The bait and water quality conditions has important influence on fish producing. In order to explore the effect of different nutrients on several important physiochemical factors of water and the growth of plankton, the variations of dissolved oxygen (DO), ammonia (NH3-N), nitrite (NO2-N), total nitrogen (TN), total phosphorus (TP ) and plankton community were measured after treating the water with inorganic nutrients (IN), micronutrients additives (MA), complex nutritional preparation (CNP) or no fortification (NF). The results of physiochemical factors showed that the IN were extremely improved by DO, NO2-N, NH3-N and TN content (P＜0.01). However, the MA group of NH3-N was only highly significantly improved (P＜0.01), highly significantly decreased NO2-N (P＜0.01), and other physiochemical factors were not significantly different from the NF group. The prokaryotes denaturing gradient gel electrophoresis (DGGE) profiles showed that the NF group of the most bands in detected 44, secondly for the group of IN (37)and MA(36), and the CNP group at least, only 34. Shannon-wiener diversity index(H') and Pielou evenness index(J) were the highest in NF with 3.78 and 0.91, respectively, and the lowest in CNP was 3.53 and 0.85, respectively. The eukaryotes DGGE profiles showed that the CNP group with the largest number by detected 25, the second of NF group (22) and the third of MA group (21), and the CNP group was the least, only 18. H' and J of the CNP group were the largest by 3.22 and 0.86 respectively, while the IN group was the minimum by 2.89 and 0.77, respectively. The test concluded that TN and TP increased significantly in all the treatment group，hazardous substances and harmful bacteria large increased in group treat with IN, while it showed a decrease in group MA; The CNP group had the highest diversity of plankton. Under this experiment condition, the MA group had effective control on the hazardous substances and harmful bacteria of the water, the CNP group could provide the water with abundance nutrients and improve the culture environment. The experimental results will provide theoretical basis to improve the water ecological environment and health cultivation.

Endo-1,4-β-xylanase (xynA, EC.3.2.1.8), the most important member of xylanolytic enzymes, is widely used in feed, food, papermaking and bioenergy and other fields. The xynA from anaerobic fungi Orpinomyces sp. PC-2 has potential exploitation value, but its enzyme activity of heterologous expression is low. In the present research, according to the codon usage frequency of highly expressed genes in Pichia pastoris, a β-xylanase gene (xynA) derived from anaerobic fungi Orpinomyces sp. PC-2 was optimized, and the modified gene (xynAm) was chemical synthesized and constructed a yeast expression vector pPIC9K-xynAm. Then, the xynAm gene was expressed in Pichia pastoris GS115, and enzymatic properties of the recombinant β-xylanase were characterized. The results showed that the enzyme activity of the recombinant β-xylanase reached the maximum of 612 IU/mL in shake-flask culture at 108 h of induction. In 10 L fermenter, the enzyme activity and specific activity of the recombinant β-xylanase reached the maximum of 3 515 IU/mL and 2 411 IU/mg at 96 h of induction. The dry weight, wet weight of the yeast cells and the protein content in the culture supernatant were reached 324, 156 and 2.25 g/L, respectively, at this time. The molecular weight of the enzyme was about 42.7 kD revealed by non-denatured sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE). Enzymatic properties analysis showed that the optimum pH and reaction temperature of the purification xylanase were 55 ℃ and 6.0, and relatively more stable at 30~50 ℃ and pH 4.0~10.6, and the Km and Vmax were 27.86 mg/mL and 277.78 mg/(mL·min), respectively. Substrate specificity analysis showed that the xylanase could hydrolyze low and high viscosity arabinoxylans, oat spelt xylan, birch xylan and 4-O-methyl-D-glucurono-D-xylan but not barley (Hordeum vulgare) β-glucan and lichenan. The enzyme activity was slightly activated by 10 mmol/L Co2+ and K+, and inhibited by Mn2+, Fe2+ and SDS. This study lays a foundation for further industrial application of the β-xylanase from the anaerobic fungi Orpinomyces sp. PC-2.

