Seed development is directly related to crop yield and quality, although several genes that can regulate seed development have been reported in the literature, the molecular mechanism of seed development has not yet established a complete regulatory network. In order to identify the main genes regulating seed development and improve the control network of seed development, the gene PH-START1 (pleckstrin homolgy - the lipid/sterol-binding St AR-related lipid transfer protein domains 1) was identified that can regulate seed development in Arabidopsis thaliana, over-expression of PH-START1 in Arabidopsis reduced the seed yield by 61.7% and the grain weight by 80%, which indicated that PH-START1 was the main gene regulated seed development. The function of the gene in regulating the development of seed was determined by analyzing the growth, seed setting rate and grain weight of the transgenic plants with functional loss and gaining. By observing the development of seed embryo and endosperm, the specific process of gene regulation of seed development was clarified. By analyzing the tissue location of the target gene, the expression pattern was clarified. The wild-type seeds were arranged neatly and tightly and the pods were full under the microscope. The PH-START1 T-DNA insertion mutant, the PH-START1, was arranged neatly, occupying the entire inner space of the siliques, with a single silique producing 55 seeds, with a 117% seed yield. However, the seeds of PH-START1 transgene OE-1 in A. thaliana were sparse and scattered, with only 18 seeds in a single silique, and only 38.30% of the seed setting rate. Further analysis showed that PH-START1 also affected the structure of the seeds. The endosperm cells of the seeds after over-expression of PH-START1 decreased obviously, and the aleurone layer cells were arranged in disorder and embryo development was blocked. In order to deepen the mechanism of PH-START1, the expression pattern of PH-START1 was analyzed. The results showed that PH-START1 was the most expressed in seed, and was mainly distributed at the base and top of style with the growth time. In stamens, the expression in filament and anther increased with the increase of development time, and was concentrated in pollen grains. This study identified that the PH-START1 was a main gene that regulates seed development, and clarified its specific process and expression patterns that affected seed development. This result provide a theoretical basis for improving seed development regulatory molecular network and modern molecular breeding.

Linum usitatissimum ω-3 polyunsaturated fatty acids (PUFAs) exert multiple physiology functions, which has to be provided from foods in mammals. The fatty acid desaturase-3 gene (FAD3) encodes a ω-3 PUFAs desaturase and converts ω-6 PUFAs to ω-3 PUFAs. In the present study, the hFAD3 gene expression cassette C2 (5'arm-CAG-hFAD3-PolyA-3'arm) and M2 (5'arm-MAR-CAG-hFAD3-PolyA-MAR-3'arm) were successfully knocked-in NCAPG-LCORL locus of bovine (Bos taurus) fetal fibroblast cells using CRISPR/Cas9 gene editing system. In the obtained transgenic cells, the ratio of ω-6 PUFAs and ω-3 PUFAs was significantly decreased from 1.549 to 0.565 investigated by gas chromatograph attached to mass spectrometer for fatty acid determinator (P<0.01). The expressions of lipolysis associated genes including peroxisome proliferator activated receptor 1 gene (PPARg), hormone-sensitive lipase gene (LIPE) and lipoprotein lipase gene (LPL) were more up-regulated than fatty acid synthesis associated genes including acetyl CoA carboxylase gene(ACC), stearoyl CoA desaturase gene (SCD) and fatty acid synthase gene (FASN) in the obtained transgenic cells detected by qRT-PCR. 59 monoclones were obtained by flow cytometry, in which 3 strains were knocked-in cells. The knock-in efficiency was 5.1%. In the number 89 knocked-in monoclonal cells, the ratio of ω-6 and ω-3 PUFAs was significantly decreased from 1.589 to 0.575 compared with the negative monoclonal cells number 21 (P<0.01). The expressions of lipolysis associated genes (PPARg, LIPE, LPL) were more up-regulated than fatty acid synthesis associated genes (ACC, SCD, FASN) in the knocked-in monoclonal cells. The expressions of fatty acids and fatty acids metabolism related genes in the monoclonal cells were in consistent with the routine transgenic cells. In addition, the hFAD3 expression of transgenic cells mediated with matrix attachment region (MAR) was 5.91 times as much as without MAR (P<0.01), thus the MAR significantly increased hFAD3 expression than that of without MAR in transgenic cells. In conclusion, the hFAD3 gene could successfully knock-in at NCAPG-LCORL locus of bovine fetal fibroblast cells through CRISPR/Cas9 system and expressed in transgenic cells. The involvement of MAR element significantly increased the hFAD3 expression. This study may provide an alternative protocol to construct transgenic vector and probably be useful to produce transgenic animals.

