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2014 Vol. 22, No. 12  Published: 05 December 2014
Cloning and Expression Analysis of CiMYBJ2 Gene from Caragana intermedia
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2014, 22(12): 1514-1524  |  Full text (HTML) (1 KB)  | PDF   PDF  (860 KB)  ( 293 )
MYB transcription factor is one of the largest family of transcription factors in plants which play impotant roles in regulation of plant growth and development, and response to many environmental stimuli. In this study, a gene named CiMYBJ2 (GenBank accession No.KJ937783) was cloned by RACE technique from Caragana intermedia and its expression level along with the promoter was analysed. The full-length cDNA of CiMYBJ2 was 1 368 bp with the 3'UTR of 196 bp and the 5'UTR of 203 bp. The ORF of CiMYBJ2 was 969 bp, encoding a protein of 322 amino acids which was hydrophilia. The full-length gDNA was 1 285 bp, including two introns and three exons. The expression pattern of CiMYBJ2 analysed by qRT-PCR showed that it was altered by heat, dehydration, NaCl and drought treatments. After heat treatment, the expression of CiMYBJ2 decreased at 1 and 3 h, and then maintained a lower level expression. the change of gene expression at different time points was extremely significant compared with the 0 h (P<0.01). After dehydration treatment for 1 h, the expression of CiMYBJ2 increased shortly, and then gradually decreased at 3 h, and the expression difference was not significant compared with the 0 h (P>0.05). After 8 h , the expression of the gene showed extremely significant comapred with that of the 0 h (P<0.01), and the expression level of the gene at 24 h reached only half of that at 0 h (P<0.05). Under NaCl treatment, the transcript level of CiMYBJ2 began to increase after 1 h, and reached the maximum at 3 h. After that the overall trend kept downward, the expression dropped to the lowest level at 8 h, and expression of the gene was extremely significant at different time points except for 12 h (P<0.01). After drought treatment of 6 d, the expression of CiMYBJ2 increased slightly, and dropped significanly after compared with 0 d (P<0.01), reached the minimum expression at 15 d. The promoter sequence of CiMYBJ2 with 873 bp in length was obtained by genome walking technique and many abiotic stress-related cis-elements were found. These results indicated that CiMYBJ2 might be involved in response of C. intermedia to abiotic stresses. The research results provide a basis for the further study of gene function and expression regulation.
Cloning of Sterol 14α-demethylase Gene (HbCYP51) and Effects of Mechanical Damage and Exogenous Phytohormones on Its mRNA Expression in Hevea brasiliensis
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2014, 22(12): 1525-1535  |  Full text (HTML) (1 KB)  | PDF   PDF  (4610 KB)  ( 207 )
Sterol 14α-demethylase (CYP51), an important member of the cytochrome P450 family, is the key enzyme in sterol biosynthesis pathway. The objective of the study was to clone CYP51 gene and illustrate its function in latex production and drainage, and in defense of rubber tree against both tapping and ethephon stimulation. The target gene, encoding sterol 14α-demethylase, was separated from the rubber latex transcriptome library of elite Hevea brasiliensis material CATAS 7-33-97 and the mRNA expression difference, which stimulated by tapping and ethephon or jasmonic acid, was identified by Real-time quantitative PCR (qRT-PCR). BLAST analysis indicated that the cloned gene from Hevea brasiliensis was related to cytochrome P450 and named as HbCYP51 (GenBank No. KM203677), which had high homologies with the sterol 14α-demethylase reported in Populus trichocarpa (90%) and Malus domestica (88%). DNA sequence analysis showed that the size of the HbCYP51 cDNA was 2 305 bp, which included an open reading frame(ORF) of 1 461 bp, and encoded a polypeptide of 486 amino acids residues. The genome sequence length of HbCYP51 gene was 5 271 bp, consisting of 3 exons and 2 introns, and the first intron presented in 5'-UTR of the gene. The phylogenetic analysis suggested that HbCYP51 and CYP51 of Solanaceae (Nicotiana tabacum, Solanum chacoense, Solanum lycopersicum and Petunia×hybrida) were in the same branch group, indicating that the HbCYP51 had closer genetic relationship with other CYP51 of Solanaceae than that of Geum rivale, Fragaria vesca and Arabidopsis thaliana. qRT-PCR analysis showed that HbCYP51 was induced in the latex. HbCYP51 was regulated by tapping, ethephon and jasmonic acid, and significantly up-regulated by mechanical damage at the 7th tapping and ethephon at 48 h, and extremely significantly up-regulated by jasmonic acid at 24 h. The result confirmed that tapping, ethephon and jasmonic acid activated the expression of HbCYP51. Correlation analysis revealed that times of tapping had significantly positive correlation with expression of HbCYP51 gene (P<0.05), and with latex yield (P<0.01), but there was no significant correlation between latex yield and expression of HbCYP51 gene. In conclusion, the HbCYP51 gene might play an important role in the defense of rubber tree, and is directly or indirectly involved in the defense response against tapping and ethephon.