Xinjiang is famous for fruit in China. And the melon is not only one of the most iconic brand, but the economic pillar industries that increase the farmers' income of home at recent years. However, due to the Xinjiang melon have been threatened increasingly by virus disease, causing a large area yield reduction even total loss of the melon yield when serious. It is affected seriously the farmers' production enthusiasm and the improvement of standard of living. And Zucchini yellow mosaic virus (ZYMV) is one of the most representative virual. In order to research biological control and anti-viral melon (Cucumis melo) in different cultivars region of Xinjiang, homologous cloning method were used, the coat protein (CP) complete gene sequence of ZYMV from 19 different regions of Xinjiang were amplified. The CP complete gene sequences of ZYMV were 1 080~1 180 bp, encoding 253~301 amino acids and synthetic protein size was approximately 33 kD. The result indicated that the nucleic acid sequence of 3' and 5' terminal noncoding region were less conserved, but the coding region and the surrounding sequence were high conserved. The N terminal of the amino acids was more conserved than C terminal. According to ZYMV homology results, isolator from 19 regions belonged to the same genotype of ZYMV. Through the analysis of the evolutionary tree that constructed according to nucleic acid sequences, ZYMV were divided into 4 subgroups, and the basis of subgroups were divided compliance with viral distribution character. The physical and chemical properties of proteins, such as the kinetics of protein, chemical and thermodynamic stability results showed that CP of ZYMV belonged to stable protein. The combined analysis with average summer precipitation and mean temperature trending in Xinjiang results showed that the change trends between index of hydrophobic and fat in CP of ZYMV with the corresponding region had a certain correlation. The analysis results indicated that the stability CP of ZYMV had some differences in different region, which may be related to the particular climatic conditions and geographic environment in Xinjiang. By sequencing analysis this study obtained the nucleic acid sequence and characteristics of CP in ZYMV from 19 regions in Xinjiang. This study will offer a theoretical foundation for the biopesticide research, and cultivating anti-virus melon in future by using RNA interference technology or genomic editing techniques. Meanwhile, It can provide initial direction for conducting the pathogenic mechanism of plant viruses and the spread of the virus to explore the research environment and propagation mechanism.

Nondret002679 gene is a type of long non-coding RNA (lncRNA) in zebrafish (Danio rerio) homologous to the LNC-PHOX2B-2 gene of large intergenic noncoding RNA 682 (lincRNA) in human (Homo sapiens), which is related to tumor and might be silenced by deleting the nucleotide sequences within its promoter region through clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) system, to facilitate its function research. In the present study, the sequence positioned upstream and downstream region of its predicted promoter were used to design guide RNA (gRNA) to target its promoter region. There were 6 gRNAs located in either upstream or downstream promoter region, named as gR1, gR2, gR3, gR4, gR5 and gR6, respectively, were designed. These gRNAs were synthesized by in vitro transcription with the templates, which were made with 3 oligonucleotides by overlap PCR, a rapid method to obtain sufficient templates for gRNA. The 6 gRNAs and Cas9 mRNA were transcribed using transcription Kit and purified by LiCl precipitation and redissolved in RNase-free water. To knock out the sequences located in the predicted promoter of the lncRNA gene, a mixture of these gRNAs and Cas9 mRNA were microinjected into each zebrafish embryo at the one-cell stage with 1.76 nL. The concentration of each gRNA in injection solution was approximately 12.5 ng/μL, which also contained 300 ng/μL of Cas9 mRNA and 0.5% phenol red. Regular PCR with sequence-specific primers were used to identify individual knockout zebrafish with a deletion in the promoter region, showing a 3 643 bp PCR product in wild type zebrafish and 579~1 664 bp PCR products range in promoter deleted zebrafish. Twenty eight 2-month-old injected zebrafishes were extracted genomic DNA of their fin for PCR, out of which 11 were identified 579~1 664 bp PCR fragments range. Sequencing results, when compared to NCBI databases, demonstrated that 11 zebrafishes occurred deletion in lncRNA promoter region with 39% of knockout efficiency. The length of deletion fragments in 11 zebrafishes ranged of 1.9~3.1 kb. In addition, 33 F1 zebrafishes obtained from the breeding of one female and one male adult founders with heterozygous deletion were screened by PCR for deletion in the promoter region. The result indicated that the 6 F1 zebrafishes had promoter deletion with fragment range of 579~1 664 bp, which occurred homozygous deletion in lncRNA promoter in 3 zebrafishes. The result indicated that CRISPR/Cas9 system was an efficient approach to delete lncRNA gene promoter, which provides basic data for function study of Nondret002679 gene.

