SLAF-seq BSA, integrated traditional bulk segregant analysis (BSA) with specific-locus amplified fragment sequencing (SLAF-seq), makes it possible to quickly locate qualitative trait genes. However, little is known about the reliability of its mapping results. In this study, the green-revertible chlorina gene (grc2) in rice (Oryza sativa) was selected as a target for comparing the mapping results provided with map-based mapping and SLAF-seq BSA. Firstly, 5302 SLAF markers were identified which showed single nucleotide polymorphism between 'grc2' and 'Y58S' (a cultivar presenting normally green leaves) by SLAF-seq. Later, the wild-type bulk and the mutant bulk were established, each consisting of 100 plants from a F₂ population of 'Y58S'/'grc2'. By automatically calculating the genotype frequency in two pools, 13 SLAF markers were found associated with grc2 which accorded with an expected separation ratio of 33∶66 for paternal / maternal in wild-type bulk and 99∶1 in mutant bulk at 0.01 level. Interestingly, all of the associated markers were distributed on two neighbour regions of 0.98~1.21 Mb and 1.57~1.99 Mb on Chromosome 6 referenced as '93-11' (an indica cultivar). Furthermore, two markers named AM1 and AM6 on the first region were confirmed linkaged with grc2, just exhibiting a genetic distance of 0.625 cM and 0 cM respectively. While referenced as 'Nipponbare' (a japonica cultivar), it was observed that the first region just covered the mapping area with traditional method, indicating the result of SLAF-seq BSA is for consulting. Therefore, the results presented herein supported SLAF-seq BSA would be a useful tool for rapidly obtaining the region associated with qualitative trait gene.

Protein phosphates 2C (PP2C) genes play an important role in regulating plant growth and abiotic stress resistance. In this study, ZmPP2C3 (GenBank No. NM_001155565.2), screened from the previous drought-rewatering transcriptome analysis and encoding PP2C protein, was cloned in maize (Zea mays). Sequence analysis showed that the ORF of ZmPP2C3 gene was 1 227 bp, encoded 408 amino acids, and the molecular weight was 43.28 kD, isoelectric point was 5.92; Sequence alignment revealed that ZmPP2C3 shared high sequence homology with AIB05995.1 (99.75%) and AIG52088.1 (99.75%) in maize; Subcellular localization showed that the protein encoded by the ZmPP2C3 gene was detected in the nucleus. The qRT- PCR analysis showed that ZmPP2C3 gene had the highest expression in the root of corn seedlings, and the expression of ZmPP2C3 was positively regulated by drought, salt, high temperature and abscisic acid (ABA) treatment. Yeast two-hybrid analysis showed that ZmPP2C3 protein was interacted with GRMZM2G180625, GRMZM2G038126, GRMZM2G155242, GRMZM2G110408 and GRMZM2G069651. This study provides a reference for further biological function analysis of ZmPP2C3 gene.

Tomato (Solanum lycopersicum) is one of the most widely grown vegetables in the world. It is a model crop for studying the development of climacteric fruits. Various plant hormones are involved in regulating the growth and development of tomato fruits. Cytokinins can promote cell division of fruit, make fruit larger, increase fruit weight, delay fruit senescence, and increase fruit yield. In order to study the effect of endogenous cytokinin on tomato fruits, the expression vector for the tomato fruit-specific promoter Tfm7 to drive the Arabidopsis thaliana cytokinin oxidase 2 gene (AtCKX2) was constructed. The transgenic plants were obtained by the method of Agrobacterium tumefaciens transformation. The transgenic plants were verified by the specific primers of the promoter Tfm7 and the target gene AtCKX2. The PCR results indicated that the promoter Tfm7 and the target gene AtCKX2 had been transferred into the tomato plants. The results of qRT-PCR showed that the AtCKX2 gene was not expressed in the leaves of tomato plants and was expressed in the pollen flowers. The expression level of AtCKX2 gene was the highest in the fruit 5 d after pollination. As the fruit developed, the expression level of AtCKX2 gene gradually decreased, and it was hardly expressed in the late stage of fruit development. Studies on tomato fruits showed that compared with wild-type tomato fruits, the content of endogenous cytokinin zeatin (ZT) and isopentenyladenine (iP) in transgenic tomato fruits decreased, the single fruit weight, vertical and horizontal diameter of transgenic fruits decreased, and the content of soluble sugar and soluble protein in fruits decreased, the content of titratable acid increased. These results indicate that endogenous cytokinins can affect the growth and development of tomato fruits. This study provides new ideas for the role of cytokinins in fruit development.

