Research Journal of Biotechnology

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Vol. 9(5) May 2014

Complementary DNA Cloning, Sequence Characterization and Expression Analyses of a Novel Gene MFN1 from Chinese Banna Mini-pig Inbred Line (BMI)

Qin Caiyan, Wang Pei, Wang Shuyan and Huo Jinlong

Mitofusin 1 (MFN1) and its active GTPase domains are important for the mitochondrial fusion process. Mitochondrial fusion may regulate mitochondrial morphogenesis and underlie complementation between mitochondrial genomes in mammalian cells. In the present study, the complete CDS of BMI MFN1 was obtained and characterized (GenBank accession numbers for the nucleotide and amino acid sequences are KC505156 and AGK36094 respectively). The tissue expression profile in 30 important tissues of BMI was carried out using quantitative real-time PCR method. The full-length coding region of MFN1 from BMI tissues consists of 2226 nucleotide which encodes 741 amino acids with a molecular weight of 84.18 kD and a pI of 6.05. The putative protein of MFN1, which is located in the cytoplasm (76.7%), contains two conserved domains of Ras-like-GTPase superfamily and fzo-mitofusin superfamily, three potential miRNA targets and no signal peptide. The sequence homology analysis revealed that the BMI MFN1 protein shared 97.2%, 95.4%, 94.3%, 93.8%, 91.5% and 91.0% identity with that of cattle, bat, mouse, rat, monkey and human. The phylogenetic tree analysis revealed that the BMI MFN1 gene had a closer genetic relationship with the MFN1 gene of cattle than with those of bat, mouse, rat, monkey and human. The analysis of tissue expression showed that BMI MFN1 gene was widely expressed in the tissues examined but pancreas and colon, being high in the testis, thymus, thyroid gland, lung, heart, liver, spleen, adrenal gland, lymph node, kidney, muscle, duodenum, ileum, esophagus, cerebrum, cerebellum, pituitary gland, hypothalamus, brainstem and sublingual gland, moderate in the submaxillary gland, epididymis, rectum, skin and spinal cord, low in the jejunum, cecum and stomach. These data provide a foundation for further insight into this swine gene.

Studies on arbuscular mycorrhizal assortment in the rhizosphere of Neem and Bamboo

Srimathi Priya L., Kumutha K., Arthee R. and Pandiyarajan P.

This study was focussed on the AM (Arbuscular mycorrhizal) fungal diversity in the saline-sodic soils based on native spore density, MPN assay and identification through spore morphology showed existence of different genera in the rhizosphere of neem and bamboo. The physico-chemical analysis of the native soils revealed that they are saline-sodic with the pH ranging from 8.7±0.50 to 8.8±0.51 and habituated two different species viz. Glomus and Gigaspora.

Characterization of phosphate solubilizing rhizobacteria from rhizosphere soil of banana (Musa spp.) in South Andaman, India

Ahmad Israr and Kushwaha Neetu Singh

Rhizobacteria from organically grown banana rhizosphere in South Andaman (Port Blair) were characterized for their plant growth promoting (PGP) properties, antagonistic activity and genetic diversity using PCR-RFLP. The biochemical characterization revealed that 30 out of 57 isolates were positive for phosphate solubilization and other plant growth promotion parameters. Antagonistic activity of these rhizobacteria against Fusarium oxysporum f. sp. cubense showed inhibition from 11 to 73%. Random fragment length polymorphism (RFLP) of 16s rDNA showed diverse homogenous population distributed randomly at 51% similarity. 16s rDNA sequencing result showed that most of the isolates belonged to B. Pumilus (GU817321 & GU818016), B. Subtilis (GU817320 and GU937766) and B. Megaterium (GU817322 and GU828017). Potential isolates of phosphate solubilizing, PGP and antagonistic Bacillus spp. could be used against fusarial wilt of banana as well as bioinoculants for sustainable and organic agriculture.

