Research Journal of Biotechnology

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Taxonomic relationships and the evolution of Chinese kale and other Brassica oleracea L. subspecies based on Chs sequences

Chen Fa-Bo, Gao Jian, Huo Shi-Ping, Yao Qi-Lun and Liu Hong-Fang

For a long time, there have been two diametrically opposed views on the origin of Chinese kale. One view states that Chinese kale is a subspecies of Brassica that should be named as B. oleracea var. alboglabra while the other view holds that Chinese kale is an independent species of Brassica that should be named as B. alboglabra. Concerning the origin area, Chinese kale has been considered to have originated from the Mediterranean coast or southern China. Twenty-six chalcone synthase gene sequences were analyzed from 22 individuals to assess the origin of Chinese kale and other B. oleracea subspecies. According to a maximum likelihood analysis, the B. oleracea sequences were separated into two well-supported groups. A Splits Tree analysis indicated that both tree and reticulate evolution existed among B. oleracea subspecies but no reticulate evolution existed among B. oleracea and its related plants. The network analyses suggested that head cabbage, white kale, turnip cabbage and kohlrabi are most evolved while Chinese kale, cauliflower, broccoli, Brussels sprout and purple kale are ancestral types. Our data indicated that Chinese kale which should be a subspecies of B. oleracea originated from the Mediterranean coast.

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Comparative analysis for seed protein profiling and SSR markers in Rice cultivars (Oryza sativa L.)

Zaman Mariya S., Jadeja G.C., Parihar Akarsh and Patel Ankita

In order to study the grain protein content and the genetic diversity among 28 rice genotypes at biochemical and molecular level, the present investigation was undertaken, encompassing phenotyping for protein and protein fractions and SSR analysis. The data analyzed for crude protein, total protein and its fractions viz. albumin, globulin, prolamine and glutelin revealed significant differences among 28 rice genotypes. In case of fractions, albumin and globulin showed positive and significant correlation between them at both genotypic and phenotypic levels while prolamine and glutelin also exhibited positive and significant correlation at both the levels. Analysis of 13 SSR primers generated a total of 185 alleles, out of which all were polymorphic with an average of 14 bands per primer. The PIC values for SSR markers ranged from 0.83 (cRM 37-1) to 0.95 (eRM 33-1) with an average of 0.89 per primer. The cluster analysis categorized genotypes into three main clusters and formed distinct grouping according to their parentage. Grouping based on SSR showed goodness of fit with grouping of genotypes based on protein fraction and total protein together because SSRs used in the present study were specific to gene/QTL governing grain protein content.

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A novel therapeutic effect of Callus from Rosa damascena that suppresses Human T-cell Activation: Medicine from Cultured Cells

Sarah Albogami and Hadeer Darwesh

In the exploration for novel drugs, medicinal plants used traditional medicines are promising candidates. For the naturally rare, seasonally dependent, and slowly growing plant species, plant tissue culture offers a cost-effective, plant available all year round, and well-controlled means for mass production of the active values of medicinal plants for pharmaceutical industries. This study was aimed to generate callus from Rosa damascena flowering buds in vitro for the first time, and evaluate whether the generated callus has therapeutic effect as anti-inflammatory agent to suppress the activated T-cells from human blood. The flowering buds of Rosa. damascina were prepared and cultured in full strength Murashige and Skoog medium supplemented with kinetin and naphthaleneacetic acid. The callus were collected freshly after 40 days and, dried until dryness. Powdered callus was extracted with methanol by Soxhlet apparatus for about 6 hrs at 60◦C. The generated extract was used to treat Human PBMCs after activating T- cells with anti-human CD3 and CD28. The results from this study showed development of a new method to culture R. damascena flowering buds in vitro. The callus obtained is capable of suppressing the activated T-cells and thus can reduce the inflammation.