Purple cabbage (Brassica campestris ssp. chinensis var. comunis) leaves are purple on the front and green on the back, rich in anthocyanins and have high nutritional value. Organic acids are important components of the flavor and quality of fruits and vegetables, and play a role as signal molecules in the biosynthesis of anthocyanins.To this end, teh present study set the applied malic acid concentration of 0, 0.5, 1.5, 3, 5, 10 mg/L to determine the color parameters and anthocyanin content of purple cabbage. The BcMYB2 (v-myb avian myeloblastosis viral oncogene homolog 2), BcMYB12 and BcMYB111 genes were cloned by RT-PCR, bioinformatics analysis was carried out, and the effects of different concentrations of malic acid on the expression of BcMYB2, BcMYB12 and BcMYB111 in purple cabbage leaves were analyzed by qRT-PCR, in order to clarify the effects of external application of malic acid on the coloration and anthocyanin synthesis of purple cabbage from physiological and molecular levels. The results showed that external application of malic acid had a significant effect on the color and anthocyanin content of purple cabbage. With the increasing of external application of malic acid, the purple degree of leaves went from light to deep to light, and the anthocyanin content showed a trend of increasing and then decreasing. The gene BcMYB2, BcMYB12 and BcMYB111 were cloned by RT-PCR, and the ORF sequences were 758, 1 102, 963 bp, encoding 248, 365 and 321 amino acids, respectively. The amino acid sequences encoded by the 3 genes all had R2 and R3 conserved domain, belonging to the R2R3-MYB gene family, and had close genetic relationship with cabbage (Brassica rapa) and Arabidopsis thaliana. All 3 genes were unstable hydrophilic proteins, no signal peptides and no transmembrane regions. qRT-PCR results showed that the relative expression of BcMYB2 and BcMYB12 in the leaves of purple cabbage were the highest in 12 d, and the relative expression of BcMYB2 and BcMYB12 increased first and then decreased with the increasing of malic acid concentration. However, the relative expression of BcMYB111 was relatively low during the whole treatment period, and there was no significant difference among treatments. Above results indicated that appropriate concentration of applied malic acid could promote BcMYB2 and BcMYB12 expression in the leaves of purple cabbage, thereby deepening the leaf color, increasing the anthocyanin content and nutritional quality of purple cabbage. This study provides basic data for further exploration of the biological mechanism of purple cabbage.

Chilling tolerance is an important reflection on the ability of plant to adopt to low-temperature environment. Melon (Cucumis melo) is sensitive to low temperature, but the genetic pattern for chilling tolerance of melon seedlings remained unclear to date. In the present study, 2 melon inbred lines, which showed tolerance and sensitive to low temperature, were used to develop 6 generation populations for joint analysis, aiming to pinpoint the genetic model controlling chilling tolerance of melon seedlings and function model of the genes related to the trait. For this analysis, a mixed major-gene plus polygenes inheritance model was applied for pursuing the inheritance law of chilling tolerance of melon seedlings in spring and autumn. The results showed that chilling tolerance had typical characters of quantitative traits and tended to inherit dominantly. The optimal genetic pattern fitted 2 pairs of additive-dominance-epitasis major genes plus additive-dominant polygene model. Dominant effects played a main role for the major genes, meanwhile epistatic effects were also observed with these genes. All the gene effects were sensitive to environmental factors. Three generation populations (B_1∶2, B_2∶2 and F_2∶3) had a major gene heritability of 73.22%, 92.36% and 95.57% in spring and 49.58%, 84.15% and 83.18% in autumn, respectively. The heritability of polygene was much low, only being detected in B_1∶2 generation. Selection efficiency of major genes was much high in the early generations of F₂ and its self-bred progenies as well as the backcross progenies (B₂×sensitive parent). B₁ generation could be more suitable for selection for polygenes. The present study could provide a reference for genetic improvement of chilling tolerance of melon seedlings.