Effects of Ovariectomy on the Fatty Acid Composition and the mRNA Expression of its Related Genes in Perirenal Adipose from Female Goat
2014, 22(12): 1544-1552  |  Full text (HTML) (1 KB)  | PDF   PDF  (308 KB)  ( 239 )
Castration has been used in livestock production widely as a mature technology, but little information is available about the effects of ovariectomy on the fatty acid composition which is regulated by lipid-metabolic genes and would potentially improve mutton flavor. The aim of this study was to explore the effects of ovariectomy on the levels of blood sugar, triglyceride and total cholesterol in serum, the fatty acid composition and fatty acid metabolism related genes expression in perirenal fat. Hybrid kid of Boer goat (Capra hircus) with similar body weight at about 5-month old were treated as the experimental animals to determine the levels of blood sugar, triglyceride and total cholesterol. Then the Agilent-6890N gas chromatograph was used to determine the composition of fatty acids and Real-time quantitative PCR (qRT-PCR) was used to detect the expression levels of acetly-CoA carboxylase (ACC), fatty acid synthase (FAS), lipoprotein lipase (LPL) and hormone sensitive lipase (HSL) genes in perirenal adipose. There was no statistical significant differences on the levels of blood sugar, triglyceride and total cholesterol in serum between Ovx and Con groups, and remained stable in the process of the whole test (P>0.05). Significant decreases of the percentage of total saturated fatty acid (SFA) and 4 kinds of SFA (decanoic acid (C10∶0), pentadecanoic acid (C15∶0), palmitic acid (C16∶0) and lignoceric acid (C24∶0)) were observed in perirenal fat of ovariectomized goats compared with controls (P<0.05 or P<0.01). The differences of total mono unsaturated fatty acids (MUFA) and polyunsaturated fatty acid (PUFA) between 2 groups were not significant, with rising trend in MUFA (P>0.05), and the percentage of oleic acid (C18∶1) was significantly higher in Ovx group than that in Con group (P<0.05). The relative expression mRNA levels of fatty acid metabolism related genes were corrected by glyceraldehyde-3-phosphatedehydrogenase (GAPDH). Ovariectomy increased mRNA expression levels of LPL gene significantly (P<0.05) and increased the ACC and FAS genes very significantly (P<0.01), but reduced the mRNA level of HSL gene significantly at the same time in perirenal fat (P<0.05). The balances of blood sugar, triglyceride and total cholesterol in serum of ovariectomized goats were not influenced compared with control goats. Ovariectomy could reduce the percentage of total SFA by increasing the expression of LPL, ACC and FAS and decreasing HSL in perirenal fat, and improve mutton flavor finally. The above results provide theoretical basis for revelation of the improved effect of ovariectomy on the fatty acid composition in perirenal fat.
