Research Journal of Biotechnology

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Vol. 14(4) April 2019

Would targeted gene modification cause nucleotide variation in the internal transcribed spacer (ITS) region of Danio rerio?

Sun Yan-Lin, Sun Yong-Jun, Bai Xin-Fu, Zhang Yi-Wei, Xu Tao, Wang Xin, Kee Yun and Hong Soon-Kwan

Page No: 1-6

Abstract: Plant gene engineering based on gene transformation and gene modification is used as an important tool for improving plant yield and resistance to stress, insect pests and plant diseases. Recently, plant gene engineering was used to study gene functions. In traditional T-DNA insertion or gene modification, because the insertion location and insertion copy numbers are not regulated, the transformants are random and various. Although new tools such as transcription activator-like effector nucleases (TALENs) have been developed for genome engineering, the binding specificity can be determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors.

However, whether gene targeting modification can affect the genome was not mentioned. In this study, we selected zebrafish samples through TALEN-mediated gene modification to examine the nucleotide variations in the internal transcribed spacer (ITS) region. The results showed that the ITS sequences among 23 zebrafish derived from one parent population showed abundant nucleotide variations, especially in the ITS1 region. Gene transformation has been suggested to affect the ITS region sequence. Though these variations on the ITS gene cannot lead to the failure of transformation, the nucleotide variations may occur in other important genes causing unsuccessful transformation.

Morphological and genetic variability of Fusarium solani causing chilli wilt disease in south India

Raghu S., Benagi V.I., Nargund V.B. and Jayalakshmi K.

Page No: 7-20

Abstract: Chilli wilt is an important emerging disease and causing serious damage in terms of yield and quality in major growing areas of south India for past few years. The present study was aimed to study the morphological, pathogenic and molecular variability of among the isolates collected from different geographical locations based on diseases intensity. Among surveyed regions, maximum disease incidence was recorded from Kurnool district of Andhra Pradesh (34.90 % PDI) followed by Bellary district of Karnataka. The least disease incidence was recorded at Chickmagaluru district (6.08). The disease incidence was more during 2013-14 when compared to 2012-13 in all the surveyed locations. Almost all the varieties were highly susceptible to the disease. Morphological and pathogenicity assays resulted in showed significant variability among the isolates. Pathogenicity of 44 isolates on highly susceptible varieties like Sitara and Byadgi dabbi indicated that 29 (65.90 %) were highly virulent, 11 (25 %) were moderately virulent and 4 (9.09 %) isolates were moderately virulent.

Molecular analysis based on the fingerprints of ITS sequence showed the presence of variability within the isolates based on geographical locations. The amplification of ITS gene generated approximately 550-600 bp bands. In phylogenetic analysis, based ITS region, overall three major groups were formed. Genetic similarity coefficient matrix as revealed by ITS sequence data among ten Fusarium revealed that maximum sequence homology (96.9 %) was found between Fs-4 to Fs-8 followed by Fs-3 to Fs-4 with 96.5% and Fs-3 to Fs-8 with 96.3 per cent sequence homology. The least sequence homology was observed between Fs-6 to Fs-1. Further restriction analysis of 30 selected isolates by EcoR1 and Taq1 enzymes showed significant variations among the isolates of Fuarium. The genetic diversity is most important to formulate efficient management strategies coupled with resistant breeding programmes. This study also helps to understand distribution of pathogen in different growing regions.

Inference of Phylogenetic relations among some Red Sea coral reef fishes via Sox14 gene sequence variations compared with other teleosts

Saad Y.M., Shaikh Omar A.K.M. and Gharbawi W.M.

Page No: 21-29

Abstract: Fish resources in the Red sea habitat lack accurate documentation and identification. In addition, the evolution of these biological resources is unclear. Nuclear Sox14 gene sequence analysis has been used to explore genetic variations and phylogenetic relations (using Maximum Likelihood and Neighbor-Joining methods) among various teleosts fishes belonging to five fish families (Scaridae, Labridae, Serranidae, Mugilidae and Cichlidae). The detected DNA polymorphisms among evaluated fish taxa were informative for inferring the evolutionary variations in these aquatic biological resources.