Tyrosinase (TYR) is the one of the most important rate-limiting enzymes in animals within the signal path of melanin synthesis, which plays a crucial role in the formation of melanin. So the expression and activity level decide the speed and type of melanin. This study was conducted to reveal the relationship between genes related to pigment and body color. We obtained tyrosinase gene (TYR) sequence of Amphilophus citrinellus by rapid-amplification of cDNA ends (RACE) technique and also investigated the expression of TYR in different developmental stages and tissues using qRT-PCR. The total length of TYR was 2 683 bp which contained 246 bp 5'-UTR, 817 bp 3'-UTR, and 1 620 bp open reading frame (ORF) encoding 540 amino acids in A. citrinellus. Sequence analysis of amino acids and phylogenetic tree analysis indicated that conservation of TYR was higher in fish than that in other vertebrates. qRT-PCR analysis revealed that TYR expressed in embryonic periods was lower in the conception and began to sharply rise in the circulation of the blood period. In the process of body color changes (black to gray to bright yellow), the expression of the TYR in black period of all tissues was significantly higher than that in grey and bright yellow period (P＜0.05). TYR expression of tail fin and scale was gradually reduced in gray to bright yellow period, while there is no significant difference between these two periods (P＞0.05). However, expression of TYR in skin significantly decreased during the body color developmental stages (P＜0.05). In the bright yellow period, TYR expressed in all kinds of tissues (heart, kidney, brain, maw, gonad, tail fin and eye), which expressed the highest amount in eyes, intermediate in swim bladder and tail fin and the lowest in skin and glands. Also, TYR expression gradually decreased among three periods of body color development. It may be related to the gradual increase of red and yellow pigment cells, as well as the changes of the number and distribution proportion of melanocytes. This study accumulated data for body color genetic and modified research of the body color in fish by revealing the molecular basis of body color variations.

The final and critical step in the synthesis of triglycerides is catalyzed by diacylglycerol acyltransferase 1 (DGAT1) and DGAT2 enzymes. DGAT2 gene plays an important role in fat metabolism, lipid deposition in the organization and growing development. In order to invest the association of polymorphism of DGAT2 with production performance in chicken (Gallus gallus), the genetic polymorphism of DGAT2 gene was investigated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RELP), and the association of polymorphisms with production performance, which included the growth traits, meat quality traits, carcass traits, blood biochemical indices and muscle fiber traits, was analyzed. The results indicated that one synonymous mutation site (C→T) was detected in exon 7 of DGAT2 gene. Three genotypes (CC, CT and TT) were found at g.13955C＞T mutation locus by digesting the PCR product with TaqⅠ, and the frequency of CC, CT and TT genotypes was 0.49, 0.48 and 0.03, respectively. The results showed that the polymorphisms of the g.13955C＞T site of DGAT2 gene had significant association (P＜0.05) with birth weight, 2 and 4 week body weight, 4 and 8 week index of body measurements and intramuscular fat in different genotype, and most CC genotype character value significantly greater than those of TT genotype, while had no significant association with other traits (P＞0.05). The result demonstrated that g.13955C＞T site of chicken DGAT2 gene related to the growth and development, and C allele was dominant genes, it can be candidate molecular markers for the poultry breeding.

