The high-throughput sequencing technology provides a platform for revealing the expressed genes within a tissue at a specific time. The giant freshwater prawn,
Macrobrachium rosenbergii,
is an ...economically important species, which is surviving in a wide-range of salinity. In this study, to understand the physiological mechanism of adaptability with respect to moulting and salinity; transcriptome sequencing of larvae and post-larvae of
M. rosenbergii
was performed using the Illumina GAIIx platform. The generated raw read-data comprised 71,391,946 and 75,276,622 paired-end reads (PE) for larvae and post-larvae respectively. Using CLC bio Genomic Workbench version 7.5 (CGWB), 71.39 million and 75.27 million of each 72 base paired-end, high quality reads were assembled into 43,383 (N50 1852) and 44,960 (N50 1874) transcripts, respectively, for larvae and post-larvae. The nucleotide level annotation of both transcriptomes showed significant similarity with unigenes of closely related species. The Gene Ontology analysis suggested enrichment of transcripts involving several biological processes linked to transcriptional regulation, signal transduction, immune response, ion-binding. Differential gene expression analysis using CGWB and DESeq identified 9680 deregulated genes of which 3454 unigenes were up-regulated and 3068 down-regulated by ≥1.5 fold (
p
< 0.05) in larval stage compared to post-larval stage. However, in larval stage 938 genes were down regulated and 1599 genes up-regulated by ≥3 fold with
p
< 0.05. GO enrichment of differentially expressed genes was shown several molecular functions for maintaining homeostasis against salinity stress. To validate the expression patterns, few transcripts were chosen for quantitative real-time PCR that showed the consistency and exactness of our analysis. In addition, we also speculated the enzymatic pathway using KEGG, which depicted that up-regulated genes are involved in several significant metabolic pathways and those are critical for maintaining osmoregulation and linked with metamorphosis. Therefore, we have generated valuable information of salinity tolerant genes in the larval and post larval stage of
M. rosenbergii
during salt- and freshwater compliances, which will be further harnessed for gene targeting. The present finding would provide the basis for further screening of salt tolerant genes associated markers for selective breeding.
Immune response mediated by toll-like receptor 22 (TLR22), only found in teleost/amphibians, is triggered by double-stranded RNA binding to its LRR (leucine-rich repeats) ecto-domain. Accumulated ...evidences suggested that missense mutations in TLR genes affect its function. However, information on mutation linked pathogen recognition for TLR22 was lacking. The present study was commenced for predicting the effect of non-synonymous single-nucleotide polymorphisms (nsSNPs) on the pathogen recognizable LRR domain of TLR22 of farmed carp,
Labeo rohita.
The sequence-based algorithms (SIFT, PROVEAN and I-Mutant2.0) indicated that three SNPs (out of 27) such as p.L159F (rs76759876) and p.L529P (rs749355507) of LRR, and p.I836M (rs750758397) of intracellular motifs could potentially disrupt protein function. The 3D structure was generated using MODELLER 9.13 and further validated by SAVEs server. The simulated molecular docking of native TLR22 and mutants with poly I:C ligand indicated that mutations positioned at p.L159F and p.L529P of the LRR region affects the binding affinity significantly. This is the first kind of study of predicting nsSNPs of teleost TLR22 with disturbed ligand binding affinity with its extra-cellular LRR domain and thereby likely hindrance in subsequent signal transduction. This study serves as a guide for in vivo evaluation of impact of mutation on immune response mediated by teleost TLR22 gene.
Background: Feed cost mainly constitutes 40-60% of total aquaculture production expenses, due to the inclusion of expensive animal-derived ingredients to the feed industry. Studies suggested that ...herbivore and omnivore fish species can consume higher level of carbohydrate (150–300 g/kg starch) as compared to carnivore fish. However, lower carbohydrate utilization in fishes is less understood till date. In the present work, It was attempted to decipher the physiological changes in genetically improved rohu (Jayanti Rohu) upon inclusion of different levels of starch in the feed regime. Methods: Three isonitrogenous (24% crude protein), isolipidic (10% ether extract) experimental diets viz. TS20 (@20% starch), TS30 (@30% starch) and TS40 (@40% starch) were formulated and fed to Jayanti Rohu fingerlings (180 number with average weight of 45±3.5 g) twice daily up to satiation level for 45 days experimental period. The growth performance, nutrient utilization, histological changes and modulation of hepatic gene expressions were evaluated after completion of experiment. Result: Results indicated significantly (p less than 0.05) increased weight gain percentage, SGR and condition factor in TS30 and TS40 as compared to TS20. The histoarchitecture of hepatic cells depicted greater cell damage with increasing levels of dietary carbohydrates. The glucose membrane transporter 2 gene and carbohydrate metabolism involved genes showed a higher expression in TS40 and TS30 compared to TS20. Therefore, the present study concluded that inclusion level of 30% starch as optimal level in the diet of genetically improved Jayanti rohu.