Sequence conservation values were relatively high within estimated fish family. The relatedness among evaluated fish species was presented and discussed. Cichlidae fishes are distantly related to both Scaridae and Labridae. The relationship between Mugilidae and both Scaridae and Labridae was nearly similar. The distance between Serranidae and Cichlidae was higher than between Serranidae and Mugilidae. Reconstruction of such phylogenetic relations based on Sox14 gene sequence variations was informative for understanding the evolution of these aquatic biological resources. More molecular markers (mitochondrial vs. nuclear DNA) should be developed for reconstructing the true phylogenetic relations among the fish taxa.

Genotoxic effect of Rhazya stricta leaf extract against larvae of Aedes aegypti

Baeshen Mohammed N., Al-Attas Sanaa G., Ahmed Mohamed M.M., Anwar Yasir, Faragalla Abdul-Rahman A., Ramdan Hassan A., Elkady Ayman I., Mahyoub Jazem A., Alabdali Huda A. and Baeshen Nabih A.

Page No: 30-36

Abstract: The biocontrol methods are extensively utilized for the control of pests. For this purpose, traditional plants are extensively used in Saudi Arabia. The predestined aim was the molecular evaluation of pesticidal effects of leave extract of a desert shrub Rhazya stricta (Decne) on larvae of Aedes aegypti (L.) (Diptera: Culicidae). Larvae of Aedes aegypti (L.) (Diptera: Culicidae) were exposed to different concentrations of Rhazya stricta aqueous leave extract (RSLE). Experimental approaches including comet assays, RAPD-PCR analysis and mitochondrial 16s rRNA gene analysis were carried out.

The results showed promising lethal effects on third and fourth instar larvae by using stock solution. The percentages of adult insects emerging from their pupas ranged between 22-90% depending on the different concentrations used. The inhibitory concentration for adult mosquitoes’ emergence reached about 0.057%. Phylogenic and RAPD analysis showed that these larvae are from 23 different genotypes. In addition, the extract can also inhibit the growth of human cervical carcinoma cell line, HeLa. Comet assay also confirms about the cytotoxicity compared to the control one. The whole data suggest that the RSLE could be an effective means for the biological control and can be used as pesticide.

Scanning Electron Imaging and Quantitative Spectral analysis of colony growth on Candida tropicalis MGR1, Clavispora lusitaniae MGR5, Wickerhamomyces anomalus MGR6 and Candida albicans MGR9

Vasuki S. and Suresh K.

Page No: 37-53

Abstract: Scanning Electron imaging and Quantitative Spectral analysis have the potential to improve the quality and accuracy of experiments designed to study the growth and invasion networks underlying different morphological traits. Cell budding and elevation edge detection were shown on Scale bar (5μm, 10μm, 40μm, 50μm) by digital form of SEM image. Candida tropicalis MGR1 (KY209901), Clavispora lusitaniae MGR5 (KY209902), Wickerhamomyces anomalus MGR6 (KY209903) and Candida albicans MGR9 (KY209906) showed morphological characters of bud scars, bud margin and interconnection, but commonly in spherical shape. Number of cells and size of the colony were characterising on YEPD solid media in five consecutive days by image analysis method.

Quantitative spectral analysis by different growth rates was carried out in four liquid media (YEPD, CMA, Malt extract medium and ½ Xylose medium). The growth curves and invasion were characterized by the presence of long transition periods. Prominent comparison of different growth phases in cultures, the minor growth rates changes was made in each isolate duration of log phase in all four media. Among the growth phases of YEPD medium, highest growth spectrum observed. This work also provides a direct, simple method to estimate the metabolic yield of a colony and nutritional sensing for quality prediction in the growth progress of yeast.

