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eISSN: 1682-2978
pISSN: 1682-296x

Editor-in-Chief:  Akhtar Jamal Khan
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Research Article
Cloning and Expression Analysis of CONSTANS-Like 16 (GbCOL16) Gene from Ginkgo biloba
Lanlan Wang, Jiaping Yan, Xiangxiang Meng, Jiabao Ye, Weiwei Zhang and Feng Xu
Background and Objective: Ginkgo biloba (G. biloba) is a precious medicinal and edible plant. It has a long juvenile phase. CONSTANS (CO) and CONSTANS-like (COL) genes are key genes in the photoperiodic flowering pathway. Information on COL genes in G. biloba is relatively lacking. The aim of this study was to characterize a CONSTANS-like 16 (GbCOL16) gene from G. biloba. Methodology: In this study, GbCOL16 gene was cloned from G. biloba. The expression level of GbCOL16 gene in different tissues of G. biloba was studied by semi-quantitative RT-PCR and quantitative RT-PCR methods. Data were analyzed with one-way ANOVA using SPSS 11.0 for windows. The means were compared with Duncan’s multiple range tests. Results: Sequence analysis results showed that the full-length cDNA of GbCOL16 was 1,337 bp, it contained a 1,311 bp ORF and encoded a deduced protein of 436 amino acids. The GbCOL16 has one conserved CCT domain, without B-Box domain. GbCOL16 has a close genetic relationship with SlCOL16 and NtCOL16 and can be clustered into group IV of CO gene family. The expression analysis results showed that the highest GbCOL16 expression was in the leaves. The GbCOL16 expression was higher in male strobili than in the stems, female strobili and young fruits. The lowest relative expression of GbCOL16 was in the roots. However, GbCOL16 barley was expressed in Ginkgo buds. Conclusion: GbCOL16 was expressed specifically in the leaves of ginkgo. The flowering regulation mechanism of GbCOL16 is similar to that of CO genes. This finding lays the foundation for clarifying the flowering gene network and molecular regulation mechanism of G. biloba.
Research Article
Molecular Cloning and Expression Analysis of a AGAMOUS-like 66 Gene (GbAGL66) in Ginkgo biloba
Jinshuang Dou, Lanlan Wang, Jiaping Yan, Mingyue Fu, Xian Zhang and Feng Xu
Background and Objective: Ginkgo biloba (G. biloba) is a precious medicinal plant and has a long juvenile phase. AGAMOUS LIKE-66 (AGL66) gene, an important flowering regulatory gene, belongs to the family of MADS-box gene family. Information on AGL66 genes in G. biloba is relatively lacking. The aim of this study was to characterize a AGL66 gene from G. biloba. Methodology: According to the unigene sequences of G. biloba transcriptome, a AGL66 gene was cloned from G. biloba, named GbAGL66 (Genbank accession number is MF443205). Quantitative real-time polymerase chain reaction (qRT-PCR) method was used to analyze the expression level of GbAGL66 gene. Data were analyzed with one-way ANOVA using SPSS 11.0. Results: The full-length cDNA of GbAGL66 gene was 1,202 bp and its open reading frame (ORF) was 1,146 bp, encoding a deduced protein of 381 amino acids. A homologue search against GenBank showed that GbAGL66 protein was a homologue of MIKC-type MADS-box proteins and had two typical MADS and K domains. Using bioinformatics software to carry on the analysis, the theoretical molecular weight is 4.33 kDa and the isoelectric point is 5.96. GbAGL66 had 60, 53 and 52% homology with the AGLs from Prunus persica, Cucumis melo and Elaeis guineensis, respectively. The expression of GbAGL66 gene in roots was the highest. The expressions of GbAGL66 gene in male and female flowers were higher than that in stems and leaves. Conclusion: In this study, a GbAGL66 gene was cloned and characterized from G. biloba for the first time. GbAGL66 was strongly expressed in roots and flowers. These findings laid the foundation for the molecular regulation of flowering of G. biloba.