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Concentration and Partial Purification of Essential fatty acids as Co-products of Biofuels from Microalgal Consortium

Sujana Kokkiligadda and Srinivasa Reddy Ronda

The life cycle analysis of biofuels from microalgae shows a feasibility to recover essential fatty acids (EFA) as co-products from polar lipid components. Lipid fractionation of Spirulina platensis and Nannochloropsis gaditana showed 86 and 78% of γ-linolenic acid (GLA) and eicosapentanoic acid (EPA) distributed in glycolipid (GL) and glycolipid + phospholipid (PL) fractions respectively. Optimized solvent recovery of total lipids from the consortium and subsequent treatment with amphipolar solvent resulted in the selective separation of triglycerides and polar lipid components (PL+GL). FT-IR spectroscopic studies confirmed the partition of triglycerides and polar lipids exhibiting their respective characteristic peaks in the IR region. The EFA’s when concentrated from these organisms following winterization at +5, +10 and -20oC with 10% w/v, showed petroleum ether and hexane as ideal solvents at 20oC for effective concentration of GLA and EPA. The weight ratio optimization of EFA to the solvent showed 5% w/v at -20oC in petroleum ether followed by hexane and dichloromethane as suitable conditions for concentration and recovery of GLA and EPA fatty acids.

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Analysis of Althaea rosea seeds from different locations in Xinjiang using FTIR spectroscopy and multivariate analysis

Zhou Xiaoying, Liu Feng and Tian Shuge

This study focused on the identification and classification of Althaea rosea seeds and its extracts from different locations in Xinjiang using Fourier-transform infrared (FTIR) spectroscopy combined with cluster analysis and principal component analysis. Wave numbers between 1800 cm−1 and 400 cm−1 were selected for the establishment of all chemometric models after systematic noise reduction, 13-point smoothing and normalization of raw FTIR data. Results showed that the spectral data of A. rosea seeds and its extracts from different production areas had many similarities and differences mainly because of the six kinds of material compositions. Therefore, infrared spectroscopy combined with multivariate analysis is an effective, specific, rapid, nondestructive and nonpolluting identification method of identifying A. rosea seeds and its extracts. The method can also reflect the differences among A. rosea seed material component contents from different production areas.

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Effects of Salinity on the Photochemical Activity of Photosystems and Protein Subunits of Thylakoid in the Halophyte Salicornia bigelovii Torr.

Zhou Feng, Qiu Nianwei and Hua Chun

Salicornia bigelovii is one of the most salt tolerant plant species in the world and it actually requires high levels of Na+ for optimal growth. In this paper, the main peaks of the absorption spectra of plant under the optimal growth conditions of 400 mM NaCl were red shifted to 440 and 681 nm and compared to plants grown without salt, indicating that the microenvironment of chlorophyll molecules affected. It is shown that magnitude of circular dichroism spectra in the salt treatment was higher than in the control, demonstrating that excitonic interactions of pigments were stimulated. Interestingly, the typical positive peaks at 507 nm and 687 nm which are polymer or salt induced-type origin, increased significantly compared to the control. Furthermore, PSI activity increased with an increase in the salt concentration which might be partly due to the increased amount of Lhca1 and PsaA/B proteins. Salt treatment had no effect on CP 26 and CP 24 content while that of CP29 was stimulated.

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Difference of soil microbiota in Perennial ryegrass turf before and after turning green using high-throughput sequencing technology

Yan Zhu, Duiyuan Ai and Weibing Zhang

Turning green is very important during the growth of turfgrass; however, little is known about the differences in soil microbial communities before and after this occurs. Therefore, in the present study, the soil microbial diversity before and after turning green was studied by high-throughput sequencing technology and the corresponding microbial compositions were analyzed. The results showed that the soil bacterial and fungal communities were richer after turning green. The difference of soil microbial community at the genera level is more significant than that at phylum level. Among bacteria, a total of two dominant genera and 115 non-dominant genera were shared by the two samples while 25 non-dominant genera were present only in sample CP1 (before turning green) and 42 non-dominant genera present only in sample CP4 (after turning green). Among fungi, a total of two dominant genera and 57 non-dominant genera were shared by the two samples, 38 non-dominant genera were present only in sample CP1 and 38 non-dominant genera were present only in sample CP4. The results also indicated that both dominant and non-dominant microbial populations differed greatly before and after tuning green, as did the overall soil microbial community structure. This study provides previously unknown information regarding the impact of temperature and moisture on soil microbial communities during the period of turning green and lays a foundation for further investigations into microbiota in turfgrass soil.