Effects of Baicalin on Expression of B-cellymphoma-2 (Bcl-2) and Bcl-2 Assaciated X Protein Gene (Bax) and Apoptosis Rates of Pig Kidney Proximal Tubular (LLC-PK1) Cells Subjected to Heat Stress
2014, 22(12): 1553-1560  |  Full text (HTML) (1 KB)  | PDF   PDF  (2151 KB)  ( 276 )
The objective of this study is to explore baicalin influence B-cellymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) expression as well as apoptosis rates of heat-stressed pig kidney proximal tubular cells (LLC-PK1). Readily cultured LLC-PK1 cells were randomly divided into seven groups. The cells in groupⅠwere used as blank control cultured at 37 ℃ and cells in groupⅡwere heart-stressed at 42 ℃ for 1 h. Cells in groups Ⅲ,Ⅳ,Ⅴ,Ⅵ and Ⅶ were cultured additionally with different concentrations (0.01, 0.1, 1, 10 and 100 μg/mL) of baicalin at 42 ℃ for 1 h, respectively. Expression of Bcl-2 mRNA and Bax protein were detected with Real-time PCR and Western blot, respectively. Apoptosis rates of LLC-PK1 cells were determined with Annexin V-FITC/PI. Results showed that as compared to groupⅠ, Bcl-2 mRNA in groupⅡincreased significantly (P<0.05), and Bax mRNA and its protein increased extremely significantly (P<0.01), and both ratios between Bcl-2 and Bax mRNA and their proteins decreased significantly (P<0.01), and the apoptosis rates increased extremely significantly(P<0.01). However, there was no evident influence on Bcl-2 protein expression (P>0.05). Compared with groupⅡ, Bcl-2 mRNA expression significantly increased in groupⅣand Ⅵ (P<0.05) and extremely significantly in groupⅤ(P<0.01), but not in groupⅢor Ⅶ (P>0.05), while the Bal-2 protein expression had no difference (P>0.05). Similarly, compared with groupⅡ, both Bax mRNA and its protein expression decreased significantly in groupsⅢ, Ⅳand Ⅶ (P<0.05) and decreased extremely significantly in groupⅤand Ⅵ(P<0.01). Ratios between Bcl-2 and Bax mRNA and their proteins in all groups except groupⅢ had a significant increase (P<0.05), while the ratio between Bcl-2 and Bax protein in groupⅤ increased extremely significantly (P<0.01). The apoptosis rates decreased extremely significantly in groupⅤ and Ⅵ (P<0.01), and decreased significantly in group Ⅳand Ⅶ(P<0.05). The results indicated that certain concentration (0.1~100 μg/mL) of baicalin protectsed heat-stressed LLC-PK cells by down-regulating Bax expression, increasing the ratio between Bcl-2 and Bax and decreasing apoptosis rates. This experiment reveals some heat-resistant mechanism of baicalin on LLC-PK1 cells exposed to heat stress which will provide valuable reference for its clinical application.
Construction of Fibroblast Growth Factor 5 Gene (FGF5) Hair Follicle Specific Expression Vector and Transfected into Cashmere Goat (Capra hircus) Fetal Fibroblast Cells
2014, 22(12): 1536-1543  |  Full text (HTML) (1 KB)  | PDF   PDF  (1831 KB)  ( 301 )
The quality and yield of cashmere goat wool are closely related to the growth and development of skin hair follicles. This study aimed to construct an eukaryotic expression vector and prove whether it could specifically express fibroblast growth factor 5 gene (FGF5) in the hair follicles of cashmere goat (Capra hircus). The keratin associated protein 6-1 (KAP6-1) promoter and FGF5 gene coding sequence were obtained by PCR. The vector pEGFP-N1-KF, a hair follicles specific expression vector of FGF5, was constructed by inserting the KAP6-1 promoter into the CMV promoter-less pEGFP-N1, and then connecting FGF5 to the downstream of KAP6-1 promoter, using Porcine teschovirus 2A peptide (P2A) to link FGF5 and enhanced green fluorescent protein (EGFP) gene. The constructed vector was transfected into cashmere goat fetal fibroblast cells and two methods were used to verify its functionality including qRT-PCR and Western blot. The enzyme digestion results showed the vector was correctly constructed. The qRT-PCR results indicated that the FGF5 gene could express in fetal fibroblast cells. The Western blotting results confirmed that the fusion protein could be availably spelited into FGF5 and GFP protein by the functionality of P2A. The result showed that the vector pEGFP-N1-KF which could specifically express FGF5 gene was successfully constructed and could normally expressed in cashmere goat fetal fibroblast cells.
