Research Journal of Biotechnology

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Vol. 10(7) July 2015

Optimization of lipase production by Myroides odoratimimus SKS05-GRD and Bioremediation of Diesel Hydrocarbons

Saranya A., Divya U.K. and Suganthi Ramasamy

One of the prime objectives of industrial microbiology is the large scale production of valuable metabolites with multiple applications. The utilization of biopharmaceutical oil waste as a substrate for lipase production by Myroides odoratimimus SKS05-GRD on one hand provides alternative substrate and on the other helps in solving pollution problems. The growth rate and bioremediation ability was investigated in soil collected from hydrocarbon rich area by using the same bacterial strain. The results revealed the time required to degrade the aromatic hydrocarbons that are toxic to the environment. Although the relation of degradation with the lipase produced by the bacterium is yet to be investigated, the efficiency to degrade hydrocarbons was detectable after 40-day incubation period. The utilization of waste oil cakes for enzyme production through SSF adds value to oil mill wastes as substrate that yields lipase which in turn effectively bio-remediates hydrocarbons from contaminated soils. This study shows a viable alternative remedy in oil and hydrocarbon contaminated environment worldwide.

Expression of Two Synthetic Lectin Genes sGNA and sNTL in Transgenic Wheat enchanced resistance to Aphids

Duan Xiaoliang, Hou Qiling and Liang Rongqi

Plant lectins have been shown toxic to sap-sucking pest such as wheat aphid. In this study, sGNA (synthesized Galanthus nivalis agglutinin) and sNTL (synthesized Narcissus tazetta lectin) genes encoding mannose binding lectins were synthesized according to the codons preferable principle of wheat genes and driven by the phloem-specific promoter rbcs in the constructs, respectively. Transgenic sGNA and sNTL common wheat lines were generated by particle bombardment transformation. Twelve homzygous T4 sGNA transgenic lines and eleven homozygous T4 sNTL transgenic lines were identified by PCR. After the insect bioassays in the lab and two-year field-test, five sGNA transgenic lines and seven sNTL transgenic lines were found highly resistant to aphids compared to the corresponding control plants. Significant differences were also observed for mortality and growth rate of aphids between the transgenic lines and the corresponding controls on detached leaf tissues. Real-time PCR analyses and protein extract clotting activity tests indicated that the increased resistance to aphids was significantly correlated with the expression of sGNA and sNTL lectins in the transgenic lines. However, no significant differences in aphid mortality and aphid amount per tiller were found between the two types of transgenic lines. Therefore both sGNA and sNTL could be served as a potential choice to produce aphid-resistance wheat through genetically modified approach.

Cloning and expression analysis of self-incompatibility S1 family protein gene in Citrus reticulate Blanco cv. wuzishatangju

Wu Xiu-Lan, Qin Yong-Hua and Hu Gui-Bing

In this study, the full-length cDNA of S1 family protein gene was cloned by RACE technique from Wuzishatangju (Citrus reticulate Blanco). The results revealed that S1 family protein was 814 bp, there were 453 bp in ORF sequences and encoded a 150 amino acid polypeptide. The S1 family protein was exclusively expressed in the anthers. When Wuzishatangju was self-pollinated, the expression level of S1 family protein in the pistils reached a maximum at 72 h and decreased thereafter. While Shatangju was cross-pollinated with Wuzishatangju, relative expressional level of S1 family protein was weakened at 72 h. The southern blot analysis indicated there were two copies of the S1 family protein in genomic DNA from Wuzishatangju. This study showed that S1 family protein may involve in the reaction of self-incompatibility in Wuzishatangju which provided a candidate gene potentially useful for self-incompatibility but its function still needs further confirmation.

Relationship of Ascorbic Acid Metabolism with the Cytoplasmic Male Sterility in Pepper (Capsicum annuum L.)