Mitogen-activated protein kinase (MAPK) cascade pathway plays an important role in plant growth and development and signal transduction in response to stress. However less the role of MAPK family members were reported in Chinese wild grape (Vitis sp.). In this study, two novel MAPK genes VyMAPK2 and VyMAPK3 (GenBank No. MK942078, MK942077) were obtained from Vitis yeshanesis. VyMAPK2 and VyMAPK3 proteins belonged to the B and A subgroup of the MAPK family, respectively, which all contained 11 conserved protein kinase subdomains, TEY motifs, activation-loop, common docking domain, P-loop and C-loop. Subcellular localization analysis showed that VyMAPK2 was mainly distributed in the nucleus, VyMAPK3 was mainly distributed in the nucleus and cytoplasm. Gene expression analysis showed that VyMAPK2 was mainly expressed in root and VyMAPK3 was mainly expressed in leaves. Under exogenous hormone treatments, auxin, gibberellin, 6-benzyladenine, ethylene, abscisic acid, salicylic acid, and methyl jasmonate could significantly induced the expression of VyMAPK3, but there was no significant change of the expression of VyMAPK2 (P<0.05). Under stress, drought, high salinity and low temperature could significantly induced the expression of VyMAPK3 gene (P<0.05), while VyMAPK2 was only induced by drought. The above results showed that VyMAPK3 gene played an important role in the development and resistance to external stresses in V. yeshanesis. This study will be helpful to understand the stress resistance mechanism of wild grape in China and supply the genes for breeding.

American red maple (Acer rubrum) leaves are bright red and beautiful, and are widely used in garden plants. It is also often used as a shelter forest trees species and a scenic forest trees species. In order to understand the differentially expressed genes and molecular regulatory pathways in American red maple during the color changes of leaves, this study performed RNA-Seq sequencing and analysis on American red maple during the color changes of leaves. The total number of unigenes was 78 571, the total length was 71 788 869 bp, and the average length was 913.68 bp. 48 387 unigenes could be assigned to the protein database for annotation information. Gene Ontology (GO) analysis found that the annotated genes involved 53 functional groups, of which metabolic processes (biological processes), cells (cell components), and catalytic activities (molecular functions) were enriched to a higher degree. KEGG analysis found that differentially expressed genes mainly regulated biological processes such as ribosomal metabolism. During the color change of leaves, 7 kinds of anthocyanin-related transcription factors were selected. And they were mainly through propane biosynthesis, anthocyanin biosynthesis and flavonoid biosynthesis, which were involved in the regulation of American red maple leaf color. Meanwhile, it was found that the expression levels of 7 transcription factors were higher in red leaves or colorful leaves, which further indicated that they might had an important role in the leave color changes of American red maple. The results of this study provide a reference for exploring the molecular mechanism of the change of leaf color in American red maple.

Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is intracellular Ca²⁺ release channel protein, and could influence the mineralization and quality of eggshell in poultry by regulating the transport and release of calcium ions in the eggshell gland. In this study, the PCR product direct sequencing method and sequence alignment technology were used to identify the ITPR1 genetic variation sites of 186 Sansui duck (Anas platyrhyncha domestica), and the correlation between SNP mutation sites and eggshell quality was analyzed. The results showed that a total of 14 mutation sites were detected in the main functional domain and some introns of Sansui duck ITPR1 gene. There were 5 mutant sites, g.46455T>C, g.46459C>A, g.46564A>G, g.46581A>T, g.46649T>G, detected in the 8th intron. There were 6 mutant sites, g.55537A>G, g.55610T>C, g.55620G>T, g.55664A>G, g.55680C>T, g.55709T>G, detected in the 15th intron. There were 2 mutant sites, g.72601T>G and g.72843A>G, detected in the 30th intron. There was a synonymous mutation site g.124036C>T detected in the 42nd exon. All of the 14 mutation sites produced 3 genotypes, all of which were moderately polymorphic. The chi-square fitness test results showed that the genotype distribution of 14 mutation sites were consistent with the Hardy-Weinberg equilibrium (P>0.05). Based on linkage unbalance analysis,there were strong linkage imbalance between each two sites of g.46455T>C, g.46459C>A, g.46564A>G, g.46581A>T and g.46649T>G. There were strong linkage imbalance between each two sites of g.46581A>T, g.55537A>G, g.55620G>T, g.55664A>G, g.55680C>T and g.55709T>G. There was also strong linkage imbalance between g.72601T>G and g.72843A>G. A total of 13 kinds of haplotypes were found in 14 SNPs, of which H2 (CAGTGACTGTGGGC) was dominant haplotype with a frequency of 0.131, and H11 (TCAATGCGAC) was an inferior haplotype with a frequency of 0.060. The correlation analysis showed that 13 SNPs had significant correlation with eggshell thickness of Sansui duck except g.72843A>G site; Only g.46581A>T site had significant correlation with egg shape index (P<0.05); Except g.55610TC, g.55664AG, g.72601TG and g.72843AG, the other 10 sites had significant correlation on egg weight (P<0.05); g.46459C>A, g.55664A>G and g.124036C>T sites were significantly associated with eggshell weight (P<0.05); Except g.124036C>T site, the other 13 sites were significantly correlated with eggshell strength (P<0.05). In conclusion, the DNA sequence of the main functional domain of ITPR1 gene in the Sansui duck was relatively conservative, while intron genetic variation was abundant. Moreover, the 14 SNPs found in this study were significantly correlated with at least 2 eggshell quality indexes, indicating that the genetic variation of ITPR1 gene might affect eggshell quality and could be used as a genetic marker for the selection of eggshell quality of ducks.