A multiple pesticide resistant Bacillus cereus PR-2J isolated from paddy field - a potential strain for pesticide detoxification

Mohapatra Ranjan Kumar, Pandey Sony and Panda Chitta Ranjan

Page No: 54-64

Abstract:The soil bacteria, residing under pesticide stress, are able to resist and detoxify these toxic pesticides into nontoxic products. The objective of the present study is to isolate and identify the bacteria highly resistant to multiple pesticides from cultivated paddy field. About 20 bacterial strains were isolated and screened against four different pesticides namely endosulfan, phorate, carbofuran and carbaryl. Out of them, seven strains showed resistance to all four pesticides at a concentration of 150 mg/l and the strain PR-2J was resistant to these pesticides even at concentration >2000 mg/l. The MIC tests for all the four pesticides showed that the strain PR-2J was susceptible to 2200, 3000, 2700 and 2700 mg/l of endosulfan, phorate, carbofuran and carbaryl respectively.

The identification of the bacterium PR-2J was established on the basis of 16S rRNA gene sequencing and biochemical characterization which showed 99% 16S rDNA sequence similarity to the Bacillus cereus. This strain can not only grow in 200-500 mg/l of each pesticide without any growth inhibition, but it is also able to grow in a mixture of all four pesticides at 1000 mg/l concentration. PR-2J is also able to utilize pesticides as a carbon source in mineral salts minimal media. So, the isolated bacterium PR-2J is an excellent pesticide resistant strain of Bacillus sp. which may be deployed in the bioremediation of multiple pesticide-contaminated sites.

Anti-oxidant activity, nutritional, multi-elemental and bioactive profile of Sikkim Himalayan indigenous Chili (Dalle Khursani)

Khatoon Uzma and Sharma Laxuman

Page No: 65-72

Abstract:Dalle Khursani is known to be one of the hottest chilies belonging to Capsicum annum group. The crop is found growing in Sikkim and adjoining hills of Northeastern India. People of these hills, especially, Sikkim have gained enough expertise in organic cultivation, seed production and post-harvest processing of the crop and now it is one of the commercial crops of Sikkim. Being a constituent of the daily diet of the people in Sikkim, it is imperative to understand the nutritive value, mineral content, bioactive compounds and antioxidant activity.

The results showed the appreciable nutrient content in Dalle Khursani and most of the proximate content was higher compared to green pepper. All the elements were also present in an appreciable amount. Dalle Khursani was rich in phytochemical content and also having a good antioxidant capacity. The acceptability, pungency and the flavour of the fruit make it more appealing to the consumer and hence increased consumption can be promoted and genetic resources of such an important crop may be conserved or maintain for further improvement.

Inheritance of fruit weight and mode of gene action for yield contributing traits in tomato

Chauhan V.B.S., Kumar R., Behera T.K., Yadav R.K. and Verma A.K.

Page No: 73-78

Abstract: The objectives of this study were to know the inheritance of fruit weight and type of gene action controlling economic traits of tomato. To understand the inheritance of fruit weight, high fruit weight (48.50 g) cultivar Roma was crossed with low fruit weight (6.43 g) cultivar Cherry Orange to develop F1 and F2 populations. The F2 population of the cross was segregated in ratio of 9:6:1 (9 high fruit weight: 6 medium fruit weight: 1 low fruit weight) and indicated digenic inheritance with incomplete dominance of high fruit weight over low fruit weight whereas existence of much variability in fruit weight of F2 population apparently raises the possibility of presence of multiple allelic series for major gene associated with fruit weight determination.

To cognize the gene action, cross Roma × Cherry Orange was used for study the genetic parameters. Additive gene effects were found to be important in the inheritance of average fruit weight, fruit length and fruit size. Dominance gene effects were important in the inheritance of average fruit weight. Concerning to the positive and significant epistatic gene effects, additive × additive were recorded for average fruit weight and fruit size, additive × dominance for number of fruits per plant and dominance × dominance for number of fruits per plant, fruit length and yield per plant. Duplicate epistasis was recorded for number of fruits per plant, average fruit weight and yield per plant while complimentary epistasis was recorded for fruit length, fruit diameter and fruit size.