Wen Jinfen, Junheng L. V., Huo Jinlong, Zha Kai, Deng Minghua and Zhu Haishan

To elucidate the metabolic mechanism of Ascorbic Acid in the CMS-pepper anthers, the metabolism changes in the reactive oxygen species (ROS) contents, antioxidants contents and ROS scavenging enzymic activities were investigated in the anther mitochondria of CMS-9704A and maintainer-9704B. At the abortion stage, anthers of CMS-9704A had higher contents of ROS than those of the maintainer. Simultaneously, there were lower contents of ASA and GSH in stage 2 and 3 and the lower activities of APX and GPX in stage 3 in scavenging ROS in the anthers of the CMS line than in the maintainer. The expression level of APX and GPX in stage 3 in anthers of CMS-9704A was obviously inhibited when ROS was produced with a great deal during anther stage; however the gene expression kept normal in the maintainer. Excessive accumulation of ROS, significant reduction activities and gene expression level of ROS-scavenging enzyme were coinstantaneous with CMS.

Molecular Cloning of Betaine Aldehyde Dehydrogenase (BADH) Gene from Harvested Papaya (Carica papaya) Fruit and its response to Temperature Stress‏

Xiaoyang Zhu, Weixin Chen and Xueping Li

Betaine aldehyde dehydrogenase in plants can respond to drought, salt and temperature stress and can play a positive role in abiotic stress tolerance. In present study, a novel BADH gene was isolated from papaya fruit using in silico cloning and 3′- or 5′- rapid amplification cDNA ends (RACE). The full-length cDNA of CpBADH was 2052 bp, with a 1509 bp open reading frame (ORF) encoding a deducted 503 amino acid polypeptide. Sequence analysis showed that CpBADH contained some highly conserved regions of BADHs in the amino acid sequence. Subcellular localization prediction revealed that CpBADH contained a targeting signal peptide (QLFIDGE) to chloroplast and a targeting-signal (SKL) to peroxisome. Phylogenetic tree analysis revealed that CpBADH had a close genetic relationship with AtBADH1 (ALDH10A8). Gene expression analysis showed that both temperature stresses induced CpBADH expression during storage. High temperature caused a signiﬁcantly higher induction of CpBADH expression. Our results showed that CpBADH may be involved in diverse roles in temperature stress tolerance. This study has provided a foundation for further study on the CpBADH function and for improvement of papaya fruit stress tolerance.

Regenerative potential and phytochemical diversity among five accessions of Bacopa monnieri (L.) Wettst

Koul A., Kushwaha M., Gupta A.P. and Mallubhotla S.

Distant geographical niches have shown pronounced effect on variability in organoleptic characteristics as well as biochemical constituents between accessions of a given plant species. The present study has been designed to evaluate the regenerative potential of five accessions of Bacopa monnieri, a herb which is of high commercial potential with a legendary reputation as a memory vitalizer through morphogenetic, phytochemical and Liquid chromatography- Electrospray ionization-Mass spectra (LC-ESI-MS) analysis. Culture media supplemented with exogenous plant hormones had a varied effect on in vitro culture morphogenesis and biomass production in the accessions. Maximum friable callus was regenerated in accession BM003 (GI: 54.61 ± 0.11) on B5 media supplemented with 2,4-D (1.0 mg l-1) while MS media with NAA (1.0 mg l-1) + BAP (0.5 mg l-1) resulted in maximum indirect organogenesis in BM003 (GI: 99.58 ± 0.06). Biochemical profiling proved the qualitative differences in the carbohydrate, alkaloid, saponin and terpenoid contents in leaf, shoot and root samples of all accessions. LC-ESI-MS analysis revealed that the shoot extract of accession BM002 (7236.38 µg gm-1) contains highest Bacoside content as compared to that of its root extract (1015.32 µg gm-1) and the shoots had higher Bacoside contents as compared to roots.

Application of Waste Raw and Composted recycled Paper Mill Sludge on Orthosiphon stamineus and its effects on Heavy Metals in Soil

Rosazlin, A., Fauziah, C.I., Wan Rasidah, K. and Rosenani A.B.