The eggshell matrix protein ovocalyxin-32 gene (OCX-32) is a specific matrix protein that affects eggshell mineralization. The study aim was to investigate the genetic variation effects of eggshell matrix protein OCX-32 gene on eggshell quality. The eggshell quality had been determined by using Changshun Blue eggshell chicken (Gallus gallus). The SNP of OCX-32 gene was screened by sequencing directly of PCR products to analyze the effects on eggshell quality. The predicted results of the three-level structure were consistent with those of the two-level structure, with only slight changes, which were not obvious. Three moderately polymorphic SNPs were detected in the OCX-32 gene of Changshun Blue-eggshell chicken, located at g.22592362 C>T in intron 1 and g.22599114 C>T in exon 6, respectively. Missense mutations (threonine to methionine) and g.22599127 G > A synonymous mutation, both generated 3 genotypes; The distributions of g.22599114 C>T and g.22599127 G> A mutation loci all deviated from Hardy-Weinberg equilibrium (P<0.05). There were no strong linkage disequilibrium among the 3 mutation sites, and 4 haplotypes and 10 diplotype were found. Bioinformatics analysis was found that g.22599114 C >T and g.22599127 G >A mutations could cause the changes of mRNA free energy and protein structure. The CG type free energy had changed from -339.00 kcal/mol to -338.90 kcal/mol. The TA type free energy had changed from -339.00 kcal/mol to -338.40 kcal/mol which led the stability decreased. The protein sequence analysis results had shown that the α helix had changed from 39.27% to 40.00%, the free curl had changed from 41.09% to 40.36% and the β corner and extension had no change. The results of correlation analysis showed that the effects of the 3 SNPs loci on the shell quality of Changshun Blue-eggshell chicken did not reach a significant level (P>0.05); the egg shape index of the diplotype H3H4 individuals was significantly higher than that of the diplotype H2H3 (P<0.05). Diplotype H4H4 (TTCCGG) had the advantage for improving eggshell strength and eggshell thickness and H1H2 (CTCCAG) were the key diplotype to increase the weight of the eggshell, which revealed that the OCX-32 gene mutation could affect the eggshell quality of Changshun Blue-eggshell chicken, and the detected 3 SNPs had the potential to be applied as genetic markers for eggshell quality selection. This study provides data reference and theoretical support for deeper study on the quality of Changshun Blue-eggshell chicken in future.