Reference Genes for qRT-PCR in Perennial Ryegrass (Lolium perenne L.) Under Various Abiotic Stresses
2014, 22(12): 1494-1501  |  Full text (HTML) (1 KB)  | PDF   PDF  (2809 KB)  ( 215 )
The selection of high stablely expressed reference genes is an important prerequisite for improving the accuracy of Real-time quantitative PCR (qRT-PCR). We investigated the expression of 6 candidate reference genes including eukaryotic initiation factor 4 alpha (eIF4A), Ti-binding peptide-1 (TBP-1), ubiquitin-conjugating enzyme (E2), ubiquitin (UBQ), zeitlupe (ZTL) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) under various abiotic stresses in perennial ryegrass (Lolium perenne L.). The analysis with geNorm (ver. 3.5), Normfinder (ver. 0.953), BestKeeper (ver. 1.0), Delta Ct and RefFinder revealed that under drought, salt, heat and ABA stresses, eIF4A gene was the most stable reference gene; under waterlogging stress, UBQ gene was the most stable reference gene. As a result, eIF4A and UBQ genes were selected as reference genes for perennial ryegrass under different abiotic stresses, which provides a foundation for gene expression investigation of perennial ryegrass.
Genetic Diversity Analysis of Modern Japonica Rice (Oryza satiua L.) from Taihu Area Based on Simple Sequence Repeat (SSR) Markers
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2014, 22(12): 1502-1513  |  Full text (HTML) (1 KB)  | PDF   PDF  (2350 KB)  ( 204 )
The japonica rice (Oryza satiua ssp. japonica) resources are rich in Taihu area. Along with large-scale popularization of rice varieties, the genetic basis of breeding material is became narrower and the genetic similarity is increased, thus the japonica rice breeding has became more difficult. Genetic diversity of 42 japonica rice varieties, collected from Taihu area, was analyzed by 24 pairs of simple sequence repeat (SSR) primers. The SSR primers were located at 12 rice chromosomes, and 2 pairs of SSR primers were selected at each chromosome. The results showed that 23 pairs of SSR primers performed polymorphism in the 42 materials. A total of 105 alleles loci were detected among 42 varieties using the 23 pairs of SSR primers. The average number of detected alleles by each primer pair was 4.57 with a range from 2 to 8. The effective alleles were 56.43 with average 2.45. The value of allelic polymorphism information content (PIC) varied widely from 0.083 0 to 0.807 9 with an average value of 0.496 6. The number of polymorphic loci each variety ranged from 6 to 19, and the number of allele loci ranged from 27 to 55. The genetic similarity coefficient of SSR markers among 42 japonica rice varieties ranged from 0.391 to 0.990, with average 0.610, and the genetic similarity coefficient of 74.45% tested materials ranged from 0.50 to 0.80. Furthermore, the UPGMA(unweight pair group method with arithmetic mean) clustering analysis at the genetic similarity value of 0.50 grouped the 42 japonica rice varieties into two groups. The 19 conventional japonica rice varieties could be grouped, and they were divided into 2 subgroups at the level of genetic similarity coefficient 0.58. Another group was composed of the 23 japonica hybrid rice varieties, and it also was divided into two subgroups at the level of genetic similarity coefficient 0.70. Based on the all analysis, it was concluded that the genetic background comparability of the japonica rice varieties in Taihu area was high and the diversity was low. Therefore, introducing and making use of the new genetic resources and creating breeding materials should be strengthened. The genetic diversity of japonica rice based on SSR markers can provide technical support and theoretical basis for breeding new rice varieties in Taihu area.
Molecular Cloning and Functional Maker Design of TaCO9-1A Gene Associated with Spring-winter Habit in Wheat (Triticum aestivum L.)