Novel mutations in mitochondrial DNA driving the susceptibility of bladder cancer

Mahmoud Maged Mostafa, Abdullah Layla S., Mahmoud Wael Mahmoud and Yacoub Haitham Ahmed

Page No: 79-85

Abstract: The aims of current investigation are to identify association between mitochondrial DNA mutation and bladder cancer in Saudi patients, to establish the frequency of mitochondrial DNA variation in bladder cancer in Saudi patients and to apply the mitochondrial DNA mutation as a prognostic marker in a certain disease. We identified 175 mitochondrial mutations in displacement region (D-loop) as SNPs in all patients’ tissues with (3.7 SNPs per patient). Eight of novel mutations in mitochondrial d-loop segment were characterized in bladder cancer subjects and all of them are nonsynonymous SNPs at different positions in d-loop region.

Three of those alterations were frequently mutated at these sites G200A, G189A and T150C with high proportions of 90%, 80% and 76% respectively. We also observed that the heteroplasmy proportion of the point mutations was 59% with 25% per patient. We suggested that these novel mutations are associated with susceptibility of incidence of bladder cancer and its applications remain to be defined and warrant further laboratory investigation.

Ergosterol quantification: a tool to measure fungal infection in plant tissue

Singh Yashaswi, Thakur Ajay, Varshney V.K. and Harsh N.S.K.

Page No: 86-88

Abstract: Ergosterol, a distinguishing sterol in the fungal cell membrane has been utilized to characterize and quantify fungal biomass. Dalbergia sissoo, an important timber yielding tree species of the Indian subcontinent has been facing severe mortality in plantations and natural forests and Fusarium solani f. sp. dalbergiae has been identified as one of the primary disease-causing pathogenic fungi.

Hence, to screen for disease tolerant genotypes of D. sissoo, ergosterol is being utilised as a chemical marker. An analytical protocol for ergosterol extraction and quantification from different fungus and fungal infected tissue has been standardised. Results of the study confirm the presence of ergosterol in the samples. Extracts of F. solani f.sp. dalbergiae and fungal infected D. sissoo callus had 9.8 mg/g and 1.56 mg/g ergosterol respectively. Ergosterol quantification will support the traditional disease score of tolerant genotypes so that an accurate assessment of tolerance of genotype to the pathogen could be achieved.

Analysis of synonymous codon usage bias of Dengue, Zika and Yellow fever virus and their vector - host adaptation

Mune Anjusha, Pandey Khushhali and Pandey Ajay

Page No: 89-110

Abstract:Virus vector and host relationship is a unique biological interaction that involves virus entry, escapes mechanism and hosts immune system. The change in codon usage pattern of viruses is an evolutionary event that facilitates their survival, in addition to adapting their fitness to the external environment, especially their hosts. The current work is a study of codon usage patterns of Zika virus (ZIKV) strains, Dengue virus (DENV) serotype and Yellow fever virus (YFV). It also aims to study the codon usage pattern of their primary vector Aedes aegypti and host Homo sapiens.

The study - based observed similarity is expressed in terms of choices of preferred codons in virus, vector and host. It is also observed that the codon usage bias of viruses is influenced by mutation pressure and natural selection. The adaptation of codon usage helps the virus to adapt to multiple host and vectors. The results suggest that viruses have a distinct, identifiable host and vector - specific codon usage pattern that is advantageous for efficient replication in host and transmission within vectors and hosts.

Studying Single Nucleotide Polymorphism in Insulin Receptor (INSR) Gene and its association with PCOS and Diabetes and Breast Cancer in the Iranian Population

Shirin Khaknejad Salim and Abolhasan Rezaei

Page No: 111-118

Abstract:The mutation of the insulin receptor gene is common with diabetes, PCOS and cancer in the women population. In this study, the rs52826008 in insulin receptor (INSR) gene was used to determine the relation between INSR and patients with diabetes, diabetes and breast cancer and PCOS and diabetes by examining a control group within the Iranian population. Clinical endocrinology and molecular tests were used in this study. Table 1 shows academic endocrinology including hormonal and biochemical analysis as LH/FSH ratio, Testosterone, LDL, HDL and similar testes between the control and case group. The calculated mean ± SD and Tuckey’s post hoc test were similar to the correlation between clinical characterization and the HDLC and SHBG levels between the three groups (control, diabetes and PCOS-diabetes patients). However, control, diabetes and PCOS- diabetes patients had higher BMI, HDL cholesterol, fasting insulin and fasting blood glucose with significant index and lower LDL cholesterol with insignificant index.