The paper industry plays a major role in the global economy of the world. A study was conducted on the waste paper mill sludge applied on the Orthosiphon stamineus for 4 crop cycle for 1 year growth at Glasshouse, Faculty of Agriculture, Universiti Putra Malaysia. Waste Paper Mill Sludge (PMS) and waste composted Recycled Paper Mill Sludge (RPMS) was used with nitrogen (0, 100, 200 and 400 kg ha-1) at the ratio of 1:1 (Recycled Paper Mill Sludge (RPMS): Empty Fruit Brunch (EFB). The growth parameters were measured twice a month for 6 months. Plant nutrients and heavy metals uptake were determined. The paper mill sludge has the potential to be a supplementary N fertilizer as well as a soil amendment. The application of waste RPMS with N significantly contributed to the improvement in plant dry matter (28.5 g plant-1) and improved soil physical and chemical properties. Total concentrations of heavy metals in soils were below the critical values. Hence, the waste paper mill sludge can be successfully used as soil amendment in acidic soil without any serious threat. The use of waste paper mill sludge for the soil fertility showing improvement in land application signifies a unique opportunity to recycle sludge back to the land to alleviate the potential waste management problem.

Isolation and Molecular Characterization of Listeria monocytogenes isolated from Chicken Samples in Erzurum, Turkey

Gulsah Adiguzel and Ebru Yurekli

In order to provide food safety and to control the food contamination during the process of food supplementation, a sound identification of food bacterial pathogens is essential. Two hundred and fifty chicken samples were obtained from the butchers and retail shops in the vicinity of Erzurum. In this study, a phenotypic and genotypic characterization of Listeria monocytogenes isolates isolated from the chicken samples was presented. As the result of the isolation study, the existence of 19 L. monocytogenes was determined which were then supported by the VITEK and specific-PCR analysis data. Later on, as result of the rep-PCR analysis was performed for specifying the genotypic differences between the test isolates, it was determined that the ERIC-PCR method could successfully be carried out for identifying and characterizing the L. monocytogenes bacterium on the levels of species, sub-species and even on strains. As the result of the cluster analysis performed by employing the ERIC-PCR data provided the maximum number of polymorphic bands, 3 samples contained in different clusters were selected and tested in 16S rRNA sequence analysis. Each of the 3 isolates, in consequence of this study, was determined to resemble the L.monocytogenes at a rate of ≥99 %.

Interaction of Proteus vulgaris with the mouse macrophage cell line Ana-1

Wu Yilong, Liu Keshu, Fei Rongmei and Zhou Yongkang

In recent years, the morbidity of Chinese alligator is rising obviously. As one of the pathogen, Proteus vulgaris were isolated from the pathogenetic Chinese alligator repeatedly. Here, two strains of Proteus vulgaris (virulent strain XC16-2 and avirulent strain XC46) isolated from Chinese alligator (Alligator sinensis) and Escherichia coli MG 1655 were studied to evaluate their capacity to escape uptake and lysis by the mouse macrophage cell Ana-1. Gentamicin is the optimal antibiotic for cell cultivation in susceptibility tests. The capacity of Ana-1 cells for uptake and lysis of the three strains was analyzed and was determined under optimized conditions (90 min, MOI: 1:1) in a 24-well cell culture plate. The uptake of E. coli MG 1655 by the Ana-1 cell line was 10-fold greater than that of the two P. vulgaris strains (virulent strain XC16-2 and avirulent strain XC46) under identical conditions. However, the in vitro intracellular killing rate constants (Kk) of the three strains by the mouse macrophage cell line Ana-1 were similar. We conclude that the pathogenic capability of P. vulgaris has no immediate correlation with the in vitro interactions with the mouse macrophage cell line Ana-1.

Changes in antioxidant enzyme activity during in vitro adventitious shoot from Cotyledonary node explants of Cucumis melo L.

Huijun Zhang

Plant regeneration through organogenesis of shoot bud from callus cultures of melon was established and callus having different regeneration capacity were obtained, This experimental system was used to investigate the release of hydrogen peroxide and the activities of antioxidant enzymes During the differentiation culture day, superoxide dismutase (SOD) activity increased in the early regeneration culture and decreased thereafter. Maximum SOD activity was observed on the 7th day of shoot culture during the initiation phase of shooting. CAT, MDA activity increased until the end of the induction phase. Peroxidise activity maximized on the 28th day and then declined to a minimum on 0 day in cultivars. The results of this study indicated that antioxidant enzymes (SOD, POD and CAT, MDA) protected cucumis melo L. callus. Especially for SOD, it is correlated with the morphogenetic process in melon callus and may actually serve as a messenger in the process of bud primordium formation. These results show that peroxidase activities are involved in defining the beginning and the length of particular phases of shooting.