Short season cotton (Gossypium hirsutum) usually accompanies with premature senescence, thus revealing and verifying senescence associated genes in cotton is very important to breed short season cotton cultivars without premature senescence. In this study, a WRKY transcription factor, GhWRKY11 (GenBank accession No. JN604988), was cloned from upland cotton with 1 055 bp cDNA and contained a 1 008 bp open reading frame (ORF), which encoded a putative WRKY group Ⅱd protein with 335 amino acids. Protein sequence phylogenetic analysis showed that GhWRKY11 had high similarity with AtWRKY11. In order to analyze the gene structure, GhWRKY11 was also cloned from genome DNA which contained 1 256 bp (GenBank accession No. JQ522936). Comparison results of GhWRKY11 which were cloned from cDNA and genome DNA revealed that this gene contained 3 exons and 2 introns. Gene sequence of GhWRKY11 was also used to do blast with G. hirsutum genome sequence. The identity of GhWRKY11 with Gh_D07G0023 and Gh_A07G0017 were 100% and 97%, respectively, which indicated that GhWRKY11 was Gh_D07G0023 in genome sequencing work, and Gh_A07G0017 was its homolog. A subcellular localization assay confirmed that GhWRKY11 protein was located only in epidermal cell nucleus of onion (Allium cepa). qRT-PCR for tissue specific expression analysis indicated that GhWRKY11 showed a higher expression level in leaf than that in root, stem and flower. GhWRKY11 had higher expression levels at 2 and 3 week-old cotyledons, and then the expression level decreased with the aging process. At 2 and 3 week-old cotyledons, GhWRKY11 had significantly higher (P＜0.05) expression level in Liao 4086 than that in CCRI10. Also, this gene could be induced by 2 plant senescence inhibitors salicylic acid (SA) and gibberellins (GA3). Compared with the expression level at 0 h, the expression level of GhWRKY11 significantly (P＜0.05) or extremely significantly increased (P＜0.01) after the treatment of GA3 or SA at 2, 4, 8 and 12 h. GhWRKY11 also could be induced by abscisic acid (ABA), jasmonic acid (JA), ethylene (ET) and H2O2 at some specific time points. When the seedlings were treated with JA and ET, expression level of GhWRKY11 significantly increased to the highest expression level at 4 h (P＜0.01), and then the expression level decreased; when the seedlings were treated with H2O2, expression level of GhWRKY11 significantly increased to the highest level at 8 h (P＜0.01). We also detected that GhWRKY11 could be induced by abiotic stresses including low temperature (12 ℃), drought (15% polyethylene glycol (PEG) 6000), salt (200 mmol/L NaCl) and damage. The expression level of GhWRKY11 increased with the time going under the low temperature conditions. Under salt stress, expression level of GhWRKY11 extremely significantly increased (P＜0.01) at 2 and 12 h. GhWRKY11 significantly expressed at 12 h (P＜0.01) when the seedlings were treated with PEG 6000 and it was not sensitive to damage treatment. GhWRKY11 was over-expressed in Arabidopsis thaliana. Compared with wild type Arabidopsis thaliana, the over-expression lines of GhWRKY11 showed delay senescence phenotype after sowing 45 days, which indicated that GhWRKY11 negatively controlled cotton leaf senescence. In this study, cloning and function analysis of GhWRKY11 provided the foundation to create transgenic short season cotton without premature senescent trait.

Wheat (Triticum aestivum) landraces have strong adaptability to the local natural ecological condition and corresponding production potential. Therefore, identifying good resources of production, quality, disease and insects resistance from wheat landraces, as well as expanding the current genetic basis of breeding parents, have been highly valued. In this study, the phenotypic data were obtained with 64 wheat landraces from Sichuan province which were grown in 3 environments across 2 years and 231 SSR markers were selected from meta-quantitative trait locus (MQTL) controlling yield or quality related traits, and association analysis was then used to reveal the genetic effect of wheat landraces from Sichuan province. Phenotype analysis showed that Sichuan landraces generally belong to medium-gluten or weak-gluten wheat, having the properties of floribunda, multiple seeds, high tillering ability and high earing rate. The heritability was high (＞50%) in effective tiller number, plant height, spikelet density, spike length, fertile spikelet number and sedimentation value. A total of 18 SSR markers were associated with yield and quality related traits including one (Xgwm372) simultaneously associated with yield and quality traits, 4 (Xwmc112, Xcfd5, Xwmc317, and Xgwm372) significantly stable, and 3 loci (Xcfd5, Xgwm328, and Xbarc181) newly identified to be associated with yield traits. Negative correlations were found between spike length and spikelet density, with 2 common associated markers Xgwm328 and Xcfd5. Besides, the marker region Xgwm448-Xgwm328-Xgwm372 (8.0 cM) on chromosome 2A was associated with spike length in multiple environments. These results may facilitate the excavation and utilization of elite genes carried by Sichuan wheat landraces.

