Research Journal of Biotechnology

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Vol. 12(3) March 2017

Cloning of DFR gene in Curcuma alismatifolia ‘Chiang Mai Pink’ and Agrobacterium-mediated transformation

Ratchanee Petchang, Phopgao Buddharak, Ruttaporn Chundet and Warut U-kong

Dihydroflavonol 4-reductase (DFR) gene is a key gene of anthocyanins biosynthesis pathway which represent an importance pathway for flower. In this study, cloning of DFR gene in Curcuma alismatifolia ‘Chiang Mai Pink’ and gene transformation was carried out. Nucleotide analysis revealed that the DFR gene was 850 bp in fragment and encoded a protein of 178 amino acid residues. Homology analysis revealed that the amino acid sequence of the DFR gene product was 63 to 96% identity to amino acid sequences of DFR gene products of other orchids such as Anthurium andraeanum, Citrus sinnensis, Camellia sinensis and Rhododendron simsii. Transformation of DFR gene into Kalanchoë blossfeldiana was made by Agrobacterium tumefaciens strain LBA4404 harboring the binary vector, pBI121-DFR. Transformation of these plants was confirmed by GUS histochemical assay and PCR analysis. Results showed that the transformation frequency exceeded 38.33% which is suitable for practical use. The resultant transgenic petals produced anthocyanins, their magenta color was more brilliant than that of the petals of wild-type by expressing the DFR gene. These results indicate that the DFR gene is a useful molecular tool for altering and diversifying flower color.

Photocatalytic degradation of malachite green using polyoxotungstate-titanium oxide nanocomposite

Seyed Sadjadi Mirabdolah, Rashidzadeh Solmaz and Farhadyar Nazanin

Heterogeneous photocatalysts such as 12-tungstophosphoric acid/titanium oxide nanocomposites (n-PW/TiO2) are attractive and have received great attention to degrade or remove environmental contaminants. PW/TiO2 nano- composites containing 10 and 20wt.% PW were synthesized by simple in-situ method. Characterization of nano composites samples was carried out by X-ray diffraction (XRD), Scanning electron microscopy (SEM), Fourier transform infrared (FTIR). Results indicated the formation of spherical n-PW/TiO2 nanocomposites in a particle size at around 25 nm. Photocatalytic activities of the nanocomposites were evaluated by malachite green decomposition. Malachite green removal was evaluated in dark, under UV illumination at the presence of nPW, nTiO2 and n-PW/TiO2 nanocomposites prepared in this work. The results showed that n-PW/TiO2 have higher photocatalytic activity than that of nPW, nTiO2 in removing of malachite green under UV illumination and in the presence of photo-catalyst.

Optimizing a Protein’s Expression in an Efficient Way

Zhi Li, Yi Li and Fang-Yin Dai

Prokaryotic expression is a common way for heterologous expression of proteins. For achieving high-level expression in E. coli, optimization is essential followed by the recombinant protein. The conditions optional for optimization contain host strain, expression vector design, gene dosage, codon usage, mRNA level and growth conditions/media. A series of factors tightly related to growth conditions involving incubate temperature, OD600 value, IPTG concentration, antibiotic concentration and time of induction have known effect on a protein’s expression. Generally, ‘One factor at a time’ method is used to optimize a protein’s expression based on these factors; however, this efficiency is low and the connections between factors are thoroughly ignored. Here, design of experiment (DoE) is introduced for optimization of protein expression in this study revealing an interaction between OD600 and incubate time which can significantly influence the expression level of our recombinant protein Bmattacin2 in addition to each separate one. The result of optimization verifies that DoE is an efficient way for optimizing a protein’s expression since it can significantly improve protein expression level compared with conventional ‘One factor at a time’ method.

Endophytic interactions of Trichoderma harzianum in a tropical perennial rhizo-ecosystem

Palaniyandi Umadevi, Muthuswamy Anandaraj and Sailas Benjamin

This study demonstrates the endophytic interaction of the well-known growth promoting and biocontrol agent in black pepper. Trichoderma harzianum, coupled with its rhizosphere fungal flora was evidenced from metagenomics. We employed short-term and long-term strategies to study the interactions of T. harzianum in black pepper rhizosphere. In short-term strategy, T. harzianum was co-cultivated with axenic plantlets while pot culturing of plants in soil mixed with T. harzianum was performed in the long-term strategy. The colonization was investigated by light microscopy and scanning electron microscopy (SEM). The co-cultivation of T. harzianum with black pepper showed the intercellular colonization at 24 h and formation of intracellular hyphae with vesicles at 48 h of interaction. The long term strategy inferred that T. harzianum was able to colonize the black pepper roots along with the AMF inter- and intra-cellularly. The whole genome metagenomic sequencing brings out the population abundance of the entire rhizosphere fungal flora.