Intramuscular fat deposition is a complex biological process involving muscle cell differentiation, adipocyte differentiation and triglyceride metabolism. Peroxisome proliferator-activated receptor γ (PPARγ) gene plays a central role in the differentiation of adipocytes in animals, but the relationship between PPARγ gene expression and intramuscular fat content still not clear. The aim of this study was to package the cattle (Bos taurus) PPARγ Adenovirus and analyze the effects of PPARγ overexpression on fat deposition in muscle cells. After subcloning PPARγ into the vector pAdTrack-CMV, a shuttle vector pAdTrack-CMV-PPARγ was constructed. The linearized shuttle vector pAdTrack-CMV-PPARγ and the adenoviral backbone vector pAdEasy-1 were co-transformed into the recombinant bacterial BJ5183 (Escherichia coli), and after homologous recombination, the adenoviral backbone vector pAdEasy-1-PPARγ containing the gene of interest was constructed. The linearized pAdEasy-1-PPARγ was transferred into 293A cells (Homo sapiens) to package and amplify the PPARγ Adenovirus. After infection of bovine muscle cells with PPARγ Adenovirus, the expression changes of the fat deposition related genes were detected by quantitative real-time PCR (qRT-PCR). The results showed that the PPARγ Adenovirus vector was successfully constructed. After the package and 2 times of amplification, the PPARγ Adenovirus with a titer of 2×10¹¹ plaque forming unit (PFU) /mL were obtained. Infection of bovine muscle cells with the PPARγ Adenovirus yielded an infection efficiency of more than 90%. After overexpression of PPARγ in bovine muscle cells, the positive regulation genes of fat deposition, including fat acid binding proteins 4 (FABP4), glucose transporter 4 (Glut4) and adipose-specific phospholipase A2 (AdPLA2) were up-regulated to 1.89, 1.51 and 1.56 times (P≤0.05). While the negative regulation genes of fat deposition including GATA binding protein 2 (GATA2), hormone-sensitive lipase (HSL) and nuclear receptor subfamily 2, group F, member 2 (Nf2f2) were down-regulated to 0.45, 0.57 and 0.25 times (P≤0.05). The expression level of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1a) was up-regulated to 141.41 times (P≤0.01), and the expression of myogenic factor 5 (Myf5) gene was not changed much. This study successfully packaged PPARγ Adenovirus, providing a tool for further study of the function of this gene, and proved that overexpression of PPARγ in bovine muscle cells has a tendency to promote fat deposition in muscle cells.

The majority of sheep (Ovis aries) in China are seasonal estrus, and the trait of estrus is regulated by hypothalamic-pituitary-gonadal axis related hormones. For exploring the secretion changes of luteinizing hormone (LH) and prolactin (PRL) in year-round estrous Small Tail Han (STH) sheep and seasonal estrous Sunite sheep at different photoperiods can contribute to understand the molecular mechanism of seasonal estrus in the term of hormone. In this study, the ovariectomized Small Tail Han sheep and Sunite sheep (SNT)were raised in light-controlled sheep house, keeping in short-photoperiod (SP) condition for 42 d, then transformed to long-photoperiod (LP) condition for 42 d, and jugular vein blood was collected in different light conditions. Radioimmunoassay was used to detect the concentration of LH and PRL in 2 breeds of sheep at different photoperiods. The results showed that the concentration of PRL in the blood of Sunite sheep was lower in short-photoperiod than that in long-photoperiod, and it was basically stable in different photoperiods in Small Tail Han sheep. It was significantly higher in Small Tail Han sheep than that in Sunite sheep at SP7 and SP21 (P<0.05). The concentration of PRL in Small Tail Han sheep was significantly lower than that in Sunite sheep after LP28 (P<0.05). The concentration of LH in the blood of Small Tail Han sheep was basically stable in different photoperiods. The concentration of LH in Sunite sheep was significantly lower than that in Small Tail Han sheep at SP21 (P<0.05). In summary, the change of PRL concentration in long-photoperiod and short-photoperiod was an important factor leading to the change of estrus status in seasonal estrus sheep. It was suggested that the concentration of PRL in long-photoperiod was high in Sunite sheep, which might weaken the LH response to gonadotropin-releasing hormone (GnRH) in the pituitary of the Sunite sheep, further to inhibit the follicular maturation and ovulation, and provide basic data for further exploration of the seasonal estrus mechanism of sheep in terms of hormones.