Plant glutathione S-transferases (GSTs, E.C.2.5.1.18) are a superfamily of multifunctional enzymes which play important roles in primary metabolism, secondary metabolism, signal transduction and stress tolerance, etc. In our present study, the 669 bp full-length CDS region of ZmGST23 was cloned from an elite maize (Zea mays) inbred line F83 based on the mRNA sequence in GenBank (NM_001111524) using RT-PCR methods, which was predicted to encode a protein consisted of 222 amino acids. The deduced molecular weight of ZmGST23 was 24.84 kD, together with isoelectric point of 5.68. BLAST analysis revealed that the protein contained a C-terminal domain and an N-terminal domain, as well as glutathione binding sites and substrate binding pockets, and belonged to tau class of GSTs. Phylogenetic tree analysis showed that ZmGST23 had the closest evolutionary relationship with a Sorghum bicolor GST protein, which shared 94% identity in amino acids. Promoter analysis revealed that there were multiple putative cis-acting elements that were involved in abscisic acid responsiveness, anaerobic induction, auxin responsiveness, gibberellin responsiveness, and defense and stress responsiveness, and some MYB transcription factor binding site involved in drought-inducibility were also found in the promoter region of ZmGST23 gene. qRT-PCR analysis revealed that ZmGST23 was highly induced by abiotic stresses such as dehydration, waterlogging, salinity, abscisic acid, auxin, gibberellin, as well as extreme temperatures, interestingly, the expression level in leaves was higher than in roots. Tissue specific expression analysis showed that ZmGST23 expressed in all seven tested tissue types. However, the expression level of ZmGST23 varied with tissue types, the expression level of ZmGST23 in young buds and mature leaves were higher than that in young roots, silk, ear leaves, tassel and ear. The prokaryotic expression vector of ZmGST23, pEASY-E1-ZmGST23, was successfully constructed. The expression of fusion protein was obtained by inducing with 0.5 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) and its relative molecular weight was 30 kD, which was consistent with the theoretical value. The results in our present study revealed that ZmGST23 might serve as a potential stress response gene for the improvement of maize inbred lines and cultivars under stress conditions in breeding activity.

In order to develop D-hydantoinase with high performance from brand new sources, this research regarded D-phydroxyphenyl hydantion separated primarily from marine Streptomyces library as the only N element to form the medium. Then double agar plate method and microporous rapid screening method were used to do the second screening. At last, the final screening was done with the method of molecular biology. As a result, 4 positive Streptomyces of D-hydantoinase including Micromonospora aurantiaca (GenBank No. FJ547135.1), Streptomyces aureofaciens (GenBank No. AB326923.1), Streptomyces sampsonii (GenBank No. GU238264.1) and Streptomyces sp. 7-145 (GenBank No. JQ782979.2) were obtained. Then the expression engineered strains of E.coli S1, E.coli S14 E.coli S29 and E.coli S145, by transforming Escherichia coli, secreting D-hydantoinase were built through degenerating primer and extending D-hydantoinases from the 4 positive bacterium. The 4 D-hydantoinases were purified, meanwhile the enzyme activity and kinetic parameters were also tested. The result showed that the enzyme from Streptomyces sp. 7-145 was the most active and its enzymatic compare energy was 9.7 U/mg, with Kcat=3.2×10-6/s and Km =9.5 mmol/L. Finally, the homology modeling online by Swiss-model and the simulation analysis of structure to the catalytic channel of D-hydantoinase through Caver Analyst were performed. According to simulation analysis, the length of the main catalytic channel, Tunnel_1, of D-hydantoinase was 9.1 ?. The amino acids residue of bottleneck were histidines of 59th and 181st sites, and glutamic acid of 313rd site, and neck radius was 2.18 ?. However, the length of the potential catalytic channel, Tunnel_2, was 13.6 ? long. The amino acids residue of bottleneck were the threonine of 62nd site, asparagine of 93rd site and tryptophan of 107th site, and neck radius was 1.52 ?. Conducting site-directed mutagenesis to the threonine of 62nd site, asparagine of 93rd site and tryptophan of 107th site, might be more likely to develop the better D-hydantoinase. In conclusion, this study provides a new screening system, understanding the catalyst mechanism of hydantoinase more intuitively through computer simulation, which lays a firm foundation for getting D-hydantoinase with better performance and the industrial use of the D-hydantoinase.