Efficacy of charcoal based formulations of Bacillus and Escherichia coli to enhance the growth and yield of Triticum aestivum L.

Aslam Faiza and Ali Basharat

Biofertilizers are formulations of beneficial microorganisms packed in an easily accessible carrier material that can be used as an alternative to inorganic fertilizers. In present study, we evaluated the plant growth promoting effects of water and charcoal based biofertilizers that were formulated by using different strains of Bacillus and waterborne Escherichia coli. Formulations of auxin producing rhizobacteria were used as single or mixed culture to inoculate Triticum aestivum L. under axenic and natural conditions. In vitro screening recorded auxin production for all bacterial strains in the presence or absence of L-tryptophan. Nevertheless, maximum levels of auxin were recorded for B. subtilis McR-7 and B. megaterium McR-8 with 5 and 10 folds increase respectively in the presence of 500 µg ml-1 L-tryptophan. In vitro experiments demonstrated the biological activity of bacterial strains that was evident from increases in root length (61%) and later root number (50%), over control. Under axenic conditions, mixed cultures of E. coli (N-21+N-40) and Bacillus (ZmR-4+McR-8) witnessed 20% and 26% increases respectively for water and charcoal based formulations. At final harvest, charcoal formulations recorded significant increases for spike length (46%) and number of tillers (50%) with B. megaterium ZmR-4. Similarly, seed weight was significantly enhanced up to 53% and 39% respectively by mixed cultures of OsR-3+N-35 and ZmR-4+McR-8. Finally, it was concluded that charcoal based formulations supplemented with auxin producing bacterial consortia have great potential to apply for field trials.

Effect of ATP on the Photosynthetic Physiological Characteristics in the Transgenic Rice (ORYZA SATIVA L.) expressing C4 Enzyme Genes

Tang Ning, Chen Quanzhan and Zhang Bianjiang

With untransformed rice and transgenic rice with different C4 photosynthesis genes as the materials, the activity of C4 photosynthesis enzymes, the photosynthetic rate and the indexes of active oxygen metabolism were determined. The activities of C4 photosynthesis enzymes in untransformed rice were very low while those of corresponding enzymes in different transgenic rice were all highly expressed. Compared with the photosynthetic rate of the untransformed rice, the photosynthetic rate of the PPDK transgenic rice was not increased; that of the ME transgenic rice was decreased for 6.1% ; that of the PEPC transgenic rice was increased for 38.5% and that of the transgenic rice with PEPC and PPDK genes (CK) and transgenic rice with PEPC,PPDK and ME genes(CKM) were similar with that of the PEPC transgenic rice under high light intensity. After the ATP treatment the rate of O2 evolution was significantly increased for the CK and CKM transgenic rice which reached about 80% of that of maize and exhibited photosynthetic characteristics of C4–like plants. This indicated that ATP is the key factor for constructing C4-like rice. In addition, after photooxidative treatment CKM transgenic rice was more tolerant to photooxidative stress. These results would provide a new technical approach for rice breeding with high photosynthetic efficiency and high grain yield.

Molecular Diversity and Biochemical Correlation of Wheat Genotypes against Heat Stress

Ramani H. R.