Chinese kale (Brassica alboglabra) is one of the important vegetables in the Cruciferous family. It is often subjected to various adverse effects during the cultivation process, causing great losses to the production. In order to improve the resistance of Chinese kale to stress, the specific primers were designed according to BoTCTP (translationally controlled tumor protein cloned from Brassica oleracea) in this research. The interest fragment was amplified by PCR, and then the overexpression vector was constructed and transferred into Chinese kale by Agrobacterium tumefaciens mediated. The transformants were confirmed by molecular methods and biological assay. Four transgenic plants were obtained, and then taken to design the experiments associated with growth and different stress, such as high temperature, low temperature, salt, heavy metal and so on. The results showed that the transgenic plants grew faster, stronger and had bigger leaves than non-transgenic plants. In the different stress condition, the transgenic plants had obviously stronger resistance than the control, which showed that the BoTCTP was closely related to the growth and had a regulatory effect on the stress resistance of Chinese kale. These results provide some scientific basis for the future research on the stress resistance of Chinese kale.

To provide an important tool for molecular marker-assisted breeding of wood quality traits, a new nuclear genome SSR marker was developed from functional genes related to Cunninghamia lanceolata wood formation. The SSR locus was detected through analyzing the sequences of 10 genes involved in the formation of C. lanceolata. The availability and polymorphism of the designed SSR primers were examined by capillary electrophoresis. Linkage disequilibrium of pairwise loci and the association analysis between developed SSR loci and wood density were performed by using TASSEL (trait analysis by association, evolution and linkage) GLM (general linear model) program. A total of 15 SSR loci were identified in 7 functional genes. There were di-, tri-, tetra- and hexa- nucleotide repeats, with trinucleotide SSRs being the most frequent,accounting for 46.7% of the total loci. Fourteen pairs of PCR amplification primers were designed according to conserved sequences on both sides of SSR loci. Among them, 7 pairs of primers showed polymorphism. A total of 644 SSR loci combinations were found to have a certain degree of linkage disequilibrium (D'≠ 0), with D' values of 183 combinations greater than 0.5. The association analysis revealed that the CLSSR4 locus from the 5' untranslated region (5' UTR) of ClCesA3 (C. lanceolata cellulose synthase 3) was extremely significantly associated with wood density (P<0.01). The wood density variance explained by this locus was 26.07%, and the corresponding alleles of A201 and A205 showed the positive and negative phenotypic effects, respectively. In this study, seven polymorphic SSR primer pairs were developed from genomic sequence of functional genes, and a SSR locus derived from the 5' UTR of ClCesA3 gene was significantly associated with wood density of C. lanceolata. These results will provide an important tool for molecular marker-assisted improvement of C. lanceolata wood quality.

The B3 family is a family of transcription factors that contains the B3 domain, which plays an important role in the growth and development of plants. In order to analysis the structure and expression pattern of Phyllostachys edulis B3 family genes, in this study, a total of 78 P. edulis B3 family genes were identified using bioinformatics techniques and methods. These genes could be divided into 4 evolutionary branches of the families, including LEAFY COTYLE-DON2[LEC2]-ABSCISIC ACID INSENSITIVE3[ABI3]-VAL (LAV), auxin response factor (ARF), related ABI3-VP1 (RAV) and reproduction meristem (REM). The transcriptome analysis of 7 materials in moso bamboo showed that the expression patterns of the 78 B3 family genes of could be divided into 5 groups, and B3 family genes had different expression level in 7 materials in moso bamboo. The first 15 genes were mainly expressed in panicles; 20 genes in the second group were mainly expressed in roots; 23 genes in the third group were mainly expressed in young shoots; 7 genes in the fourth group were mainly expressed in leaves; 13 genes in the fifth group were mainly expressed in bamboo rhizome. So, the B3 family of P. edulis plays a important role during the growth of moso bamboo. Two genes were selected from 4 subfamilies to detect the expression patterns of B3 family of P.edulis in different parts of bamboo shoots by qRT-PCR. The results showed that the P. edulis family genes were diversely expressed in different parts of bamboo shoots. Most of B3 family members had high expression in most internode tissues. For example, PH01000024G2370, PH01000297G0830, PH01001094G0220, PH01000114G0050, PH01000548G0300 and PH01000528G0920 had high levels of expression in the 2-4 node internode (internode 2-4). Only 2 genes (PH01000198G0380, PH01001094G0220) had high levels of expression in interval. There were 6 genes (PH01000297G0830, PH01000198G0380, PH01000114G0050, PH01000548G0300, PH01000528G0920 and PH01000469G0440) with high levels of expression in sheath, and one gene (PH01000024G2370) was almost no expression. Only one gene (PH01000024G2370) had a high level of expression in roots. Results suggested that the ARF family (eg PH01000114G0050, PH01000548G0300) played an important role in the rapid growth of bamboo shoots. According to the expression patterns of RAV family (e.g. PH01000024G2370, PH01000297G0830), it was presumed that the growth of shoots in roots and near roots was relatively inhibited during the rapid jointing growth of bamboo shoots. This study systematically investigated the structure, phylogenetic relationship and the expression pattern of B3 family genes of P. edulis, which provides basic data for the further study of B3 family gene function.