SDA1 mutant of wheat (Triticum aestivum) which is incomplete development in spike, narrow development in leaves and dwarf development, is controlled by a recessive pleiotropic single gene Triticum aestivum spike development atrophy 1 (TaSDA1) and it is discovered in the experiment field, belonging to natural mutation. To further research the mutation mechanism of TaSDA1 gene, the present study investigated some major agronomic traits, such as the heading time, flag leaf width and length, the number and length of the spike, dissecting the differences of flag leaves expressed-protein through two-dimensional electrophoresis (2-DE) between SDA1 and wild-type during the heading time, and performed the qRT-PCR verification of the differentially expressed proteins. According to the t test, there were significant differences of the flag leaves' width and length between SDA1 and wild-type. Also the total height of the SDA1 was much lower than that of the wild-type. SDA1 was later in heading than the wild-type. Twenty-seven differentially expressed protein spots were selected by PDQuest (version 8.0.1) image software, among which 23 protein spots were successfully identified. The Mascot software was used to search against the NCBInr database and Uniprot database for protein annotation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to explore further biological functions of the 2-DE proteins. Among these differentially expressed proteins in SDA1, SSP7101, SSP7110, SSP7112 and SSP8418, related to the Rubisco holoenzyme composition, which were the key enzymes of the Rubisco holoenzyme in photosynthesis, were down-regulated and one of the Rubisco holoenzyme (SSP213) composition showed deficiency. Glyceraldehyde-3-phosphate dehydrogenase A (GAPA, SSP8000), one of the important proteins of glyceraldehyde-3-phosphate dehydrogenase (GAPD), was down-regulated. Peroxiredoxin (Prx, SSP7320), which was important to the oxidation resistance, also showed down-expressed in SDA1. One RNA-binding protein (SSP1309) was down-regulated in SDA1, leading to the down-regulation of 2 translation elongation factors (eEF-Tu, SSP4614 and SSP4802), and finally made the protein biosynthesis blocked. Some of 23 proteins, participating in the adversity stress response, were down-regulated (SSP3738, SSP5209) and showed defiency (SSP3719) in SDA1. qRT-PCR was used to validate ribulose-1,5-bisphosphatecarboxylase/oxygenase large subunit (chloroplast) (SSP7101), chloroplast-localized PtrToxA-binding protein1 (SSP7303), translation elongation factor G(SSP4614) and triosephosphate isomerase (SSP5209) genes expression at the transcriptional level, which were consistent with the protein expressed amounts detected by 2-DE. The mutation phenotype of SDA1, might be related to the flag leaves' differentially-expressed proteins in the decline of the photosynthetic capacity, disorder of the energy metabolism, the decline of oxygen stress resistance and coercive capabilities, process of mRNA editing and the blocked protein biosynthesis. The development of spike and leaves is of great importance to the grain yield of wheat, thus a better understanding of TaSDA1 gene will lay foundation to the theory of genetic improvement of spike and leaf development.