Uncoupling protein 3 (UCP3), as an important regulatory protein, is mainly involved in the regulation of the body's energy metabolism and plays an important role in lipid metabolism, fatty acid oxidation and fat deposition. It plays a decisive role in the production of ATP in oxidative phosphorylation. In order to study the relationship between UCP3 gene and pork quality traits, 11 meat quality indexes of 165 Jiangkou Luobo pigs (Sus scrofa) were determined. SNPs loci were screened by sequencing all exons of UCP3 gene amplified by mixed DNA pool. Single sample DNA was amplified and sequenced for validation and typing. At the same time, the difference of meat quality among individuals with different genotypes was analyzed. The expression of UCP3 gene of individuals with different genotypes was detected to study the relationship between meat quality and UCP3 gene expression. This study identified 6 SNPs, they were g.3127A>G, g.3205T>C, g.10439C>T, g.10441C>A, g.10466G>A and g.10479A>G/T. Population genetic parameters analysis showed that the heterozygosity except g.10479A>G/T locus was high , which indicated that the distribution of alleles in the population was relatively uniform; the number of effective alleles was high, which indicated that the degree of variation in the population was large; and the content of polymorphic information was more than 0.25, which indicated that the loci could provide some genetic information. χ² test showed that the genotype distribution of g.3127A>G and g.3205T>C loci was in Hardy-Weinberg equilibrium, while the genotype distribution of g.10439C>T, g.10441C>A and g.10466G>A loci deviated significantly from Hardy-Weinberg equilibrium. Linkage disequilibrium analysis showed that g.3127A>G and g.3205T>C loci, g.10439C>T and g.10466G>A loci were completely linkage disequilibrium, g.10441C>A, g.10466G>A and g.10479A>G/T were strongly linkage disequilibrium. The correlation analysis between SNPs loci and meat quality showed that marbling scores of TT and GG individuals at g.10479A>G/T loci were significantly higher than those of AA and AG individuals, whereas shear stress was the opposite. Genotype-expression correlation analysis showed that g.10439C>T, g.10441C>A, g.10466G>A and g.10479A>G/T loci were significantly higher than those of others. UCP3 gene expression was negatively correlated with shear stress and positively correlated with intramuscular fat content. The results showed that g.10439C>T, g.10441C>A, g.10466G>A and g.10479A>G/T sites were important SNPs sites affecting the meat quality of Jiangkou Luobo pigs, and the increase of UCP3 gene expression was beneficial to the deposition of intramuscular fat. The results provide a reference for the screening of molecular markers that contribute to intramuscular fat deposition in Jiangkou Luobo pig.

C1q tumor necrosis factor-related protein 6 (CTRP6) differentially regulates the metabolism of porcine intramuscular and subcutaneous fat, thus it has potential value in high quality lean pig (Sus scrofa) breeding. However, the effect of CTRP6 deficiency on animal health is still unknown. CTRP6 knock-out (KO) mice (Mus musculus) was constructed, and litter size, body weight, food intake, behavioristics of stressful situations, tissue sections, related physiological markers and gene expression were analyzed. The purpose is to explicit effects of CTRP6 deficiency on mice health. The results showed that average litter size was 5.4±0.3 in KO mice and 5.7±0.7 in wild-type (WT) mice. The survival rate was (92.1±3.2)% in KO mice and (93.1±4.5)% in WT mice. Two genotypes showed no significant difference. Weight analysis showed that the body weight of 8-week-old KO and WT mice were (25.08±0.34) and (28.34±1.06) g, respectively. The body weight of KO mice was significantly lower than that of WT mice (P<0.05), so does the weight of white adipose tissue (P<0.05). However, two genotypes showed no difference in average daily food intake. In tail suspension test and forced swimming test, the immobility time of KO mice was longer than that of WT mice, which indicates that KO mice were inclined to give up struggling under pressure conditions. In open field test, the number of crossing scores in KO mice was significantly less than in WT mice, which suggests that KO mice lack of interests in exploring a new environment. In sucrose preference test, no difference was observed between 2 genotypes in the sucrose preference. In morris water maze experiment, KO and WT mice exhibited similar learning ability in the first 5 days of place navigation test, but the frequency per min of passing through the plague position was less in KO mice than in WT mice on the 6th day of spatial probe test, which indicated the memory ability of KO mice was decreased. Further analysis of tissue section and inflammatory marker genes in brain were conducted and KO mice exhibited a higher level of inflammation than WT mice. The expression of memory related genes FBJ osteosarcoma oncogene (c-fos) in hippocampus was analyzed and it was down-regulated in KO mice. In conclusion, CTRP6 deficiency has no significant effect on mice litter size and food intake, but it reduces the body weight and adipose accumulation of mice. A negative impact on stress tolerance, interests of exploration and memory ability are detected, which may be related to the enhancement of brain inflammation. This study provides a certain theoretical basis for the further application of CTRP6 in high-quality lean pig breeding production. This study provides a theoretical basis for further revealing the function of ctrp6 gene.