Rhododendron henanense subsp. lingbaoense belongs to Ericaceae, Rhododendron, and is a rare and endemic species in Henan Province. Because of its narrow distribution and poor natural regeneration, the population size has been shrinking, and systematic research and protection has become increasingly urgent. In order to develop SSR (simple sequence repeat) molecular markers for conservation genetics research on R. henanense subsp. lingbaoense, simplified genome sequencing by restriction-site associated DNA sequencing (RAD-seq), SSR search and primer design were performed. A total of 6 107 SSR loci were detected in 120 970 assembled sequences. Among them, the proportion of di-nucleotide motifs was the highest, accounting for 63.01% of the total loci; followed by tri-nucleotides with 765, accounting for 12.53%. 3 843 pairs of primers were successfully designed for these SSR loci using software Primer Premier 3.0. A total of 100 pairs of primers were randomly selected and verified with 20 individuals of R. henanense subsp. lingbaoense, and 87 were able to amplify the target bands. Fifteen pairs of polymorphic SSR loci were further analyzed using Cervus 3.0, and the polymorphic information content ranged from 0.463 to 0.893 with an average of 0.649. Among them, there were 13 highly polymorphic loci, no deviation from Hardy-Weinberg equilibrium was detected in the 15 loci. Above results could provide data support for the study of SSR markers in R. henanense subsp. lingbaoense and Rhododendron.

Indole-3-acetic acid (IAA) as a plant hormone participates in the regulation of many plant growth and development processes and plays an important role in the symbiosis between arbuscular mycorrhiza fungi (AMF) and land plants. But the regulation mechanism of AMF infection on auxin metabolism is unclear. In this study, the effects of Glomous mosseae (G.m) infection on the tobacco (Nicotiana tabacum) shoot height, leaf area, the IAA content, activities of enzymes involved in IAA metabolism and related gene transcript levels in tobacco leaves were analyzed, aimed to elucidate the relationship between the AMF induced growth improvement and the metabolism of endogenous IAA. The results showed that G.m inoculation promoted tobacco growth displayed by improvement in increase rates in shoot height and leaf area compared with those of control (without inoculation). The IAA content, activities of peroxidase (POD) and indole-3-acetic acid oxidase (IAAO), enzymes participate in the degradation of auxin, decreased in inoculated plants. G.m infection caused up regulation in transcript levels of IAA synthetic enzyme gene (NtAMI1), IAA responsive factor genes (NtARF1) as well as auxin transcriptional regulator gene (NtIAA9), depressed the expression of auxin-repressed protein (NtARP1;1). Therefore, it can be deduced that G.m infection improved tobacco growth by promoting NtAMI1-dependent IAA production, inhibiting POD and IAAO-dependent IAA degradation, as well as increasing IAA function by up-regulating NtARF1 and NtIAA9 expression and down-regulating NtARP1;1 expression. These results provide the scientific basis lights for the deep understanding of the promoting effect of AMF on plant growth.