At present, there are few studies on sheep (Ovis areis) populations in Southwest regions. To analyze genetic variability, population structure and evolutionary relationships of Chinese indigenous sheep in Southwest regions, the complete sequences of mitochondrial DNA (mtDNA) cytochrome (Cytb) gene of 183 individuals in 10 sheep populations were determined with primer pairs which were designed by Oligo 6.0 based on the sheep mitochondrial genome. We analyzed the genetic diversity and population structure of 8 local breeds and 2 imported breeds using softwares of MEGA 5.2, DnaSP 5, Phylip 3.695 and Network 4.0. Results of genetic diversity showed that 62 haplotypes were defined by 153 polymorphic sites. The mean haplotype diversity, the average nucleotide diversity and the average number of nucleotide differences were 0.792, 0.002 43 and 5.054, respectively. The range of the nucleotide diversity (Pi) among 10 sheep breeds was from 0.001 28 (Zhaotong sheep) to 0.006 55 (Guidehei sheep). The haplotype diversity (Hd) varied from 0.582 (Tengchong sheep) to 0.924 (Xizang sheep). The results showed that 3 sheep populations in Yunnan Province had a relatively lower level of genetic diversity, while the genetic diversity level of several other sheep populations was higher. From the results of analysis of molecular variance (AMOVA) analysis, selected 10 sheep populations in China had maintained a high level of within-population genetic differentiation (87%), with remainder explained by differentiation among populations (13%). The neighbor-joining tree and network analysis indicated that there were 3 main branches in Chinese indigenous sheep, however, lineages A and B were still main lineages of Chinese indigenous sheep in southwest China. These results may provide theoretical reference for developing a scheme of preservation and utilization of sheep genetic resources in Southwest China.

Pluripotent stem cells (PSCs) include embryonic stem cells (ESCs) derived from the inner cell mass of an embryo, epiblast stem cells (EpiSCs), embryonic germ cells (EGCs) from primordial germ cells (PGCs) and induced pluripotent stem cells (iPSCs). These cells have unlimited developmental potentiality which can give rise to all cell types of body. Thus, these cells provide an efficient route to promote the study of developmental biology, establish animal models for human genetic diseases, obtain organs for xenotransplantation and generate high quality and disease-resistant new breeds. Up to date, most studies on PSCs were mouse (Mus musculus) and human. While, the ESCs and subsequently iPSCs of livestock also are undergoing effective development, which was not only used for keeping and promotion inherent selected quality of special livestock, but also production of chemical drugs and antibodies from livestock, even preclinical association of human diseases, because there were less limitations of organ physiology and immune systems in livestock than mouse, which may block research. However, the isolation and enrichment of livestock ESCs were difficult, so as the cell line establishment in vitro. As the persistent explore of livestock ESCs and iPSCs, they have become an innovation research field in life science and high-tech agriculture and biology. In this article, we review the progress and application of livestock PSCs.

Aiming at improving the detection efficiency, a novel loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) method for rapid detection of Meloidogyne camelliae was developed, mostly based on the nucleotide amplification by a LAMP and visual detection by a LFD. Three pairs of primers were designed targeting the conserved region of ribosomal DNA-internal transcribed spacer (rDNA-ITS) and used in the biotinylated Real-time fluorescence LAMP. The optimized LAMP was of 63 ℃ 15 min. LFD, Real-time fluorescence curve and agarose gel electrophoresis were separately used for LAMP products detection, and the practicability of LFD was comparatively evaluated. The results indicated that LAMP products were hybridized by a fluorescein isothiocyanate (FITC)-labeled probe ITS-HP and visually detected in LFD in 5 min. It just needed 25 min from the beginning of LAMP reaction to the judgment of results, which was approximately 2 h time-savings compared with the conventional PCR method. The results demonstrated that LAMP-LFD could specifically detect M. camelliae with a detection limit of 4 pg/μL M. camelliae genomic DNA, which was lower than the conventional PCR of 100 times, and the detection sensitivity was 1/1 000 for a single juvenile J2 genomic DNA. Therefore, this rapid, sensitive and specific LAMP-LFD was an expected option in the inspection and quarantine for M. camelliae.