Recent advances in high throughout DNA sequencing
technologies have revolutionized for better understanding
of structure-functional relationships of genes in identifying
trait-associated ...transcriptomes and their regulated gene
expressions. Subsequent breakthroughs in gene editing
technologies such as zinc finger nucleases, transcriptional
activator-like effect or nucleases (TALEN) and CRISPR
(clustered regularly interspaced short palindromic repeats)
determined chromosomal loci so as to understand gene
functions in vivo. Such editing technologies are now being
implemented in many laboratories due to an affordable
cost and easiness of techniques. Targeted gene delivery
and disruptions are now not only restricted to standard
cell lines or stem cells, but also primary cell lines and nonmodel
agriculturally important species. Progress and
implications of gene integration and disruptions in food
fishes like salmon, carps, etc. will be highlighted. The
positive impacts on myostatin gene (negative growth
hormone regulator) disruption mediated muscular growth
have been documented. Transposon mediated gene
integration technologies for value-additions to small
indigenous aquarium fishes by expressing attractive
fluorescent color genes could be the future of rainbow
revolution. Issues linked with further-tuning with regards
to improved efficacy and specificity, while reducing offtarget
effects of gene editing tools will be addressed. There
are health and environmental concerns with genetically
modified organisms (GMOs). CRISPR/Cas9 mediated
editing generates indels and hence supposed to be free
from transgene-nontoxic and non-allergen. Scientific
progress regarding to generate genetically modified carps;
those could well be cultivated in a confinement and at the
same time economically profitable; will be highlighted.
Emphasis should be given for transfer these technologies
from the laboratory to land for the development of a
consumer-friendly sustainable farming system.
Objective: To identify white spot syndrome virus (WSSV) entry into the host-cells of the cultured shrimp Penaeus monodon, we have attempted to localize PmRab7 (Ras-related in brain) which is playing ...a vital role in the WSSV internalization. Methods: In this study, we have cloned PmRab7 and expressed in Escherichia coli, further purified rPmRab7 was used for antibody production, isolation of lysosomal sub-cellular fractions and western blot against lysosomal protein. Moreover, high fold-change in PmRab7 regulation with increasing copy number of WSSV has been studied by using real-time PCR. Results: 651 bp amplicon size gene was successfully amplified, ligated amplicon with pTZ T-tail vector confirmed by colony PCR and retriction enzyme digestion on agarose gel. Subcloned (pRSET-B) 651 bp gene transformed successfully in Rosetta and after 6 h of induction expressed rPmRab7 was on SDS page, furthermore soluble fraction of rPmRab7 (26 kDa) was purified by ni-NTA column. AntiPmRab7 antibody was received by Merk Pvt. Ltd., and western blot analysis revealed that PmRab7 is present in the lysosomal sub-cellular fraction. Copy number of WSSV was increased 5 fold in 24 h and 20 fold in 72 h of infection and subsequently transcrtipt of PmRab7 was Ct = 1.0 to Ct = 8.5. Conclusions: Presence of PmRab7 on lysosome clearly indicating PmRab7 participating in lysosomal maturation, other hand WSSV may follow the same route of entry. WSSV internalization has directly linked with regulation of PmRab7.
Objective:To identify white spot syndrome virus (WSSV) entry into the host-cells of the cultured shrimpPenaeus monodon, we have attempted to localize PmRab7 (Ras-related in brain) which is playing a ...vital role in theWSSV internalization. Methods:In this study, we have cloned PmRab7 and expressed inEscherichia coli, further purified rPmRab7 was used for antibody production, isolation of lysosomal sub-cellular fractions and western blot against lysosomal protein. Moreover, high fold-change in PmRab7 regulation with increasing copy number ofWSSV has been studied by using real-timePCR. Results: 651 bp amplicon size gene was successfully amplified, ligated amplicon with pTZ T-tail vector confirmed by colonyPCR and retriction enzyme digestion on agarose gel. Subcloned (pRSET-B) 651 bp gene transformed successfully inRosetta and after 6 h of induction expressed rPmRab7 was onSDS page, furthermore soluble fraction of rPmRab7 (26 kDa) was purified by ni-NTA column. AntiPmRab7 antibody was received by Merk Pvt. Ltd., and western blot analysis revealed that PmRab7 is present in the lysosomal sub-cellular fraction. Copy number ofWSSV was increased 5 fold in 24 h and 20 fold in 72 h of infection and subsequently transcrtipt of PmRab7 was Ct = 1.0 to Ct = 8.5. Conclusions: Presence of PmRab7 on lysosome clearly indicating PmRab7 participating in lysosomal maturation, other handWSSV may follow the same route of entry.WSSV internalization has directly linked with regulation of PmRab7.
PDF