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2014, 22(12): 1471-1481  |  Full text (HTML) (1 KB)  | PDF   PDF  (2056 KB)  ( 255 )
Photoperiod plays an important role in triggering the change from vegetative growth to reproductive growth. CO (constans) plays a significant role in the photoperiod pathway in the model plant Arabidopsis. To learn more about the CO-like gene in wheat, the homologous cloning method was used to isolate the related gene. Here a novel gene TaCO9-1A (GenBank accession: KM236233) that encoding a CO-like protein was cloned from hexaploid wheat (Triticum aestivum L.) Ningchun 4. The results showed that the coding sequences (CDS) of TaCO9-1A was 876 bp in length, encoding 291 amino acids including a classic CCT domain, but not the B-box domain. A phylogenetic analysis showed that TaCO9-1A was classified into the same branch with Ghd7 in rice (Oryza sativa L.) and HvCO9 in barely (Hordeum vulgare L.), which were negative regulators of flowering. TaCO9-1A protein with a predicted molecular weight 31 kD was succesfully expressed by the plasmid pEASYE1-TaCO9-1A in Escherichia coli BL21 (DE3). The expressions of TaCO9-1A were detected in root, stem, leaf and young spike in common wheat at heading stage by Real-time quantitative PCR (qRT-PCR), and the expression level was highest in leaf and lowest in root. Comparing the sequence of TaCO9-1A in winter and spring varieties, it was found that 6 bp deletion in the second exon existed in winter varieties. Thus, a codominant marker was developed based on the 6 bp deletion of TaCO9-1A in winter varieties. In 25 wheat varieties with different winter and spring habits, two kinds of bands (511 and 517 bp) were amplified with this marker. The codominant marker was highly correlated with not only spring and winter habit but also the heading, flowering time of wheat cultivated at Yangling in two years. Our results revealed that TaCO9-1A played an important role in wheat vernalization and photoperiod pathway. These results provide basic data for the further research of TaCO9-1A regulating development in wheat, and will help to reveal the molecular mechanism of spring-winter habit and maturity traits in wheat by TaCO9-1A regulation.
Completely Sequencing and Gene Organization of the Anser cygnoides Mitochondrial Genome
2014, 22(12): 1482-1493  |  Full text (HTML) (1 KB)  | PDF   PDF  (2764 KB)  ( 469 )
Mitochondrial genome (mtDNA) has advantages in rapid evolution, rich polymorphism and maternally inheritance without gene recombinations, which has become an ideal molecular markers of population genetics, phylogenetics, molecular ecology and taxonomy. In this study, the primers were designed based on the mitochondrial genome sequence of Bean goose (Anser fabalis) which was a closely related species of Swan goose (Anser cygnoides). Swan goose mitochondrial genome sequence was analysed by direct sequencing techniques. The results showed that whole mitochondrial genome sequence was 16 739 bp(GenBank accession No. KJ124555) in Swan goose, including 22 tRNA genes, 2 rRNA genes, 13 protein-coding genes and a D-loop region. Base composition of T, C, A and G were 22.49%, 32.24%, 30.21% and 15.06%, respectively. Besides, the base preference of AT was not determined. 22 kinds of tRNA were all typically cloverleaf structures. Compared to 12SrRNA of Red Junglefowl (Gallus gallus) and Mongolian Ground Jay (Podoces hendersoni), we found the secondary structure of 12SrRNA included 4 domains, 37 stem-loops and 13 salients in the Swan goose, and LSP/HSP, ETAS1-2, goose hairpin, E-box, F-box, D-box, C-box, Bird similarity-box, CSB1-box, CSB-like and OH in the D-loop control region. Finally, taken Red Junglefowl as an outgroup, the phylogenetic tree was constructed based on mitochondrial genome sequences using Neighbor-joining (N-J) algorithm, Maxium-likelihood (ML) algorithm and Bayesian model. The results showed that Swan goose, Greylag goose, Bean goose, white-fronted goose and Canada goose had close genetic relationship. The findings enrich the ducks mitochondrial genome sequences and provide a theoretical basis for the study of geese phylogeny.
Applications of DNA Technologies in Agriculture
2014, 22(12): 1463-1470  |  Full text (HTML) (1 KB)  | PDF   PDF  (289 KB)  ( 396 )
With the development of molecular biology, some DNA-based technologies have showed great potentiality in promoting the efficiency of crop breeding program, protecting germplasm resources, improving the quality and outputs of agricultural products, and protecting the eco-environment etc., making their roles in modern agriculture more and more important. To better understand the application of DNA technologies in agriculture, and achieve the goals to promote their utilities in modern agriculture, this paper describes, in some different way, the applications of molecular markers, transgenic engineering and gene's information in agriculture. Some corresponding anticipations for their development prospects are also made.