Research shows that Liver kinase b1 (Lkb1) plays key roles in mouse (Mus musculus) skeletal muscle development and energetic metabolism processes. Yak (Bos grunniens) is regarded as the specific livestock in Qinghai Tibet Plateau and its adjacent regions, whose growth and development of skeletal muscle is one of most important factors effecting on meat performance. To explore the underlying functions of Yak Lkb1 regulation on skeletal muscle development, growth and energy metabolism, its CDS region was firstly cloned using cDNA as template from longissimus dorsi muscle (L-muscle) by PCR. Next, the physic-chemical properties of Lkb1 amino acid sequence and evolutionary conservation among species were predicted by bioinformatic methods. Lastly, the tissues expression pattern in Jinchuan Yak was detected by quantified PCR and subsequently the expression difference between Jinchuan and Maiwa Yak in skeletal muscle was analyzed. The result showed that the CDS of Yak Lkb1 gene was 1 317 bp (GenBank No. MH412713), encoding 438 amino acids. ProtScal and TMpred software predicted that Lkb1 was a hydrophilic protein with characters of nuclear/cytoplasm translocation, existed transmembrane domain in the region of 231~253 (from inside to outside) and 238~255 (from outside to inside) amino acids. Phosphorylation/dephosphorylation sites were analyzed by NetPhos2.0 Server showed that Lkb1 had 6 phosphorylation/dephosphorylation sites in the N-terminal of this protein, including Ser13, Ser18, Thr7, Tyr8, Tyr19 and Tyr23. Further, functional prediction by Protfun2.2 Server showed Lkb1 might regulating energy and fatty acid metabolism. In addition, the secondary structure of Lkb1 was mainly α-helices and random coil, α-helices with 33.79% and random coil with 44.75%, respectively. Moreover, the conservation analysis showed that Yak Lkb1 gene has highest homology comparison of sheep (Ovis aries) and goat (Capra hircus), both with 97.5% comparability of nucleotide sequence and 99.54% of amino acids sequence. The amino acid homology of Lkb1 comparing to chicken (Gallus gallus), pig (Sus scrofa), dog (Canis lupus familiaris), zebrafish (Danio rerio), panda (Giant panda), gorilla (Pan troglodytes), human (Homo sapiens) and chimpanzee (Rattus norvegicus) were 79.73%, 95.89%, 84.05%, 83.31%, 92.71%, 81.51%, 99.54% and 87.47%. Therefore, the Lkb1 protein had high homology among species. However, the comparability of Lkb1 between Yak and mouse just reached at 45.48% and 44.19% of nucleotide and protein sequence, respectively. Interestingly, mRNA level of Lkb1 gene was enriched in skeletal muscle, compared to that of in liver and fat tissues. Furthermore, the differential expression detection in L-muscle and semitendinosus muscle (S-muscle) from Jin chuan and Maiwa Yak revealed that Jinchuan Yak had significantly higher mRNA level of Lkb1 gene in S-muscle than that of Maiwa Yak, but its mRNA level was indistinguishable in L-muscle between these 2 strains (P<0.05). Thus, it's speculated that Lkb1 might be involved in skeletal muscle growth and energy metabolism. Collectively, these data will provide reference to elucidate the function and mechanism in Yak skeletal muscle biology in the further study.

Bone morphogenetic protein (BMP)/Smad family member (SMAD) signaling pathway plays important role in the regulation of mammalian cell proliferation and differentiation. In order to study the effects of BMP4 and BMP/Smad signaling pathway on litter size of Small Tail Han (STH) sheep (Ovis aries). The present study used qRT-PCR to detect the expression of BMP4 gene and its downstream gene SMAD4 in the BMP/Smad signaling pathway in the 14 tissues (heart, liver, spleen, lung, kidney, brain, cerebellum, hypothalamus, ovary, pituitary, uterus, oviduct, thyroid, and adrenal gland). These tissues were collected from STH sheep (3 polytocous sheeps and 3 monotocous sheeps) in the follicular phase. In previous work, a missense mutation g.63454744T>G in BMP4 coding region was found by sheep genome resequencing. Then the genotypes of g.63454744T>G were detected by Sequenom MassARRAY^® SNP technique in 380 STH sheep and the association between the mutation and litter size were analyzed. The results of qRT-PCR showed that these two genes expressed in all 14 tissues in follicular phase. The BMP4 gene highly expressed in spleen, liver, heart, and uterus. SMAD4 gene were highly expressed in cerebellum, hypothalamus, pituitary, and ovary. The expression of BMP4 gene in uterus (P<0.001), hypothalamus (P=0.002), pituitary (P=0.001), and ovary (P<0.001) of polytocous ewes were extremely significant higher than that of monotocous ewes. The SMAD4 gene expression in hypothalamus (P<0.001), cerebellum (P=0.001), ovary (P<0.001), pituitary (P=0.001), brain (P=0.001), uterus (P=0.007) and oviduct (P=0.005) of polytocous ewes were extremely significant higher than that of monotocous ewes. The SNP g.63454744T>G in BMP4 gene had a low polymorphism (polymorphism information content<0.25) in STH sheep. The χ² test indicated that the locus of g.63454744T>G of BMP4 gene was under Hardy-Weinberg equilibrium (P>0.05) in this STH sheep population. The polymorphisms of g.63454744T>G in BMP4 was significantly associated with each parity litter size in STH sheep (P<0.05), and the litter size of mutant homozygotes was higher than that of wild types and heterozygotes. Therefore, it could be concluded that there was a positive correlation between BMP4 and SMAD4 gene expression and litter size in STH sheep during follicular phase, and the SNP locus of g.63454744T>G in BMP4 might have significant association with the litter size in each parity of STH sheep. The present study provides a reference basis for lambing traits selection of STH sheep.

