NAC (NAM/ATAF/CUC) family is a class of transcription factors unique to plants, and plays crucial roles on developmental regulation, hormone signal transduction, biotic and abiotic stress responses. In the present study, a stress-responsive NAC gene, named as GhSNAC1 (GenBank No. KU759894), was isolated from upland cotton (Gossypium hirsutum) via reverse transcription PCR (RT-PCR). The sequence analysis indicated that GhSNAC1 was 1 104 bp in length and encoded a protein of 299 amino acids with a relative molecular mass of 33.9 kD and an isoelectric point of 6.18. GhSNAC1 contained a conserved NAC domain in the N-terminus, indicating that it was a typical NAC transcription factor. The phylogenetic analysis indicated that GhSNAC1 had extremely high similarity with ATAF1 (Arabidopsis transcription activation factor 1) from Arabidopsis thaliana. qRT-PCR anaylsis showed that GhSNAC1 was induced by treatments of drought, salt, cold and Verticillium dahliae which suggested that GhSNAC1 might be involved in responses to biotic and abiotic stresses. GhSNAC1 was then overexpressed in transgenic tobacco (Nicotiana tabacum) plants. Transgenic lines and wild type (WT) were used to evaluate the germination and seedling growth under either salt (100 mmol/L NaCl) or drought (200 mmol/L mannitol) stress. The results indicated that drought and salt stresses significantly inhibited the germination of WT seeds, and the germination rate of WT was below 50%, while the germination of transgenic lines was above 80%. The WT seedlings were small and weak. However, the transgenic lines were vigorous. The fresh weight per plant and root length of transgenic lines were more than twice and 1.5 times of those of wild type, respectively. Collectively, the above results suggested that overexpression of GhSNAC1 in tobacco could improve tolerance to drought and salt stresses. The present study laid a foundation for further exploring the molecular mechanism of drought and salt tolerance of GhSNAC1.

Telomerase is a ribonucleoprotein complex with reverse transcriptase activity in eukaryotic cells, telomerase reverse transcriptase (TERT) is the major component of telomerase. AtTERT is the first plant telomerase reverse transcriptase TERT gene cloned from Arabidopsis thaliana. In order to clarify the structure and function of AtTERT, bioinformatics tools and software were used to analyze the physicochemical properties, protein structure and functional domain of AtTERT, and the genetic relationship of TERT genes among different species. Moreover, the telomerase activity and AtTERT gene expression patterns under salt and oxidative stress were analyzed. The results showed that the AtTERT belonged to a hydrophilic protein without a signal peptide, and had no obvious transmembrane domain. There were 125 phosphorylation sites, of which 87, 28 and 10 were serine, threonine and tyrosine sites, respectively. Irregular crimps and α-helices were the major structural elements of secondary structure of AtTERT. Conserved domains were the TRBD (telomerase RNA binding motif) domain and the RT (telomerase reverse transcriptase motifs) domain. Three-dimensional structure modeling analysis revealed a 74% similarity of the main-chain conformation between AtTERT and the Tetrahymena thermophila telomerase reverse transcriptase c6d6vA. Phylogenetic analysis using the TERT protein sequences of Gramineae, Leguminosae, Liliaceae, Rosaceae, etc, revealed that TERT was highly conserved in the same plant group. For example, TERT was highly conserved in the branches of Prunus mume and P. persica (bootstrap>99), and they had a confidence index cluster of 100, and all of the above belonged to Rosaceae. The phylogentic tree constructed by TERT conforms the right phylogenetic relationships of the plant species. Leguminous plants such as Glycine max, Phaseolus vulgaris and Solanaceae plants such as Solanum lycopersicum, S. tuberosum, Nicotiana tabacum also reflected a similar situation. Furthermore, intron and exon sequence analysis was carried out using the TERT sequences from different families. It was found that the number of exons and introns of TERT from different plants were different. Functional network prediction indicated that AtTERT might interact with some important functional proteins such as the non-homologous end-joining protein 70 (Ku70), Ku80, telomerase protective protein 1a of Arabidopsis thaliana (AtPOT1a), TER1, nucleolar protein (NAP57), recombinant DNA repair protein 50 (RAD50) and suppressor with morphogenetic effect on genitalia 7 (SMG7), which involved in telomere repair or non-telomere functions. To reveal the potential non-telomere function of AtTERT, telomerase activity and AtTERT expression pattern were detected simultaneously during salt and H₂O₂ treatment. At 3 d of 200 mmol/L NaCl treatment, telomerase activity increased obviously. However, the telomerase activity decreased at the 7 d. Accordingly, the Arabidopsis seedlings had been whitened at the 7 d. Further, the telomerase activity increased when treated with 0.4 mmol/L H₂O₂, and decreased with the further increase of H₂O₂ concentration from 0.8 mmol/L to 2.0 mmol/L. The AtTERT expression also increased first and then decreased under salt and oxidative stress. In summary, the present research results indicated that TERT genes are highly conserved in the same group of plants. Telomerase might have non-telomeric functions, such as regulation of gene expression, and cell repair in response to abiotic stress. The results provide new reference data for further study of plant TERT structure and function.