With the rapid development of intensive aquaculture, the accumulations of ammonia nitrogen in the culture water environment threaten the ability of ammonia excretion of fish and lead to oxidative damage and immune suppression, physiological structure and glycogen metabolism injured, even the death of fish. This study aims to explore the effects of ammonia-nitrogen stress on the glycogen mobilizing ability of great blue-spotted mudskippers (Boleophthalmus pectinirostris). The blood glucose levels, glycogen contents, glycogen synthase and phosphorylase activities as well as their gene expression levels of great blue-spotted mudskippers were investigated under ammonia-nitrogen stress. In this study, fish were accumulated in 10‰ seawater for 2 weeks 5 d and then transferred to 10‰ seawater (the control group) or to 10‰ seawater + 8 mmol/L ammonium chloride (the ammonia stress group) for 72 h. Under ammonia-nitrogen stress conditions, the blood glucose levels of great blue-spotted mudskippers at 1, 3, 6, 12, 48 and 72 h were significantly higher than those of the control group (P<0.05), and the liver glycogen contents at 1, 3, 6, 24, 48 and 72 h were significantly lower (P<0.05). While the contents of muscle glycogen and gill glycogen were not significantly changed, indicating that great blue-spotted mudskippers mainly mobilized their liver glycogens, rather than the muscle or gill glycogens, to maintain glucose homeostasis under ammonia-nitrogen stress. Gene expression and enzyme activity analysis showed that liver glycogen phosphorylase (GP) activities at 1, 3, 6, 12, 48 and 72 h and their mRNA abundances at 1, 3, 12, 24, 48 and 72 h were significantly increased under ammonia-nitrogen stress (P<0.05), while liver glycogen synthase (GS) activities and their mRNA abundances showed no significant change, indicating increased liver glycogen catabolism, rather than anabolism, under ammonia-nitrogen stress. Liver glycogen, not the gill glycogen, is the main carbohydrate mobilization site for great blue-spotted mudskippers to cope with ammonia-nitrogen stress. In this condition, great blue-spotted mudskippers mobilize the liver glycogen, mainly through accelerating the catabolism, rather than suppressing the anabolism, to maintain blood glucose homeostasis. The ORF of its liver GP (GenBank No. XM_020932431.1, XM_020935772.1) was 2 544 bp in length with 44.54% AT content, and encoded a peptide of 847 amino acids with molecular mass of 97 kD and a theoretical isoelectric point of 5.94. The liver glycogen phosphorylase of great blue-spotted mudskipper contained one phosphorylase pyridoxal-phosphate attachment site (consensus: E-A-[SC]-G-x-[GS]-x-M-K-x(2)-[LM]-N), 8 N-glycosylation sites (consensus: N-{P}-[ST]-{P}) and 11 protein kinase phosphorylation sites (consensus: [ST]-X-[RK]), one of which was cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-dependent protein kinase phosphorylation site (consensus: [RK](2)-X-[ST]). This gene showed 80.36% in homologous similarity with human (Homo sapiens) and 81.11%~86.89% with other fish homologous, indicating the high conservation for GP genes. Molecular evolutionary analysis detected 3 positively selected sites (686G, 715N, 807G) on great blue-spotted mudskipper's liver GP gene, indicated the unique adaptive evolution for great blue-spotted mudskippers. This study enriches the molecular biological information of the liver type glycogen phosphorylase gene and glycogen metabolism for great blue-spotted mudskippers under ammonia-nitrogen stress.

Wheat (Triticum aestivum) is an important staple crop in the world. The stable and high yield wheat production is fatal for crop security in China. Fusarium head blight (FHB) is a major spike disease, which threatens wheat production and food security. The research advances of the damage of FHB, screening and identification of the germplasm resources resistant to FHB, resistance types, heredity and mapping of the resistance genes, disease resistance molecular mechanism, breeding, and transgene are summarized in this paper. It is also proposed that it's necessary to discover the molecular requirements for pathogenicity of Fusarium spp. and the molecular mechanisms of host resistance against FHB for a breakthrough in future wheat resistance breeding. The paper will provide a theory basis for developing more effective novel techniques using new strategies for future wheat disease resistance breeding, which based on absolutely or highly preventing Fusarium invading.