Research Progress in the Intestinal Health About Genetically Modified Organisms
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2014, 22(12): 1561-1566  |  Full text (HTML) (1 KB)  | PDF   PDF  (271 KB)  ( 317 )
The gastrointestinal microflora play an important role in maintaining the physiological balance, and has an important relationship with nutrition metabolism, immune regulation of host. The gastrointestinal health reflects the body's health directly. Exogenous substances may influence the gastrointestinal health by altering intestinal microflora, intestinal permeability, mucosal immunity, intestinal metabolites. Safety assessment of gastrointestinal health about genetically modified organisms (GMOs) is used to monitor the unintended effect, which provides guarantee to the safety of GMOs. This paper summarized the researches on the unintended effect of gastrointestinal health caused by GMOs. The safety assessment of gastrointestinal health will continuously improve and perfect the system of safety assessment of GMOs.
Rapid-Sexing Poultries via a New Pair of Universal Primers
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2014, 22(12): 1567-1574  |  Full text (HTML) (1 KB)  | PDF   PDF  (1413 KB)  ( 324 )
Poultry sex can be identified by anal swelling or other conventional methods. However, there's a problem that individuals can be injured effortlessly by anal swelling and with higher error rate for geese and pigeons especially. A new pair of primers named gCHD was designed in this study aiming at chromodomain helicase DNA binding protein 1 (CHD1) gene which links to sex of poultries and CHD1 fragments of chickens (Gallus gallus), ducks (Anas platyrhynchos), geese (Anser anser) and pigeons (Columba livia) were amplified. Via agarose gel electrophoresis, the production showed one band for male individuals and two bands for female individuals with the longer band location similar to band of males. Cloning sequence of production and sequence alignments between productions of chickens showed that the longer bands was located in Z chromosome and the shorter bands in W chromosome, named CHD1-Z and CHD1-W, respectively. In these 4 identified species, the length of CHD1-W was about 150 bp shorter than that of CHD1-Z, and the deficiency was in the 14th intron for CHD1-Z mRNA and in the 7th intron as to CHD1-W mRNA. Compared to molecular method, the correct rate of anal swelling measure for chickens, ducks, geese and pigeons were 100%, 93.75%, 87.50% and 87.50%, respectively. These results indicated that the new pair of primers gCHD could be the powerful universal primers to identify sex of poultry by counting the number of bands different between 2 sexes of poultry. This molecular sexing method can not only identify the sexes of poultry in advance for enhancing cultivation efficiency, but also provides a reliable sexing tool for specific study on specific gender of embryos.
Reference Gene Selection for Quantitative Real-time PCR in Pandora neoaphidis
2014, 22(12): 1575-1583  |  Full text (HTML) (1 KB)  | PDF   PDF  (939 KB)  ( 335 )
Selection of a suitable reference gene is an important prerequisite for precise gene expression analysis by Real-time quantitative PCR. Pandora neoaphidis, an obligate aphid pathogenic fungus, can induce a drastic epidemic to cause the collapse of aphid populations on crops. To determine potential reference genes for normalization of Real-time PCR data on P. neoaphidis, the transcript levels of 3 traditional housekeeping genes including 18SrRNA(18S), 28SrRNA(28S) and elongation factor 1 alpha-like protein(EF1), were measured in this study. We investigated the expression stability of 3 candidate reference genes in P. neoaphidis ARSEF 5403 in different developmental stages including conidia stage, germ tubes stage, early hyphae stage and elongated hyphae stage, as well as under different nutrient conditions including OS-SDB medium, GLEN medium and Grace's insect cell culture medium. The expression stability of candidate reference genes was calculated using 3 algorithms including geNorm, NormFinder and BestKeeper. Results from Real-time PCR revealed that designed primers had a good proliferation efficiency and specificity. The analysis with geNorm algorithms revealed that the stability value (M value) of candidate reference genes was 18S(0.457)>28S(0.534)>EF1(0.749) under different developmental stages, and 18S(0.389)>28S(0.557)>EF1(0.607) under different nutrient conditions. Additionally, the analysis with NormFinder algorithms revealed that M value of candidate reference genes was 18S(0.084)>28S(0.264)>EF1(0.509) under different developmental stages, and 18S(0.118)>28S(0.355)>EF1(0.403) under different nutrient conditions. 18S was ranked as the most suitable reference gene of the 3 candidate reference genes analyzed by geNorm and NormFinder. However, the analysis with BestKeeper algorithms revealed that 28S was the most suitable reference gene under all conditions examined, followed by 18S, and EF1 was the most unstable reference gene. Comprehensively, the mean rank based on stability banking analyzed by the three algorithms showed that 18S was the most suitable reference gene under all conditions examined. Conclusively, candidate reference gene 18S has the most stable expression levels among all the samples of P. neoaphidis from different developmental stages and different nutrient conditions. Meanwhile, 18S can also be used as a stable reference gene for normalization of Real-time quantitative PCR, and provides supports for the further studies for P. neoaphidis on the expression stability of the developmental or virulence relative genes.