Small Tail Han sheep (Ovis aries) is a sheep breed with both excellent meat and fur in China, and famous for its fast growing speed and high lambing rate. microRNA (miRNA) is post-transcriptional regulator factor which is involved in the regulation of development of skin and hair follicles in various animals. In the present study, in order to illustrate this regulation in Small Tail Han sheep, the miRNA expression profiles of the skin and hair follicles during adult (XWHY_1), postnatal 30 d (XWHY_2) and newborn (XWHY_3) were established and analyzed by using high throughput sequencing technology. The results showed that total 1 062 miRNAs, including 160 known miRNAs and 902 new miRNAs, were identified in these 3 developmental periods. By statistical analysis of these miRNAs, it was found that there were 69, 127, and 73 specifically expressed miRNAs in XYHY_1, XYHY_2 and XYHY_3, respectively. Total 666 miRNAs were commonly expressed in the 3 periods. Statistical analysis of differential expression of miRNAs was conducted by Fisher exact test and Chi-square test, and meet the condition of |log₂(Fold_Change, FC)|≥1 and P≤0.05. By comparing XWHY_1, XWHY_2 and XWHY_3, 339 differentially expressed microRNAs were screened, 226 differentially expressed microRNAs were obtained after removing the repetition, and 2 645 target genes were predicted. Differentially expressed miRNAs were enriched by Kyoto encyclopedia of genes and genomes (KEGG) Pathway, results showed that 2 645 target genes were annotated to 181 signaling pathways. Differentially expressed of miRNAs was enriched by gene ontology (GO) and showed to be mainly participated in transcriptional regulation, formation of cell membrane and ion binding. The present study provides basic data to analyze the molecular mechanism of fur/velvet formation at miRNA level, and also provides a new thought to improve the yield and quality of wool/velvet of Small Tail Han sheep by means of manual regulation while maintaining its existing excellent quality.

The genetic quality control research is very necessary for specific pathogen free (SPF) Yorkshire (Sus scrofa) and Landrace pig populations used as the important experimental animals. To analyze the genetic diversity of the mitochondrial DNA (mtDNA) displacement-loop region (D-loop region) and reveal the maternal genetic structure of experimental specific pathogen free (SPF) Yorkshire and Landrace population, genomic DNA of SPF Yorkshire and Landrace pigs were extracted, the mtDNA D-loop region was amplified by PCR, and the sequences were aligned. The local Bama miniature pigs and Rongshui miniature pigs were used as control. A total of 40 variable sites were identified in 107 samples, including 13 haplotypes. Compared with the local breeds, the fewer haplotypes were found in the Yorkshire and Landrace, which contained 3 and 2 specific haplotypes, respectively, and one shared haplotype H4. There was one dominant haplotype in each of the two breeds. Analysis result of the haplotype diversity (Hd) showed that the Rongshui miniature pigs was the highest (0.767) and Landrace was the lowest (0.429). Yorkshire and Bama miniature pigs were 0.566 and 0.503, respectively. In addition, the results of network and phylogenetic tree showed that the genetic relationship between Yorkshire and Landrace populations was closely related to European pig breeds. The study indicated that there was less haplotypes and lower genetic diversity in SPF Yorkshire and Landrace populations. Only one dominant haplotype for each breed was observed. This study will be beneficial for the further research of experimental animal standardization and genetic quality control.