PCR products were sequenced and aligned using DNAMAN Demo Software Version 8.0. Figures 1, 2, 3 and 4 visualized by Chromas software showed no SNP on the position of rs52826008 (CGG was mutated to CAG). On the other hand, according to table 2, one-way ANOVA followed by Tuckey's post hoc test showed no significant variation between control, diabetes, PCOS and diabetes and diabetes and cancer patients (p>0.05). Finally, the results concluded that there is no significant variation between INSR gene and diabetes, diabetes and breast cancer and PCOS and diabetes patients. The current study concluded that no variation exists between the control and case groups based on rs52826008 in the insulin receptor (INSR) gene.

Screening and Characterization of Potent Tanninolytic Bacterial Isolate from Rhizospheric Zone of Cycas Species

Dhiman Sunny and Mukherjee Gunjan

Page No: 119-123

Abstract:The objective of present research work was to screen and characterize the potent tannin hydrolysing bacteria from the rhizospheric soil of Cycas species. Tannin hydrolysis leads to gallic acid production which has its utilization in a plethora of applications in various sectors ranging from therapeutics and food industry to agriculture and photography. Tannin hydrolysing bacterial isolates were screened from the soil samples collected from rhizospheric zone of plants belonging to Cycas species. Soil samples were enriched with enrichment media containing tannic acid (1% w/v). Enriched samples were plated on Tannic Acid Agar (TAA) plates.

Zone of tannic acid hydrolysis around bacterial colonies confirmed their tannin biodegradation potential. The quantitative evaluation of tannase activity of potent tannin hydrolyzing bacterial isolates was done under submerged conditions. Bacterial isolate B-2 exhibited maximum tannase activity (0.83 U/ml) at temperature 370C, pH (5.5) upon 36 hours of incubation under submerged conditions. The bacterial isolate B-2 exhibiting maximum tannin hydrolysis and maximum tannase production was identified as Bacillus sp. based on morphological characteristics, microscopic examination, gram staining and biochemical tests.

Gene delivery by Non-viral vectors: An attempt to get through barrier in gene transfer

N. Lalitha Jyotsna and N. Saritha

Page No: 124-129

Abstract:Vectors for gene transfer are available extensively. But in spite of numerous advances in the field of molecular biology for delivering genes into the cells, none of these fulfills all requirements of gene transfer. The non-viral methods of gene transfer appear to be valuable theoretically but found to be lagging behind practically. The artificial non-viral vectors have to cross the intracellular and extracellular barriers and to increase the efficiency of gene transfer. These barriers included the manufacture, preparation and preservation of the carrier.

The transfer of gene to the targeted cells and delivery of genes remains prime object to be concentrated. The new vectors are incessantly synthesized and scrutinized. We made an effort to detail the different types of non-viral carriers by emphasizing the improvement and current advances that had enhanced the non-viral gene delivery effectively and confer the safety concern of the non-viral vector.

Molecular phylogeny of mullet fishes using Sox14 and COI gene sequence variations

Saad Y.M., Mohamed J.S. and El-Domyati F.M.

Page No: 130-146

Abstract:The present study was designed for testing the utility of Sox14 gene sequences to identify the molecular variations among three mullet fishs (M. seheli, L. carinata and M. cephalus) comparatively with COI barcoding system. DNA polymorphism within each estimated genus was presented as single nucleotide polymorphism, haplotype diversity and nucleotide diversity. Overall distance values within evaluated Mugilidae fish samples were 0.122 and 0.007 based on COI and Sox14 sequence variations respectively.

The results indicated that M. seheli is distantly related from M. cephalus and L. carinata. The M. cephalus is closely related to L. carinata. Mugilidae evolutionary variations were estimated also comparatively with other ray finned fishes. Both nuclear and mitochondrial DNA markers consistently separated the estimated fish species. The genetic information based on mitochondrial COI and nuclear Sox14 genes has a value for the future fish conservation strategy.