Pathogenic variability in different tomato varieties (Solanum lycopersicum) based on the ITS nrDNA region

Yan-Lin Sun, Ho-Min Kang, Young-Sik Kim and Soon-Kwan Hong

Tomato (Solanum lycopersicum) is one of the major vegetable crops in the world. To satisfy various demands of people, more than 500 tomato varieties have been bred out. Most of tomato varieties are highly susceptible to some diseases such as the tomato powdery mildew. Recently, the critical and important work is the prevention and treatment of disease. In this study, 26 healthy tomato plants covering with 24 tomato varieties were collected to test the infection situation of pathogens and endophytic fungus species in order to induct effective disease management. The results suggested that most of tomato varieties were infected with 1~3 pathogens/endophytic fungi which could be classified into four types including the tomato powdery mildew (Oidium neolycopersici and other uncertain Erysiphaceae species), tomato leaf mould pathogen (Cladosporium fulvum and Passalora fulva), tomato leaf spot pathogen (Botrytis fabae) and uncultured fungus species. The tomato powdery mildew could accrete with the leaf mould pathogen and Sclerotinia sclerotiorum. All detected fungus species showed that the characteristics of pathogens and endophytic fungus species having potential inhibitive pathogens had not been detected in this study. Based on the disease tolerance analysis, our results indicated that Nice Def and Max Thailang are considered to be the perfect tomato variety for cosmically planting and popularization. This work provides an efficient way of being able to detect infected pathogens, even in the early stage of infection of plants.

Transient expression of chicken gamma interferon in lettuce

Tao Wang, Yong Jun Wu and Xi Luo

We constructed a novel expression vector for transient gene expression using Agrobacterium tumefaciens infiltrated into leaves of commercially available lettuce (Lactuca sativa L.). The vector co-expressed the marker β-glucunidase (GUS) and chicken gamma interferon (ChIFN-γ) in lettuce leaves while retaining its native conformation. ChIFN-γ expression ranged from 1.0% to 1.2% of the total soluble protein. The molecular weight of the protein was approximately 30 kDa and it exhibited antiviral activity (2×103 IU/mg in vitro) by inhibition of vesicular stomatitis virus replication on chicken embryo fibroblast. In summary, transient expression of avian cytokine by Agrobacterium-mediated vacuum infiltration in lettuce provides an efficient, inexpensive and small-scaled plant expression system for studying gene expression in vitro with a final aim for production oral plant vaccine in bioreactor.

Improved Consumption of Phosphorus by Ariopsis seemanni (Colombian Shark Cat Fish) from Plant based Fish Feed using Hypocrea lixii SURT01

Thyagarajan R., Karthick Raja Namasivayam, Masilamani Selvam M. and Narendrakumar G.

The mechanism of enzyme Phytase (myo-inositol hexakisphosphate phosphohydrolase) is to catalyse the hydrolysis of phytic acid (myo-inositol hexakisph- osphate) which is an indigestible, organic form of phosphorus present in grains and oil seeds and releases a usable form of inorganic phosphorus.. The effect of pretreated plant-based supplementary fish feed with a partially purified fungal phytase produced by Hypocrea lixii SURT01 was evaluated on Ariopsis seemanni fingerlings. The plant-based supplementary feed (soybean, groundnut cake and wheat flour in 1:1:1 ratio) was treated with partially purified phytase at the rate of 300 U/g. The pretreatment of feed with phytase resulted in a significant increase in the breaking phosphorus attached with phytate. The growth performance and chemical composition of fingerlings fed with phytase treated diet were found to be better than fishes fed on the control diet without affecting the survival rate (100%). The results of the present study indicate that phytase treatment of supplementary feed can be effective for improving the growth of the cat fish fingerlings.

Production of Daucus carota hairy root for in vitro culture of arbuscular mycorrhizal fungi

Srinivasan M. and Kumar K.

Induction of hairy root by two different strains of Agrobacterium rhizogenes-MTCC- 532 and 2632 grown in YEMA culture media was studied in carrot (Daucus carota).The maximum hairy root induction was observed when carrot was simmering with 48 hours old culture of A. rhizogenes MTCC-532. With different concentrations of acetosyringone (ACS) tested, addition of 150 µM/l acetosyringone was found to enhance the transformation frequency up to 75 % and 60 % by A. rhizogenes MTCC-532 and MTCC 2364 strains respectively. Transformation efficiency is highly dependent on the type of bacterial strain used and carrot genotype. Freshly harvested carrots showed better hairy root induction. A. rhizogenes transformed hairy roots had the ability to form quickly numerous lateral roots as well as a negative geotropic growth habit. The MS medium was found to be the best medium for hairy root mass multiplication that supports the high root biomass production and rapid root tip elongation.