The experiment was carried out using fourteen wheat genotypes of that seven were heat tolerant and seven were heat susceptible. Seeds were grown in germination bag filled with soil for 10 days. Total 12 Operon series RAPD primers were amplified to generate the 105 fragments. The percent polymorphism obtained for RAPD primers ranged from 71.4% to 100% with an average value of 92.33% per primer. Subcluster A1 (b) of cluster- I consisted of only one heat tolerant genotype J-2010-06 with more than 66% of similarity. Subcluster A2 of cluster-I consisted of only one heat susceptible genotype J-2010-13 similarity of more than 85%. Cluster-II consisted of two genotypes J-2010-05 and GW-190 showing similarity of more than 85% that belongs to heat tolerant groups. Pearson correlation was carried out between six parameters like relative water content, membrane stability, membrane injury, lipid peroxidation, hydrogen peroxide content and chlorophyll stability index. Fourteen wheat genotypes were used for correlation analysis including six screening parameters as listed above. From the results, it was concluded that genotypes J-2010-05 and GW-190 belong to same cluster and both are heat tolerant genotypes. As well from biochemical correlation, membrane stability and relative water content were potent parameters for screening of heat tolerant and heat susceptible wheat genotype against heat stress.

Synthesis of core-shell nanomagnetic iron oxide for loading theophylline

Fathi Fereshteh, Seied Sajadi Mir Abdollah and Farhadyar Nazanin

Magnetic nanoparticles are used in medicine and pharmaceutical applications because of their unique properties such as high magnetic saturation, stability and biocompatibility. They act as carriers for drugs and achieve them to tissues via the human circulation system. In magnetic targeted drug delivery, magnetic nanoparticles(e.g magnetite, Fe3O4) must be cauted with biocompatible materials. In this study we synthesized iron oxide(IO) nanoparticles and coated them with PVP, SiO2 and Au to combine drug (theophylline). Then we demonstrated synthesis of these carrier nanoparticles by FT-IR, XRD, TEM and SEM and EDX and showed drug well loaded on iron oxide nanoparticles.

RAPD and ISSR based Intra-specific molecular genetic diversity analysis of Cymbopogon flexuosus L. Stapf with a distinct correlation of morpho-chemical observations

Saikia Debajit, Dutta Sukriti, Ghosh Sneha, Lal Mohan and Bhau Brijmohan Singh

Cymbopogon flexuosus L. Stapf is a valuable medicinal plant that belongs to the family Poaceae. In this report, genotypic and phenotypic variations among 12 genotypes of C. flexuosus were assessed based on three marker systems namely morphological, biochemical and molecular markers i.e. Random Amplified Polymorphic DNA (RAPD) and Inter-simple sequence repeat (ISSR). Through this study we are trying to develop a fine relationship among the germplasm by correlating the genotypic and phenotypic data with respect to betterment of essential oil quality and quantity. The UPGMA dendrogram constructed from compiled ISSR and RAPD analysis shows highest dissimilarity between genotype RLJ-M3 and RLJ-M10 (0.57) and the highest similarity between genotype RLJ-M7 and RLJ-M9 (0.90).

Construction and Analysis of Genes expressed during Sogatella furcifera Horvát Wing Dimorphism by Suppression Subtractive Hybridization (SSH) method

Liu Jia-Ni, Li Zheng-Yue, Yu Lei, Xu Sheng-Guang, Chen Ze-Bin and Gui Fu-Rong

The aim of this project was to identify the genes associated with the formation and development of wing dimorphism of white-backed planthopper Sogatella furcifera (Horvath). Suppression subtractive hybridization (SSH) libraries were constructed from macropterous (long wing morph, LWM) and brachypterous (short wing morph, SWM) individuals of S. furcifera. From these SSH libraries, the tester library was prepared from macropterous insects whereas the driver library was prepared from brachypterous ones. From the SSH library, we obtained 176 high quality differentially expressed sequences, of which 47 expressed sequence tags (ESTs) in macropterous S. furcifera were screened by the reverse northern blot. 34 sequences were compared against non-redundant NCBI databases using BLAST. Of the unique sequences, 76% shared sequence identity with known genes of other organisms whereas 24% shared no significant similarity to any databases entries. Many biological processes such as metabolism, transcription, signal transduction, cell growth and division, cell structure, cell fate, protein synthesis or degradation, development, defense etc. might be involved in wing dimorphism regulation. Their functions will be guidance for studying wing dimorphism mechanism of S. furcifera and the SSH approach has proven to be useful in identifying the differentially expressed sequences on S. furcifera as well.