Blocks＇ analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes （QDNY）.

关键词： agglutinating activity insecticidal activity molecular clone sequence analysis Zephyranthes candida agglutinin.

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Cloning and Identification of A Novel Variant of Human Vascular Endothelial Growth Factor

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2001年第3期21卷 191-194页

作者：过健俐屈伸 Department of Biochemistry School of Basic Medical Sciences Tongji Medical College Huazhong University of Sciences and Technology Wuhan 430030

A novel variant of human vascular endothelial growth factor (h'VEGF165) cDNA was amplified by nested PCR method from the HL60 1 cells and was cloned into a eukaryotic expressing vector pcDNA 3 to construct a recombinant plasmid pCD-h'VEGF165. The amplified h'VEGF165 cDNA fragment was identified by enzyme digestion and DNA sequencing methods. Also, wild-type hVEGF165 cDNA was obtained, identified and cloned into a eukaryotic expressing vector pcDNA3 by using the same methods. The results of DNA sequencing showed that h'VEGF165 cDNA cloned from HL60 1 was 600 bp in size with 8 % of the base sequence in h'VEGF165 cDNA being changed as compared with the base sequence in the wild-type hVEGF165 cDNA. The results of sequencing of hVEGF165 which was cloned from HL60 by us were consistent with the reports completely.

关键词： human vascular endothelia growth factor nested PCR variant molecular clone

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molecular Cloning and Expression Analysis of a Hexokinase Gene,MdHXK1 in Apple

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Horticultural Plant Journal 2016年第2期2卷 67-74页

作者： ZHAO Jin SUN Meihong HU Dagang HAO Yujin State Key Laboratory of Crop Biology MOA Key Laboratory of Horticultural Crop Biology(Huanghuai Region) and Germplasm Innovation College of Horticulture Science and Engineering Shandong Agricultural University

A hexokinase gene named MdHXK1(MDP0000309677) was cloned from ‘Gala' apple(Malus × domestica Borkh.). Sequence analysis showed that the MdHXK1 gene was 1 497 bp long and encoded 499 amino acids. The predicted molecular mass of this protein was 54.05 kD, and the pI was 5.76. A phylogenetic tree indicated apple MdHXK1 exhibited the highest sequence similarity to Pyrus bretschneideri PbHXK1. Analysis of the functional domain showed that the MdHXK1 protein included two conserved kinase domains. The prediction of subcellular localization suggested that the MdHXK1 protein was mainly localized in the cytoplasm. There was an indication that MdHXK1 existed as one copy in the apple genome by Southern blotting. Silico analysis suggested that the promoter sequence contained several typical cis-acting elements, including defense, sugar signaling and phytohormone responsive elements. Quantitative real-time PCR analysis demonstrated that the MdHXK1 gene was mainly expressed in stem and flower tissues. During the development of apple fruits, the expression of the MdHXK1 gene initially increased and then decreased. The changes on Glc phosphorylation relative activity and glucose concentration showed the same trend. In addition, the expression of this gene was induced by salt stress, low temperature, and abscisic acid(ABA). Finally, we obtained and purified the fused MdHXK1 protein by recombinant prokaryotic expression. Studies have demonstrated that MdHXK1 may participate in sugar metabolism in apple fruits. Enzyme encoded by MdHXK1 is a key factor in the mediation of sugar accumulation. Recently,researchers on hexokinase at home and abroad mainly focused on model plants, such as Arabidopsis, tobacco and rice, but orchard fruit like apple were underresearched. Our research established the foundation for the further study of the functions of MdHXK1.

关键词： apple hexokinase MdHXK1 molecular clone expression analysis prokaryotic expression

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Identification and characterization of three FT-like genes in sugarcane

Identification and characterization of three FT-like genes i...

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第十九届中国作物学会学术年会

作者： Lin Xiuqin Lu Xin Li Xujuan Mao Jun Liu Hongbo Zi Qiuyan Liu Xinliong Sugarcane Research Institute of Yunnan Agriculture Science Academy

FLOWERING LOCUS T(FT) is an important of flowering genetic pathways and plays crucial roles in flowering transition, which encode proteins with high similarity to phosphatidy1 ethanolamine-binding proteins, function as flowering promoters or repressor. The sugarcane(Saccharum spp.) is a valuable sugar and biofuel crop, but the molecular mechanisms controlling flowering are still unclear. In this study, three FT-like genes, designated as ScFT-1, ScFT-2 and ScFT-3, were isolated and characterized from sugarcane cultivated variety(YueTang93-159). The FT-like genes display a common exon-intron organization. Sequence comparisons and phylogenetic analysis with homologous genes from other plant species group sugarcane FT genes into two groups: ScFT-1 and ScFT-2 were cluster into FT-like group Ⅰ, ScFT-3 were cluster into FT-like group Ⅱ. Expression analysis indicates differences in the expression patterns of these three genes at the temporal and spatial levels. ScFT-1, the sugarcane FT homolog, is induce expressed in the mature leaf of inflorescence initiation stage and booting stage, it was mainly related to photoperiod sensitivity of sugarcane, and could be a florigen gene of sugarcane, function as flowering promoter;ScFT-2, is mainly expressed in spica, it may related to sugarcane floral development;ScFT-3, is mainly expressed in the mature leaf of sugarcane vegetative growth, and it’s expression level was down regulation in flower transition stage, it may acts as flowering inhibitor. The differential expression of he FT-like gene family in saccharum suggests that they plays multifaceted roles in plant growth and development. Ectopic over-expression of ScFT-2 in Arabidopsis caused very early flowering in transgenic Arabidopsis plants. Over-expression of ScFT-1, which has greatest similarity to the florigen-encoding FT, also caused a early in flowering, but not as obviously as ScFT-2. Constractly, over-expression of ScFT-3, delayed the flowering time in the transgenic Ara

关键词： sugarcane FT-like gene molecular clone expression pattern analyses over-expression

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