The study on anthocyanin regulatory genes as a visual marker in transformation of common wheat (Triticum aestivum)

He Yanli, Yang Fengping and Zhang Xiaodong

Anthocyanin is a kind of water-soluble flavonoid which makes the plants show red, blue, purple, red purple or other colors. Expression of the metabolic regulatory gene causes the anthocyanin pigmentation in plant cells and makes the plant organs change color and easy to be observed. Screening of transgenic tissue on the basis of the anthocyanin pigmentation has been studied in wheat. In this paper, isolated scutella of wheat was transformed by Agrobacterium with a genetic construct(pBAC9075) consisting of the two anthocyanin biosynthesis regulatory genes, B1 and C1 and a herbicide glyphosate resistant epsp gene, each under the control of the CaMV35S constitutive promoter. 132 transgenic plants were obtained. The results of PCR analysis showed that 5 of the 132 T0 transgenic plants were positive. The anthocyanin pigmentation was observed in the shoots and leaves of some positive transgenic plants. The results showed that it is possible to use this visual trait as a tracking system for wheat transformation and permits the selection of the transgenic plants at an early growth stage.

Genetic Diversity of Soft Rot Pathogens in Potato of South Indian Origin

Ponvizhi Ramya V., Gunasekaran S. and Senthil Kumar M.

Totally 25 isolates were obtained from spoiled potato, among those isolates virulent strains were screened by pathogenecity. Diversity of soft rot pathogens in spoiled potato was identified by 16 S rRNA sequencing revealing Cellulosimicrobium funkei, Enterobacter asburiae, Kocuria palustris, Pectobacterium carotovorum subsp. carotovorum, Bacillus subtilis subsp. inaquosorum, Bacillus aryabhattai, Arthrobacter nicotianae and Klebsiella oxytoca. Among those pathogens Pectobacterium carotovorum subsp. carotovorum has higher polygalacturonase, pectinolytic and cellulolytic activity. It has higher rot severity and broad host range infecting various families of vegetables like Alliaceae (Onion), Apiaceae (carrot), Brassicaceae (cabbage, broccoli, radish), Cucurbitaceae (Ivy gourd) and Solanaceae (green pepper, capsicum, potato, tomato).

Direct and indirect somatic embryogenesis from immature zygotic embryos in Robinia pseudoacacia L.

Yang Xi, Huan Wang, Yuhan Sun, Junqi Zhang, Xiaofang Luo, Yuling Wang, Jinquan Zhang, Ling Wang and Yun Li

The optimum developmental stage of explant for somatic embryogenesis in Robinia pseudoacacia L. was achieved using immature zygotic embryos from 10 open-pollinated plus trees as explants for three consecutive years (2010-2012). The trend of explant age on callus induction rate was parabolic and the highest frequency (95%) of callus induction was obtained in 55-day-old explants incubated on Murashige and Skoog medium supplemented with 5.0 mg•L-1 2,4-Dichlorophenoxyacetic acid and 0.5mg•L-1 6-Benzyladenine. Controlled crosses were conducted with five plus trees in 2012 to study the parent genotype influence on somatic embryogenesis capacity of explants. The variation in callus induction rate between families was 73-97% and the maternal effect was significant at this stage. The frequency of direct and indirect somatic embryogenesis varied among families on optimum medium and only maternal genotypes were statistically significant. The mean number of somatic embryos per family and embryo maturation rate was affected by the mother×father interaction. More than 98% of somatic embryos were converted into complete plants after mature culture and more than 95% of regenerating plants were successfully transferred to soil.

Self-crosslinking and Grafting Reactions in mTGase-mediated Modification of Wool Fibers

Li Xiaoli, Wang Qiang, Fan Xuerong, Wang Ping, Yuan Jiugang and Cui Li

The mechanism of enzymatic acyl-transfer reactions of wool by microbial transglutaminases (mTGase) was revealed in terms of the release of ammonia, DSC curves and analysis of amino acid in this paper. It was first demonstrated that mTGase-mediated self-crosslinking and grafting reactions of wool concurrently occurred. Furthermore, the released ammonia in the homogeneous wool keratin system was larger than that in the heterogeneous wool system, indicating that the former was easier. 2,4,6-trinitrobenzenesulfonic acid (TNBS) was used to mediate the two reactions of the proteinous substrates. The results showed that TNBS could seal the free amino groups of the substrates and prevent self-crosslinking of the substrates effectively.