Visual Detection of Streptococcus iniae Based on Loop-mediated Isothermal Amplification Combined with a Lateral Flow Dipstick
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2014, 22(12): 1584-1594  |  Full text (HTML) (1 KB)  | PDF   PDF  (3031 KB)  ( 391 )
Streptococcus iniae is a species belonging to Gram-positive coccus and produces beta hemolysis, which can infect a broad range of freshwater and marine fish species and lead to seriously economic losses in the aquaculture industry worldwide. Thus, a diagnostic method for rapid and accurate detection of S. iniae was urgently needed to effectively control the spread of streptococcicosis caused by S. iniae. In the present study, a LAMP-LFD method was developed to rapid detection of S. iniae, based on the loop-mediated isothermal amplification (LAMP) and visualization by a lateral flow dipstick (LFD). Three pairs of primers, i.e., gyrB-F3, gyrB-B3, gyrB-FIP, gyrB-BIP, gyrB-LF and gyrB-LB, were designed to recognize the gyrase subunit B (gyrB) gene of S. iniae; and among which the forward inner primer (gyrB-FIP) was biotinylated. A DNA probe (gyrB-HP) labeled by fluorescein isothiocyanate (FITC) was also designed specifically to recognize the gyrB gene. The biotinylated LAMP was performed and the LAMP product was hybridized with FITC-labeled probe and detected by LFD. The optimized assay could detect S. iniae by incubation at 65°C for 30 min, and under such conditions, the initiation time for positive amplification gradually increased as the concentration of template decreased, showing a typical linear relationship. Starting from isothermal amplification, the whole detection procedure needed only 40 min, which reduced by 2 h compared to conventional PCR assay. The LAMP-LFD could specifically detect S. iniae isolate and no amplification was observed from other pathogenic bacteria, e.g., the Streptococcus (i.e., Streptococcus dysgalactiae subsp., and et al), multiple Gram-positive bacteria (i.e., Staphylococcus aureus, Nocardia seriolae, and et al), and multiple Gram-negative bacteria (i.e., Vibrio harveyi, Pseudomonas aeruginosa, and et al). The detection limit of LAMP-LFD for pure culture of S. iniae was 8.70×101 cfu/mL (1.74 cfu/reaction), which was 10 fold higher than LAMP and 100 times higher than the conventional PCR assay. It was all positively detected when taking pure gDNA of S. iniae of 8.70×101 cfu/mL (the detection limit) as template in triplicate, which indicated good reproducibility. In the case of artificially contaminated liver tissue of Lateolabrax japonicus, the detection limit was 4.35×103 cfu/mL, which was 100 times higher than the conventional PCR method as well. Infection by S. iniae was successfully detected from field samples by LAMP-LFD, which was quite coincident with the results obtained by conventional culture methods. Therefore, LAMP-LFD was an accurate, time-saving and easy-to-use method for detection of S. iniae, and can be used to on-site screening of S. iniae.
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