Effect of inductive factors on synthesis of paracin C— a bacteriocin against Alicyclobacillus spp.

Pei Jinjin

Paracin C is anti-Alicyclobacillus bacteriocin which showed favorable potential application as an industrial food preservative in acid fruit juices. The study explored the exogenous factors that affect the synthesis of paracin C by a resting cell system. The resting cell medium contained (g/L): Lactose 20 g/l, Sodium acetate 5.0 g/l, MnSO4 0.25 g/l, MgSO4 0.5 g/l, Ammonium hydrogen citrate 1.0 g/l and KH2PO4 1.0 g/l. In this work, synthesis of paracin C was self-induced with a threshold at 10 AU. Inducing effectiveness was observed when paracin C was added as an inducer at the beginning of the exponential phase. Gly, vitamin B1, vitamin B2, Glycerol and pyruvic acid were able to be used as inducers of paracin C synthesis. Besides, sensitive strains greater than 103 CFU/ml co-cultured with Lactobacillus paracasei CICC 20241 successfully induce the synthesis of paracin C. Resting cell system has been reported as a means to study and screen for the effects of inductive factors on bacteriocin production. The findings are of importance for the bacteriocin biosynthesis regulation and improvement of bacteriocin production yields which will facilitate the application of this bacteriocin in food industry.

Combined effect of Arabian coffee, cardamom and cloves on obesity associated insulin resistance in high-fat diet (HFD)-induced C57BL/6J MICE

Ghedeir M. Alshammari

The objective of this study was to investigate the antihyperglycaemic and antihyperlipidemic efficacy of Arabian coffee, Cardamom and Cloves on HFD induced diabetic mice. C57BL/6J mice that were fed a high fat (HFD) developed obesity, hyperglycemia and hyperinsulinemia compared with those that were fed a Standard pellet diet (SPD). Mice fed with HFD increased the levels of plasma glucose, insulin, glycosylated hemoglobin and a decrease in hemoglobin. The activities of carbohydrate metabolic enzymes such as glucose-6-phosphatase and fructose-1,6-bisphosphatase increased while glucokinase activity and glycogen level decreased in HFD mice. Treatment with Arabian coffee, Cardamom and Cloves significantly reduced the plasma glucose, insulin and HbA1c and it also elevated the levels of Hb. Arabian coffee, cardamom and cloves supplementation to diabetic mice reverted back the activities of carbohydrate metabolizing enzyme in a significant manner and increased the liver glycogen level. The increased levels of lipid profile such as total cholesterol (TC), triglycerides (TG), phospholipids (PL) and free fatty acids (FFA) were observed in both the plasma and tissues of HFD-fed C57BL/6J diabetic mice. The plasma lipoproteins, very low density lipoproteins-cholesterol (VLDL-C) and low density lipoproteins-cholesterol (LDL-C) were significantly increased and the plasma levels of high density lipoproteins-cholesterol (HDL-C) were decreased in HFD-fed C57BL/6J diabetic mice. An HFD-induced C57BL/6J diabetic mouse treated with Arabian coffee, Cardamom and Cloves signiﬁcantly reversed all these parameters towards normalcy. Moreover, combined effect of Arabian coffee, cardamom and cloves has more pronounced effect than their individual treatment. These ﬁndings suggest that Arabian coffee with cardamom and Cloves is having better antihyperglycaemic and antihyperlipidemic effect on HFD-fed C57BL/6J diabetic mice.

Toll-like receptor 2 gene polymorphism and mastitis resistance in indigenous and crossbred cattle

Haris Girish, Sivaselvam S.N. and Karthickeyan S.M.K.

Exon 1 and exon 2 of TLR 2 gene were amplified by PCR. The sequence analysis revealed 24 mutations: one in exon 1 and the rest in exon 2. Out of these 24 mutations, 22 were identified as SNPs of which seven were non-synonymous mutations. The allele frequencies were found to differ between healthy and mastitic cows in 11 SNPs. In exon 1, the variation was found at position199C>A with C being the major allele and A the minor allele with respective frequencies of 0.64 and 0.36. Highly polymorphic exon 2 contained 23 variations within 2508 bp region. At position 11538 of the TLR 2 gene, the Bos taurus sequence has T allele which is completely replaced by C in both crossbreds and native cattle. In SNP at position 11594, the Bos taurus sequence has G allele while the native animals showed A and at position 11613 the Bos taurus had C allele while the native cattle had T allele. Among the three genotypes in 10095G>T, GG and GT genotypes were found to have lower mean SCS (5.207 and 6.633) than the TT genotype (10.380). GG genotype had the lowest SCS and it could be a mastitis-tolerant genotype.