Assessment of Genetic Fidelity in Terminalia bellirica (Gaertner) Roxb Plantlets regenerated from Nodal Culture using RAPD Markers

Sahu Bharti and Koche Vijaya

The present work describes assessment of clonal fidelity in T. bellirica plantlets regenerated through nodal culture using RAPD marker. Micro shoots were developed form nodal explants of T. bellirica on the medium containing 0.5 mgl-1 6-benzylaminopurine and rooting was achieved on 2 mgl-1 NAA. Random amplified polymorphic DNA (RAPD marker) was used to assess the genetic fidelity of regenerated plants. Among the regenerated plants ten no. of plants were randomly selected for molecular analysis. RAPD profiles of clone of 10 plants prepared with MAP 1 - 20 primer amplified 104 bands were all monomorphic. The genetic fidelity testing of micro-shoots, based on a RAPD analysis indicates a strong genetic purity like the parent.

Occurrence of Diverse Fungal Communities in decaying Vegetable Waste

Mathew. L., Potty V.P. and Nair G.M.

The study was intended to isolate and identify indigenous fungal flora from decaying vegetable waste. The morphological characterization was based on properties like colony diameter, culture characteristics, texture, surface and reverse colouration, zonation and sporulation of selected test fungi on the growth medium. The fungal strains identified were Penicillium aurantiogriesum, Fusarium verticillioides, Aspergillus versicolor, Aspergillus niger, Rhizopus stolonifer, Penicillium oxalicum, Penicillium sp, Penicillium expansum, Sporulating hyaline form, Alternaria dianthicola, Non sporulating hyaline form and Fusarium semitectum. Of the 12 fungal cultures isolated, profuse growth on the culture medium was reported by Fusarium verticillioides, Aspergillus niger, Rhizopus stolonifer, Penicillium sp, Fusarium semitectum and Non sporulating hyaline forms. Heavy sporulation was shown by Aspergillus niger, Rhizopus stolonifer,and Penicillium oxalicum and moderate sporulation by Aspergillus versicolor, P.aurantiogriesum and Penicillium expansum. Pigmentation was also exhibited by sporulating hyaline form of fungi.

Optimization of lipase production by a new fungal strain Cladosporium cladosporioides NK-LF36 isolated from oil contaminated soil

Kalyani N. and Saraswathy N.

In the present study, a total of 20 fungal isolates were obtained from oil contaminated soils. Screening of lipolytic fungal isolates on selective media enabled the isolation of potential isolate LF1 based on the zone of clearance. The isolate LF1 exhibited greater clearance zone and higher lipase activity. It was identified as Cladosporium cladosporioides strain NK-LF36 based on morphological and 18S rRNA sequence analysis. The production medium was optimized to increase the lipase production conditions. Maximum enzyme production (8.82U/ml) was obtained in a basal medium containing 3% karanja oil as sole carbon source at pH 6.0 and 30°C after 6 days of incubation. Lipase activity was further enhanced (11.65 U/ml) on addition of 4% yeast extract, 0.2% ammonium tartarate as nitrogen sources and 0.01% KCl. The lipase production was slightly enhanced in the presence of 10% n-butanol and was highly stable in the presence of n-propanol, toluene and n-hexane. The results of the study show that the new fungal strain NK-LF36 was found to be inducible, mesophilic and solvent tolerant indicating its potentiality in industries.

Production and partial characterization of extracellular xylanase from acidophilic Aspergillus flavus MTCC 9390 grown in SSF mode

Bhushan Bharat, Pal Ajay and Kumar Satish

Multi-step screening comprising of qualitative and quantitative assays was carried out to isolate hyper-secreting xylanolytic microbial strain. The enzyme super-secreter Aspergillus flavus MTCC 9390 was selected for optimizing xylanase production in solid state fermentation where various process variables like temperature, carbon source and moistening agents were optimized using conventional ‘change one-variable-at-a-time’ approach. A significant increase in enzyme production was achieved by change in fermenting substrate and switching it over to horticultural waste material. A further increase in xylanase production was achieved when the spent mass was agitated at warm temperature with minimal volume of extractant such as tween-80. The extracellular xylanase was fractionated with ammonium sulfate and characterized with respect to reaction parameters. The crude xylanase was active at 60°C and pH 6.0 and stability experiments revealed its better biochemical characteristics for its commercial adoption in juice industries.