Histone methylation is key epigenetic regulatory feature that have important function in many biological processes which include heterochromatin formation, X-chromosome inactivation and transcriptional regulation. Among them, histone H3 lysine specific demethylase 6A (KDM6A) has functions of regulating embryonic development, cell reprogramming and cell differentiation. At present, the research on KDM6A mainly focused on experiments in a non-cellular environment, and there were few studies in cells, and no animal model of KDM6A overexpression is observed. In the present study, total RNA of 293T CELL was extracted and reversely transcribed into cDNA. Specific primers were designed according to human (Homo sapiens) mRNA sequence of KDM6A gene. The coding sequences of KDM6A gene was cloned in vitro and was constructed the overexpression vector pcs2-KDM6A by genetic engineering. Then, the vector pcs2-KDM6A was transfected into mouse (Mus musculus) embryonic fibroblasts via lipofectin transfection method. The gene expression patterns were detected by qRT-PCR. The qRT-PCR assay showed that the mRNA expression level of KDM6A in transfected group was significantly higher than control group (P<0.05). In contrast, and there was no significant difference between expression of KDM6B in transfected group and control group. Furthermore, H3K27me3 expression was detected by laser confocal immunofluorescence (IF) and western blot (WB) technology respectively. These results demonstrated that the expression of H3K27me3 level after transfection of pCS2-KDM6A vector both were significantly lower than control group. Taken together, the expression vector pCS2-KDM6A was successfully constructed, and was proved to had correct enzyme activity at the cell level. The pCS2-KDM6A vector can be used to study the function of KDM6A in the development and related diseases both in vivo and in vitro. Moreover, an animal model of overexpression of KDM6A can be prepared by this vector, and a basic material for analyzing the research mechanism of KDM6A and H3K27me3 in biological events such as X chromosome inactivation, embryo development and cell reprogramming is provided.

In addition to the regulation of lipid metabolism, miR-33 is also reported to be involved in glucose metabolism, inflammatory response, and cell cycle. However, the role of the chicken (Gallus gallus) miR-33 and its relationship with the predicted target gene choline phosphotransferase 1 (CHPT1) are still unclarified. The present study was conducted to investigate whether CHPT1 was the target gene of miR-33. The bioinformatics methods were used to predict the target genes of miR-33, and 378 genes from 3'-UTR database were predicted and CHPT1 gene was included. Then, the miR-33 overexpression vector and CHPT1 luciferase reporter vector were constructed and co-transfected into the mouse (Mus musculus) C2C12 myoblast cell, dual-luciferase reporter assay showed that the expression of luciferase reporter gene linked to the 3'-untranslated region of CHPT1 mRNA was down-regulated by miR-33 overexpression in C2C12 cells (P<0.01). Furthermore, the down-regulation was completely abolished when the predicted miR-33 target site in CHPT1 3'-UTR was mutated. Then mir-33 antagonists LNA (locked nucleic acid)-antimiR-33 was designed and synthetised, after transfecting chicken primary liver cells, the expression level of miR-33 decreased by 44%, while CHPT1 mRNA increased with a certain degree. By qRT-PCR, it was found that miR-33 highly expressed in muscular stomach and heart muscle, and the miR-33 expression in muscular stomach was significantly different from that in spleen, kidney, brain and glandular stomach (P<0.05), and very significantly different from that in liver and thigh muscle (P<0.01). In liver and abdomen fat tissue of 4 week lean and fat line chicken, miR-33 was significantly higher in the fat line than that in the lean line (P<0.05). The CHPT1 expression in the abdominal fat tissues was significantly higher in fat line chicken than that in lean line chicken (P<0.05). The above data indicate that miR-33 might play an important role in lipid metabolism in the chicken liver by negatively regulating the expression of CHPT1. The present study provides new clues for lipid synthesis.