Diversity of rhizospheric Bacillus sp. isolated from Corn and Wheat fields and their use in growth promotion of plants

Nawaz Huma and Faisal Muhammad

This work deals with the diversity of Bacillus in Wheat and Corn rhizospheric soil from different areas of Punjab, Pakistan. Strains in corn rhizosphere are Bacillus subtilis, Bacillus cereus, Bacillus axarquiensis, Bacillus safensis, Bacillus endophyticus andBacillus pumilus whereas Bacillus subtilis, Bacillus anthracis, Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus cereus and Bacillus subtilis are found in wheat rhizosphere. The traits which are important for plant growth promotion are studied for all strains. Bacillus subtilis, Bacillus cereus strains were involved in siderophore production whereas about nine strains from both rhizospheres were phosphate solubilizers. CSH27 (82 µg ml-1) and CSH4 (112 µg ml-1) were highest auxin producers among corn strains whereas WSH8 (108 µg ml-1) and WG1 (68 µg ml-1) were highest auxin producers among wheat isolates. CMS17 showed more ACC deaminase activity whereas majority wheat isolates showed more activity of ACC deaminase. Some of these isolates were also involved in nitrification and denitrification. These plant growth promoting traits may be helpful to use these isolates as biofertilizer.

Soil microbiota variation in Perennial ryegrass turf and Festuca elata turf using illumina sequencing

Zhang Weibing and Yao Tuo

Soil bacteria and fungi play key roles in ecosystem functioning and the maintenance of soil fertility. Many studies have been carried out to assess the effects of plant on microorganism communities in different environments. However, little is known about whether turfgrass species affect the microbial community in soil. Therefore, in the present study the microbial diversity in two kinds of turf-grass soil was studied by high-throughput sequencing technology and the corresponding analysis of microbial composition was conducted. The results showed that the bacteria and fungi in the soil of Perennial ryegrass turf were richer than that in the soil of Festuca elata turf. The difference of soil microbial community at the genera level is more significant than that at phylum level. Among bacteria, a total of seven dominant genera and 119 non-dominant genera were shared by the two samples while 19 non-dominant genera were present only in sample CP1 (Perennial ryegrass turf) and 25 non-dominant genera were present only in sample CP2 (Festuca elata turf). Among fungi, a total of six dominant genera and 90 non-dominant genera were shared by the two samples, 12 non-dominant genera were present only in sample CP1 and 16 non-dominant genera present were only in sample CP2. The results also indicated that both dominant and non-dominant microbial populations differed greatly in the two samples as did the overall soil microbial community structure. This study provides previously unknown information regarding the impact of turfgrass species on soil microbial communities and also lays a foundation for further investigations into microbiota in turfgrass soil.

Genetics of post flowering stalk rot resistance (PFSR) genes caused by Macrophomina phaseolina (Tassi) Goid. in maize

Suneetha P. and Anuradha G.

Maize is one of the most important economic cereal crops and an ideal forage crop. The productivity of maize is influenced by both biotic and abiotic factors. More serious diseases are leaf blight; stalk rots, downy mildews and rusts. Post flowering stalk rot (PFSR) is the complex disease caused by Macrophomina phaseolina. To study the genetics of post flowering stalk rot caused by Macrophomina phaseolina, maize inbred lines JCY3-7-1-2-1-b-1 (resistant) and 5238 (susceptible) were crossed to produce F1. F1 was selfed to produce F2 population and crosses to produce BC1F1population. F1, F2 and BC1F1 populations derived from the parents were inoculated with the pathogen M. phaseolina which causes post flowering stalk rot. Field data revealed that F1’s was resistant indicating that the resistance is governed by dominant gene and the ratio of resistant to susceptible plants was 3:1 in the F2 population and 1:1 in the BC1F1 population indicating that the resistance to M. phaseolina was controlled by a single dominant gene.

Optimization of dansyl chloride derivatization conditions for biogenic amines determination and survey of biogenic amines in some fermented foods

Cai Chenggang and Sha Ruyi

Biogenic amines are mainly formed by decarboxylation process of amino acids in different foods especially some fermented products. Dansyl chloride had been utilized as derivatization agents for BAs determination. The optimal derivation conditions varied between several studies. In this study, an experimental design was used to optimize the variables involved in the dansylation of the following amines: histamine, tyramine, phenylethylamine, tryptamine, cadaverine, purtrescine, spermidine and spermine. The optimal experimental conditions for forming the dansyl derivatives are pH value of 9.45, 62.4 ºC and 46.8 min. Under the optimized conditions, BAs contents in several Chinese fermented products of rice wines, fermented soy jams, fermented sausage, hams etc. were analyzed. The results showed that BAs ranged from 18 to 2179.3 µg/g in different samples.