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Molecular cloning and sequence analysis of the primary amine oxidase2 (MmPAO2) in mulberry and patterns of MmPAO2 gene expression under abiotic stress conditions

Chen Dandan, Dominic Kwame Kotoka and Zhao Weiguo

A full-length cDNA sequence coding a primary amine oxidase 2 (PAO2) of mulberry tree which we designated MmPAO2, was cloned based on mulberry expressed sequence tags (ESTs) and rapid-amplification of cDNA ends (RACE) technology. Sequence analysis showed that the MmPAO2 is 2364 base pairs (bp) long, its open reading frame (ORF) is of 2364 bp and encoding 787 amino acids with a predicted molecular weight of 87.53 kD and an isoelectric point of 6.54. Phylogenetic analysis based on MmPAO2 with other species showed that mulberry had a closer relationship with Chuansang (Morus notabilis XM_010104187.1), strawberry (Fragariavesca XM_004300959.2), plum (Prunusmume XM_008241099.1), apple (Malusdomestica KM067898.1), pear (Pyrus XM_009364674.1). The expression patterns of the MmPAO2 gene under conditions of drought, salt and ABA stresses were quantified by qRT-PCR. The results of quantitative PCR analysis showed that the transcriptional level of MmPAO2 mRNA changed significantly under the abiotic stress conditions compared to the normal growth environment. Overall, these results showed a better understanding of the molecular basis for signal transduction mechanism during the stress responses in mulberry trees.

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Expression, purification and characterization of a cholesterol oxidase from Arthrobacter simplex

Jing Zhang, Yang Han and Fuping Lu

Arthrobacter simplex CPCC 141369 was industrially used for steroid drug bioconversion which showed high conversion performance, especially for cholest-4-en-3-one production from cholesterol. To investigate the underlying biochemical basis, one of the cholesterol oxidase (AsCHO) encoding genes was cloned from the strain and heterologously expressed in Escherichia coli BL21. The recombinant protein exhibited optimal activity at pH 8.0 and 35 °C, was stable at pH 7–8 and 20–50°C and showed a specific activity of 12.5 U/mg at 35 °C and pH 8.0. The Vmax and Km values of AsCHO for cholesterol were determined to be 0.91 mM min-1 and 0.28 mM respectively. Compared with previously reported cholesterol oxidases, AsCHO showed significantly higher catalytic efficiency indicating its potential biotechnological applications.

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Molecular and Field based study to understand the Genetic Diversity for High Temperature Tolerance at Flowering in Pearl Millet (Pennisetum glaucum L.)

Jukanti Aravind, Manga Vinod and Pathak Rakesh

Pearl millet is emerging as an important summer cereal in Asia and Africa. High temperatures (≥42°C) during summer coincides with flowering resulting in poor seed set and reduced grain yield. At present, there are very limited options for summer cultivation of pearl millet. Field and molecular screening identified several inbred restorers (CZI 2002/6, CZI 2010/11 and CZI 2011/5) and populations (CZP 2K-9) tolerant to high temperature at flowering. Further, heat susceptibility index (HSI) and heat tolerance index (HTI) also confirmed the tolerance and susceptibility reactions of inbred restorers and populations. CZI 2010/11 showed the highest HTI (2.13), low HSI (0.69) and geometric mean (202.0 g/plot) among the inbred restorers while CZP 2K-9 (1.48, 0.70, 540.9 g/plot) showed amongst the populations. The percent yield reduction during summer as compared to kharif among inbred restorers and populations ranged from 32.8 to 57.1 and 25.9 to 44.0 respectively. Significant genetic diversity among the different genotypes was observed as demonstrated by high polymorphism (~73.0%). Polymorphism information content (PIC), Nei's gene diversity and Shannon's information index ranged from 0.25 to 0.80, 0.20 to 0.41 and 0.32 to 0.59 respectively. Cluster analysis and principal coordinate analysis (PCoA) were largely consistent. The separation of clusters in the dendrogram and PCoA plot was mostly similar and degree of grouping of genotypes was largely based on reaction to high temperature at flowering (tolerance/susceptible) as revealed by both the analysis. The high temperature tolerance sources identified here can be used for developing improved cultivars and their parents.