Disease outbreak from Streptococcus agalactiae infection has become the most serious bacterial disease for tilapia (Oreochromis spp.) aquaculture in China, while effective prevention and control methods have not yet been established. It is an important approach to breeding the most S. agalactiae -resistant hybrid tilapia for better control and prevention S. agalactiae in tilapia. 4 family groups of hybrid tilapia F₁ with different parents were got. They were O. niloticus Egypt strain ♀×O. aureus Xia'ao strain ♂ (NE ♀×AX ♂, EX), O. niloticus Xianghu strain ♀×O. aureus Xia'ao strain ♂(NX ♀×AX ♂, XX), O. niloticus Xianghu strain ♀×O. aureus Egypt strain ♂ (NX ♀×AE ♂, XE), O. niloticus Egypt strain ♀×O. aureus Egypt strain ♂ (NE ♀×AE ♂, EE). The offspring were strengthened rearing for 100 d in the ponds and taken back to the laboratory. All fish were acclimated for 14 d in the laboratory then artificially infected by S. agalactiae intraperitoneally. Fish were cultured on 33±1 ℃. The relative survival rate and serum biochemical parameters of 4 hybrid tilapia infected by S. agalactiae were investigated. The relative survival rate was recorded at different times post infection; Blood samples were respectively collected and analyzed. The relative survival rate at 7 d after infection was as follows: EX hybrid tilapia (50%) > EE hybrid tilapia (37.5%) > XE hybrid tilapia (33.3%)>XX hybrid tilapia (20.8%). Alanine transaminase (ALT) of EX hybrid tilapia showed no significantly difference (P>0.05) before and post injection, while that of other 3 species was significantly higher after infection(P<0.05);The serum electrolytes of 4 hybrid tilapia showed significantly difference after infection by S. agalactiae (P<0.05), but Na⁺, Ca²⁺ and Cl^- of EX hybrid tilapia almost returned to control level at 3 d after infection. Globulin (GLO) of 4 hybrid tilapia increased statistically at 3 d after infection, and peaked at 7 d, while the albumin/globulin (A/G) index of 4 hybrid tilapia declined significantly at the time (P<0.05); Lactic dehydrogenase (LDH) of all 4 hybrid tilapia increased significantly on the progress of disease (P<0.05), and peaked with approximately 1.81-fold, 6.1-fold, 2.42-fold and 3.77-fold when compared with control samples prior to infection, respectively. Superoxide dismutase (SOD) activity of EE hybrid tilapia was significantly lower than that of the other species prior to infection, but SOD activity of EX hybrid tilapia was the lowest at 1 d and 7 d after infection. The triglyceride (TG) of 4 hybrid tilapia decreased significantly at 3 d after infection by S. agalactiae (P<0.05), while the total cholesterol (TC) of EX, XX and XE decreased significantly at 3 d after infection (P<0.05). Biochemical indexes had shown the 4 hybrid tilapia manifested liver and kidney damage, muscle injury and lipid metabolism after infection, while EX hybrid tilapia diserved less liver damage and muscle injury. And EX and EE hybrid tilapia showed less injury on metabolic activity. These results indicated that EX hybrid tilapia was the most resistant to S. agalactiae. In this study, the potential effects of the different tilapia orthogonal combinations were evaluated from the perspective of anti-streptococcosis. The expected results provide theoretical basis and potentially effective for genetic improvement of tilapia S. agalactiae -resistant breeding.

Several novel gene editing techniques have been applied in scientific research and livestock breeding in recent years, including zinc finger protein nuclease (ZFN), transcriptional activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats/CRISPR associated proteins (CRISPR/Cas) system. These gene editing techniques site-directly edit genomes by recognizing the target sites according to the specific structure followed by endonuclease dissection,which is characterized by high efficiency, accuracy, and simple production. In this paper, we demonstrated the basic principles, differences and potential main problems of above three gene editing techniques, summarized the application in the main livestock pigs, cattle and sheep, and provided an outlook of the future of these techniques in application.

Genome-wide association study (GWAS) is an effective way to study the relationship between genetic variation and complex traits or diseases. The idea of GWAS is to find the correlation between molecular variation and target phenotype. With the development of the high-throughput, and the improvement of bioinformatics and statistical methods, it promotes researches on fine mapping of complex traits. On the basis of genome-wide association study, this review introduces the research progress of GWAS on genome, metabolomics as well as proteome, including haplotype-based GWAS, metabolic GWAS and gene expression-based GWAS. We finally make a prospect for GWAS study in the future.

Rice (Oryza sativa) G6H1 is a new transgenic rice line that has the feature of insect-resistant and herbicide-tolerant. It is developed by Chinese scientists with independent intellectual property rights. The previous studies showed that G6H1 had good performance of insect and glyphosate resistance, and it met the requirement of application level for insect-resistant and herbicide-tolerant, and had a good and broad application prospect. At present, there was lack of methods for accurate quantitative detection of G6H1. In this study, based on the droplet digital PCR (ddPCR) platform, a duplex ddPCR accurate quantification method for G6H1 was established. First, the specificity of primer and probe designed for the G6H1 event was verified by qRT-PCR. The fluorescent signal was not observed in other transgenic crops and non-transgenic crops and only was observed in G6H1 event, these results indicated that designed primer and probe had high specificity for G6H1; then, the suitable different rice endogenous genes for duplex ddPCR detection method were screened and selected, the sucrose phosphate synthase gene (SPS) with 122 length was chosen as the candidate endogenous gene in this study. Moreover, the concentration and annealing temperature of the primer and probe for duplex ddPCR were optimized. The final concentration of primers/probes as 0.5/0.25 μmol/L combined with 58 ℃ annealing temperature were determined as the final optimal condition for the duplex ddPCR. The established duplex ddPCR method for analysis of transgenic rice G6H1 showed that the limit of detection for endogenous and exogenous genes reached 3 copies, and the limit of quantification of endogenous and exogenous genes was 25 copies, and the relative standard deviation (RSD) of quantitative results was less than 25%. Finally, the quantitative results of samples content of 5%, 1%, and 0.5% of transgenic rice G6H1 were compared using simplex qRT-PCR, simplex ddPCR, and duplex ddPCR. All the results showed that the established duplex ddPCR method had good specificity, high sensitivity, accuracy and reliability, and was suitable for precise quantitative analysis of transgenic rice G6H1 event. The establishment of this method provided an alternative method for quantitative detection of transgenic rice G6H1, and improved the detection technology system for transgenic rice components of China. Meanwhile, the established method also can work as new technical reserves for the safefy threshold regulation of genetically modified agricultural organisms.