Maize (Zea mays) Bt11 is a genetically modified (GM) maize strain that has the feature of insect-resistant and herbicide-tolerant. At present, there was no accurate quantitative detection methods for Bt11. Droplet digital PCR (ddPCR) is the third generation of PCR technology emerged in recent years, and has great application prospect in quantitative field of genetically modified products. In order to solve the implementation problems of current genetically modified food standards and improve the inspection and supervision of genetically modified foods in China, the application of ddPCR in the screening detection of genetically modified organisms was established. A quantitative detection method for exogenous and endogenous gene of Bt11 was established based on duplex ddPCR. The maize endogenous gene Zein and the specific gene Cry1A(b) of Bt11 were selected as quantitative target sequences. The method of duplex ddPCR for quantitative detection of GM maize Bt11 was evaluated by stability, specificity, quantitative linear range, quantitative detection low limit and accuracy analysis. The results showed that both Bt11 event-specific sequence and endogenous gene could be amplified specifically with good stability. It had an excellent linearity when the DNA concentration is in the range of 0.05~10 ng/μL, and the correlation coefficient R² was 0.999. The limit of quantitation (LOQ) of endogenous and exogenous genes were 11.14 and 3.96 copies, respectively. The results of quantitative accuracy test showed that the relative standard deviation (RSD) was between 5.68% and 10.31%, meeting the requirement of less than 25%. In conclusion, the quantitative detection method for exogenous and endogenous genes in genetically modified maize Bt11 was established in this study and had strong specificity, good stability, high accuracy and wide quantitative range, which could be used for quantitative detection of the components of GM maize in food.

P38 mitogen-activated protein kinase (p38MAPK) plays an important role in the process of biological autoimmunity regulation. To explore the role of p38MAPK in the immune response of Nile tilapia (Oreochromis niloticus), the p38MAPK partial coding sequence was prepared by gene synthesis. It was cloned into pET-28a to construct prokaryotic expression plasmid pET-28a-p38MAPK. And expression was successfully induced in Escherichia coli after purification by Ni-NTA chromatography column, the p38MAPK polyclonal antibody was prepared by immunizing New Zealand white rabbits (Oryctolagus cuniculus) with purified fusion protein. Using the purified polyclonal antibodies, the p38MAPK protein expression was detected in different tissues of Nile tilapia. The results showed that the fusion protein obtained by the expression induction of the prokaryotic vector had a molecular weight of about 23 kD, and mainly existed in the form of inclusion body. The titer of antibody was estimated as high as 1∶256 000 dilution ration detected by ELISA and Western blot, and it could specifically recognize the p38MAPK protein. The tissue expression analysis showed that p38MAPK was expressed in most tested tissues, with the highest levels in ovary, brain and muscle; moderate levels in head kidney and blood; low levels in skin and gill; the lowest levels in liver, spleen, intestine and heart. This study provides foundation for further exploring the disease-resistant immunity of Nile tilapia.

The Pratylenchus species are important plant-parasitic nematodes and seriously damage the agricultural production worldwide. The establishment of a rapid method for detecting Pratylenchus species is significant to the integrated management of root-lesion nematode diseases. Based on the comparisons of mitochondrial cytochrome oxidaseⅠ(mtCOI) gene sequences from Pratylenchus species, primers were designed, including 3 forward primers Pc-m193-F, Pn-m280-F and Ps-m66-F specific to P. coffeae, P. neglectus and P. scribneri, respectively, and one common reverse primer Pcns-m-R. The PCR products were 225, 137 and 351 bp, respectively, and the PCR sensitivities were 1/1 000, 1/10 000 and 1/100 of individual adult nematode, respectively. The multiplex-PCR system was established successfully after optimizing the concentration ratio of different primers and the annealing temperature. Three Pratylenchus species can be simultaneously and specifically detected using the multiplex-PCR by the combination of primer Pc-m193-F, Pn-m280-F, Ps-m66-F and Pcns-m-R with concentration of 0.4, 0.4, 0.8 and 1.2 μmol/L, respectively, and the annealing temperature with 54 ℃. The detection of 18 Pratylenchus samples using the multiplex-PCR was carried out, and the results were consistent with previous identification. The established multiplex-PCR system could be used to quickly detect the P. coffeae, P. neglectus and P. scribneri either in the samples of single or mixed species infection. This technique could provide the support for diagnosis and integrated management of Triticum aestivum root-lesion nematode diseases in China.