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Isolation and characterization of biocontrol fungi capable of infecting Amethystea coerulea- like tea garden weeds

Hong Yongcong and Xin Wei

Using of microbes to control weeds is becoming a new weed control method applied in the tea gardens. In this study, potential biocontrol fungi capable of infecting a tea garden weed Amethystea coerulea were isolated from the weed and their pathogenicity, host range and growth characteristics were also characterized. Eleven potential biocontrol fungus strains were isolated from the A. coerulea leaves with the symptoms of infection by the fungi. Of the 11 strains, the strain WYSJZ-B2, belonged to Curvularia sp., had strongest pathogenic capacity to A. coerulea and can infect all 8 tested weeds. Its optimal growth temperature was 28 ºC while the optimal initial pH value was 7. And its optimal lighting condition was 12 h light/12 h dark. In conclusion, Curvularia sp. WYSJZ-B2 has the potential to be developed as a herbicide for biological control of tea garden weeds.

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Variability in Biochemical and Yield Performance of Okra (Abelmoschus esculentus L.) Germpalsm under yellow vein mosaic virus infection

Meena Rakesh Kumar, Chatteerji Tanushree, Kumari Sweta, Verma Ambresh K. and Thakur Sanket

A comprehensive field study was carried out with ninety okra genotypes germplasm lines at Adithya Biotech Lab and Research Pvt. Ltd., Raipur along with the check VRO-6 (susceptible to yellow vein mosaic virus) to assess the variability in physiological parameters like phenol content, chlorophyll content and total sugar content along morphological and seed yield contributing traits during kharif 2013. The results revealed that out of ninety okra genotypes viz. AO:109, AO:119, AO:118, AO:133: AO:190, AO:189 and AO:190 were found to be resistance to YVMV infection over the check for seed yield and have higher phenol and chlorophyll content. The phenol and chlorophyll content are found to be significantly positively correlated with seed yield. It can be summarized that for Yellow Vein Mosaic Virus infection condition, the okra genotypes need to have not only high seed yield but also higher phenol, chlorophyll and lower sugar values.

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Cyclo-Pro-Phe effects on Staphylococcus aureus growth and energy metabolism

Duiyuan Ai

Cyclo-Pro-(D)-Phe-(D) and cyclo-Pro-(L)-Phe-(L) are cyclic dipeptides with broad-spectrum antibacterial activity. We assayed antimicrobial activity, growth, cell viability and total oxidation-reduction capacity (resazurin test). The effect on energy metabolism was estimated by its effect on the NADH/NAD+ ratio and ATP concentration. Cyclo-Pro-Phe did not have antibacterial activity but significantly reduced ATP level and increased NADH and NAD+ compared with controls. Cyclo-Pro-Phe was found to influence growth and energy metabolism of Staphylococcus aureus.

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Micro-propagation of Fir (Abies pindrow Royle.) from Juvenile Material

Bhat S. J. A.

An experiment was conducted to develop a protocol for micro-propagation of fir (Abies pindrow Royle.) from juvenile material. The sterilized explants (internodal segments) were inoculated in the MS medium supplemented with different concentrations of growth hormones and there was highest shoot proliferation (22.00%), number of shoots per explant (4.00) achieved on MS medium supplemented with BAP (3.00 mg l-1) and highest length of microshoots (13.20 mm) by MS medium supplemented with GA3 (0.10 mg l-1). The in vitro raised shoots were transferred for rooting to half strength MS medium containing different concentrations of IBA and NAA alone and in combination with activated charcoal. The highest rooting (10.66%) was achieved on half strength MS medium containing IBA (4.00 mg l-1) and activated charcoal (200 mg l-1). The rooted plantlets were kept for hardening under shade and irrigated with ¼ MS liquid medium for one month.

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