General control non-derepressible-2 (GCN2) regulates protein synthesis by phosphorylation of eukaryotic translation initiation factor 2 (eIF2α) and responding to amino acid deficiency and various stresses. At present, the action mechanism of GCN2 in yeast and animals has been relatively clear, but the regulation mechanism of plant GCN2 is not clear. In order to further study the function of GCN2, 2 special primer sequences were designed to amplify the full-length NtGCN2 and N-terminus (1~437 aa) gene sequences, and then inserted them to pET-15b and pCzn1 vectors, respectively. The prokaryotic expression vectors pET15b-NtGCN2 and pCzn1-NtGCN2 (1~437 aa) were transformed into Escherichia coli BL21-CodonPlus-(DE3)-RIPL, BL21-CodonPlus-(DE3)-RPL and BL21(DE3) Plys for expression, respectively. The full length NtGCN2 and N-terminal NtGCN2 (1~437 aa) proteins were successfully expressed in E.coli BL21-CodonPlus-(DE3)-RIPL and BL21(DE3) Plys, respectively. The expressed protein were identified by Western blot using 6×His mouse antibody as a primary antibody. The prokaryotic expression condition of full-length NtGCN2 including induction concentration of isopropyl β-D-1-thiogalactopyranoside (IPTG), culture temperature and culture time were optimized. These results showed that soluble NtGCN2 protein was obtained in the supernatant of BL21-CodonPlus-(DE3)-RIPL under the conditions of 16 ℃, 0.5 mmol/L IPTG and 13 h induction. The protein was purified by affinity chromatography on anion exchange column Hitrap Q and Ni²⁺-NTA agarose and the NtGCN2 protein was not well separated and purified. Furthermore, the purified NtGCN2 and NtGCN2(1~437 aa) proteins were obtained by inclusion body renaturation, and a single band of NtGCN2(1~437 aa) pure protein was obtained by further Ni²⁺-NTA agarose affinity chromatography. Finally, The purified recombinant protein was used as immunized rabbit antigen and polyclonal antibody against NtGCN2 was successfully prepared, the titer and specificity of the antibodies were analyzed. Enzyme-linked immune sorbent assay (ELISA) assay showed that the antibodies titer reached over 1∶50 000. Western blot analysis showed that the prepared NtGCN2 full-length and NtGCN2(1~437 aa) polyclonal antibody could specifically recognize the recombined NtGCN2 protein. In this study, the prokaryotic expression and purification of NtGCN2 protein were achieved, and the antibody against NtGCN2 was prepared, which would provide data for the further exploration of the function of plant GCN2.

Bactrian camels (Camelus bactrianus) is a endangered species in China's northwest region. It has special physiological characteristics and has widely uses. Induced pluripotent stem cells (iPSCs) has important application value in protecting the germplasm resources of this species. However, the traditional methods for the isolation and identification of iPSCs are complicated and cumbersome. For the rapid and efficient isolation and identification of iPSCs, the CRISPR/Cas9 system was used in this experiment. The single guide RNA (sgRNA) was designed near the stop codon in reference to the Nanog gene expressed only in the embryonic stem cells (ESCs) stage of Bactrian camel, and the cleavage vector was successfully constructed and screened; the whole camel genome was used as a template to obtain upstream and downstream homology arms, and two reporter genes of green fluorescent protein gene (GFP) and neomycin resistance gene (Neo) were inserted between the two homologous arms, the expression vector was successfully constructed. At the same time, the minimum lethal dose of geneticin (G418) of Bactrian camel fibroblasts was obtained by the minimum lethal dose experiment. After sequencing and verification of the above two vectors,the Bactrian camel fibroblasts were co-transfected with the expression vector and the cleavage vector and cultured for 48 h, and then cultured for two weeks in a selection medium containing G418; then, the normal culture medium was used to expand the cells after the drug screening, the highly viable stable transfected cell line was successfully obtained. This experiment will provide biomaterials for the correct screening of iPSCs by sequencing the reporter gene after the Nanog gene.