Research Article
Characterization and Expression Analysis of an AP2 Gene from Ginkgo biloba
Xian Zhang, Lan Lan Wang, Jia Ping Yan, Ming Yue Fu, Jin Shuang Dou and Feng Xu
Background and Objective: APETALA 2 (AP2) gene is an important transcription factor in plant flower development and involved in signal transduction of plant growth, development and physiological and biochemical reactions. The aim of this study was to characterize a AP2 gene from Ginkgo biloba (G. biloba). Materials and Methods: The specific primers were designed based on AP2 unigene sequence of transcriptome data. The full-length cDNA of homologous genes were cloned from G. biloba by RT-PCR. Tissue expression analysis was estimated by quantitative RT-PCR methods. Results: Sequence analysis results showed that the full-length cDNA of GbAP2 was 2018 bp and contained a 1974 bp open reading frame, which encoded a 657 amino-acid protein. The predicted molecular weight and isoelectric point were 72.03 kDa and 5.91, respectively. Multiple alignments showed that the GbAP2 protein had high homology with the AP2 protein of other plants. The GbAP2 contains two AP2 domains and belongs to the AP2 subfamily of the AP2/ERF family. Phylogenetic tree analysis revealed that GbAP2 had closer genetic relationship with Cycas revolute from Cycadaceae. Tissue expression analysis showed that GbAP2 gene was expressed in roots, stems, leaves, male strobili, female strobili and fruit of G. biloba and strongly expressed in the leaves and female strobili. Conclusion: In this study, a novel AP2 gene (GbAP2) was cloned and characterized from G. biloba for the first time. The results of expression pattern of GbAP2 in different tissues suggested that GbAP2 might be involved in whole plant growth and development in G. biloba.
Research Article
Mycorrhiza Modulates Morphology, Color and Duration of Flowers in Hyacinth
Miao- Miao Xie and Qiang- Sheng Wu
Background and Objective: Hyacinth, one of world-famous bulbous ornamental plants is widely planted in various habitats. The present work tried to evaluate whether and how inoculation with arbuscular mycorrhizal fungi (AMF) regulates flower morphology and flowering duration of hyacinth. Materials and Methods: Three AMF species, namely, Diversispora spurca (D. spurca), Diversispora versiformis (D. versiformis ) and Funneliformis mosseae (F. mosseae ) were inoculated into potted hyacinth (Hyacinths orientalis L. Anna Marie) plants for 130 days. Flower morphology, flowering duration, nutrient status, indole acetic acid (IAA), chlorophyll and anthocyanin concentration were measured. Data were analyzed by one-way analysis of variance (ANOVA) with SAS. Significances of treatments were compared by the Duncan’s multiple range tests at p<0.05. Results: Root mycorrhizal colonization varied from 38.0-48.7%. Amongst three AMF species, only F. mosseae significantly (p<0.05) has increased flower biomass, opening flower number, flower stem height and floret diameter. Diversispora spurca and F. mosseae, respectively prolonged flower duration for 1.4 and 3.3 days. Mycorrhizal plants with F. mosseae had significantly (p<0.05) higher IAA concentration of flowers and roots, higher anthocyanin concentration of flowers, higher chlorophyll b and total chlorophyll levels of leaves and greater N, P and K levels of flowers and roots. Diversispora spurca colonized plants possessed significantly (p<0.05) higher flower IAA levels and leaf chlorophyll b and total chlorophyll concentrations. Diversispora versiformis generally did not affect these variables. Conclusion: Mycorrhizas could modulate morphology, color and duration of flowers in hyacinth, which is closely related with nutrient status, IAA levels and AMF species. Meanwhile, F. mosseae exhibited the best stimulated effects and can consider to be used in hyacinth cultivation.
Short Communication
Cross-linked Enzyme Aggregates of Pig Liver Esterase Evaluated in Kinetic Resolution of Racemic Clopidogrel
Yasser Gaber and Mohamed Ismail
Background and Objective: Immobilization of enzymes as cross-linked aggregates is one of the cheapest, simplest and effective techniques for improving their stability and reusability and even avoiding contamination of the product with the catalyst. Clopidogrel is a widely used antiplatelet drug, only S isomer is the biologically active enantiomer produced by resolution of the racemic compound. In the current study, cross-linked aggregates of pig liver esterase were prepared and evaluated for kinetic resolution of racemic clopidogrel. Materials and Methods: Cross-linked Enzyme Aggregates (CLEA) of the commercially available crude pig liver esterase cPLE were prepared using glutaraldehyde at concentrations of 12.5-125 mM as crosslinker either in presence or absence of Bovine serum albumin (BSA). cPLE-CLEA was used for kinetic resolution of racemic clopidogrel and compared to the performance of soluble cPLE. Light microscopy and scanning electron microscopy SEM were used to examine cPLE-CLEA. Results: Soluble cPLE showed ability to resolve racemic clopidogrel at enantioselectivity (E) of 9.2. The resolution of clopidogrel was found to be optimal at 30°C. The cPLE-CLEA preparations showed reduced enzymatic activity. The kinetic resolution experiments showed also lower E values (E = 1.3-4.5) compared to soluble cPLE. Microscopical examination of cPLE-CLEA showed wide size variation and SEM revealed the shape of cPLE-CLEA before and after use in the kinetic resolution experiments. Conclusion: Crude PLE was able to resolve racemic clopidogrel, the effects of different temperatures were studied and the highest E value recorded was 9.2 at 30°C. Increasing concentrations of glutaraldehyde as a cross-linker adversely affected PLE activity. The cPLE-CLEA showed lower enantioselectivity compared to the free cPLE.

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