Establishment of a rapid and accurate method for genetic sexuality identification in half-smooth tongue sole (Cynoglossus semilaevis) is the basis of sex control technology and high-female breeding of half-smooth tongue sole. This fish harbors ZZ/ZW sex determination system, in which female contains ZW heteromorphic chromosomes compared to male (ZZ). In this method, a pair of primers (CS-SEX-F and CS-SEX-R), which were located in the different regions between Z and W chromosome, was firstly designed according to the whole genome sequencing data. Then genomic DNA templates were extracted from the fin by boiling method as follows: 30~50 mg of fin samples were picked into 1.5 mL eppendorf tube and 400 μL NaOH (50 mmol/L) was added, then they were mixed thoroughly and centrifuged at 12 000 r/min for 10 min. The supernatant was used as template in PCR reaction system, which contained 5.0 μL 10×Buffer, 4.0 μL dNTP (2.5 mmol/L), 1.0 μL CS-SEX-F/CS-SEX-R (10 μmol/L), 0.5 μL rTaq (5 U/μL), 5.0 μL DNA supernatant. The program was 95 ℃ 10 min; 95 ℃ 30 s, 60 ℃ 30 s, 72 ℃ 30 s for 35 cycles; 72 ℃ 10 min. The PCR products were analyzed by electrophoresis in agarose gel at 150 V and 20 min. Genetic sexuality could be distinguished via different band patterns, where ZZ male exhibited single band (366 bp), and ZW female harbored double bands (366 and 253 bp). The advantages of this newly established method lie in its simplicity and time-effective compared to the previous ones, so it has tremendous potential in the future for application to accurate identification of genetic sexuality and efficient screening of pseudo-male fish in aqua farm.

The purpose of this study was to establish a transgenic insect cell line containing Helicoverpa armigera cadherin (HaCAD) for subsequent study on the interactions between Bacillus thuringiensis (Bt) insecticidal crystal protein and its receptor cadherin. Based on the specific membrane-adhering characteristics of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) envelope protein GP64, PCR primers were designed based on AcMNPV gp64 gene sequence. A 172 bp DNA fragment of gp64 signal peptide (gp64sp) and a 135 bp fragment of the gp64 C-terminal transmembrane domain (gp64ctd) were amplified by PCR, respectively. Both gp64sp and gp64ctd fragments were ligated and an additional DNA sequence containing 6 restriction digestion sites was inserted. This 301 bp fragment was replaced into the multiple cloning sites of expression vector pIZ/V5-His. A surface display vector named pIZ/V5-gp64 that allowed the exogenous gene to be expressed on the cell membrane was successfully constructed. The amplified 3 882 bp of the cadherin repeat (CR) and the membrane proximal region (MPR) of HaCAD and recombinant plasmid pIZ/V5-gp64 were digested with restriction enzymes, respectively, and then ligated. A recombinant vector named pIZ/V5-gp64-HaCAD was constructed. This vector was transfected into cotton bollworm cell line Ha-E-1 via liposome-mediated transfection. The transfected cells were screened on the selective culture medium containing zeocin and the selected cells were further cloned. A transgenic cell line Ha-T-CAD containing the recombinant HaCAD gene was obtained. A majority of transgenic cells were circular in shape while a few cells exhibited short spindle shape. Compared with the original parent Ha-E-1 cells, no obvious changes in the both morphology and size of the transgenic cells Ha-T-CAD were seen. PCR verification results showed that 3 882 bp fragment of HaCAD gene was successfully integrated into these cells. The results of immunofluorescent visualization revealed that in the transgenic cell line Ha-T-CAD, the red fluorescent dye recognizing cadherin protein was mainly distributed in cells. Western blot analysis indicated that 140 kD of cadherin protein expressed and displayed on the membrane of Ha-T-CAD cells. This established transgenic cell line Ha-T-CAD could be used to study the interactions between Bt insecticidal crystal protein and its receptor cadherin and will provide an important research tool for studying the action mechanism of Bt insecticidal crystal protein and the mechanisms underlying the resistance of insects against Bt toxin.