Isolation, optimization and purification of endoxylanase from newly isolated Bacillus sp.

Shanthi V. and Roymon M.G.

A new endoxylanase producing bacteria was isolated from forest soil of Bhilai-Durg region of Chhattisgarh (India) and was identified as Bacillus sp. MCC2728. Optimization of both cultural and nutritional factors was done for maximizing xylanase production. Maximum xylanase production was observed after 72 hours of incubation. The optimum pH and temperature were 9.0 and 55°C respectively. 5% inoculum size with a shaking speed of 150 rpm produced optimum levels of xylanase. Wheat bran served as the best carbon source, a combination of peptone and yeast extract proved to be the optimum nitrogen source. Pre-treatment of wheat bran using 0.5N NaOH increased the enzyme yields. Among the different supplements tested, xylose was found to be the most suitable supplement capable of inducing maximum xylanase production. K2HPO4 was the best metal salt. The enzyme was purified by a two-step purification process using 80% saturated ammonium sulfate solution. The total activity was 4430 U. The specific activity was determined as 518.13 U/mg and the purification fold was 5.47 with yield 34.50%. SDS-PAGE analysis showed two protein bands of 25-30 KD and 38-40KDa. The ability of purified xylanase from Bacillus sp. MCC2728 to hydrolyze birch wood xylan was confirmed by zymogram analysis.

Partial molecular characterization of the Fig latent virus 1 (FLV-1) infecting figs in Western Saudi Arabia

Elbeshehy Esam K.F., Aldhebiani Amal Y. and HassanWael M.

A virus with filamentous particles ca. 700 nm long, denoted Fig latent virus 1 (FLV-1) is widespread in Western Mecca region, Kingdom of Saudi Arabia (KSA) fig orchards. RT-PCR assay was conducted on 80 fig samples collected from four fig-growing provinces including Fatima, Khulais, Rabigh and Al Shifa valleys which consisted of leaf tissues gathered from Capri fig and common fig to investigate the presence of FLV-1. Results showed that the virus was present in the survey areas with different levels of infections. The infection rate reached 33.75%, with a peak of 35% in Alshifa and Fatima valley provinces followed by 40% and 25% in Rabigh and Khulais valley provinces respectively. The specific primers CPtr-s and CPtr-a for FLV-1-cp. gene yielded 442 base pair DNA fragments. The results showed the isolate belong to characterize the FLV-1-KSA isolate. The following sequences were used in the comparison: FLV-1(FN377573.1), FLV-1(KM516762.1) and FLV-1(KM516763.1). The partial nucleotide sequence alignment showed (99%) homology between FLV-1(FN377573.1) isolate and FLV-1(KM516762.1) isolate and 98% between FLV-1(KM516762.1) isolate and FLV-1(KM516763.1) isolate. On the other hand, the sequence alignment also indicated that the FMV-1(KSA) isolate is a similarity with FLV-1(FN377573.1), FLV-1(KM516762.1) and FLV-1(KM516763.1) showing 86% according to the phylogenetic homology tree.

High level expression of soluble recombinant porcine interferon alpha 8 using cold-inducible expression systems

Xia Ye, Guo Jiahong, Ling Hongli and Miao Denian

The porcine interferon alpha (PoIFN-) exerts broad-spectrum antiviral effects on various viruses. To date, most of recombinant PoIFN- (rPoIFN-) prepared from Escherichia coli were deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant PoIFN-8 (rPoIFN-8). This was gained by codon optimization, cold induced expression vector and optimization of expression conditions. An E. coli codons optimized and synthetic mature PoIFN-8 was cloned into the cold-shock expression vectors of pCold II and then transferred into Escherichia coli BL21 (DE3) to induce by IPTG. By optimizing inducer concentrations and induction duration, rPoIFN-8 was expressed primarily as a soluble protein. Purification of the recombinant TEE-His-tagged proteins was achieved in a single step by Ni2+-charged column chromatography. The final yield of soluble rPoIFN-8 was about 100mg from 1L flask culture. The purified rPoIFN-8 was confirmed by western blot. The antiviral activity of rPoIFN-8 against VSV on PK-15 cells was up to 1.0×109 IU/mg of protein. Our study provides a feasible and convenient approach to produce large quantities of soluble and biologically active rPoIFN-8 for both research and industrial purpose.

The transcriptome and proteome of Amphibalanus amphitrite

Vibin Jessy and Kumar Pradeep

Amphibalanus amphitrite, an acorn barnacle, found worldwide is a model organism for the study of biofouling and ecological research. This mini-review focuses on the recent transcriptome and proteome studies that have been carried out on this organism for better understanding of how marine organisms attach to a substrate causing biofouling. Still much is to be elucidated for the development of a good antifouling agent which will be cost efficient and eco-friendly.