HD-Zip (homeodomain-leucine zipper) is one of the important transcription factors, which acts important roles in regulating plant growth, development, morphogenesis, and resistance to various abiotic stress. In the present study, the plant expression vector pCAMBIA3300-Ubi-ZmHDZIV13/14-bar was succeeded constructed, the ZmHDZIV13 and ZmHDZIV14 genes were transferred into maize inbred line 'Zheng 58' by Agrobacterium tumefaciens-mediated stem dip transformation, and the transgenic maize lines were screened by Basta herbicide, and were detected by PCR and Southern blot method. After that, T₂ transgenic and non-transgenic maize lines were used to analyze the drought tolerance. Results showed that the non-transgenic/transgenic maize lines grew normal growth without stress, and there was no significant difference in the contents of the malondialdehyde (MDA), H₂O₂, proline (Pro), soluble sugar (SS) contents, and the activity of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) in roots and leaves between both maize lines; however, under drought stress, the contents of MDA and H₂O₂ decreased significantly(P<0.01), whereas, the contents of Pro and SS, and activity of POD, CAT, and SOD increased significantly(P<0.01) in roots and leaves of ZmHDZIV13 and ZmHDZIV14 transgenic maize lines. These results indicated that ZmHDZIV13 and ZmHDZIV14 genes could improve the drought tolerance in maize plants. Therefore, this study could provide the basis data for the further study of ZmHDZIV13 and ZmHDZIV14 genes and creation of transgenic drought-tolerant materials.

Indolic glucosinolates (IGS) play a crucial role in plant pathogen defense responses, MYB (myeloblastosis) 34, MYB51 and MYB122 are 3 important transcription factors that regulate indolic glucosinolate biosynthesis. This study systematically studied the temporal and spatial expression patterns of 3 transcription factor genes and their responses to hormones, which could provide data for further study on the disease resistance mechanism of indolic glucosinolates. Using Arabidopsis thaliana genomic DNA as a template, the promoter sequences of MYB34, MYB51 and MYB122 genes were amplified, plant expression vectors with GUS (β-glucuronidase coding gene) expression driven by the promoters of MYB34, MYB51, and MYB122, respectively were constructed and transferred into Arabidopsis thaliana. PMYB34::GUS, PMYB51::GUS and PMYB122::GUS transgenic lines were confirmed. Through the detection of GUS activity, the temporal and spatial expression patterns of 3 genes and the response to several hormones were analyzed. Gene expression analysis showed that the temporal and spatial expression patterns of MYB34, MYB51 and MYB122 genes were similar. All the 3 genes generally expressed in various stages of growth and development and in various tissues, in the cotyledons, hypocotyls and radicles of seedlings, and the vascular bundles of roots, stems, leaves, flowers, and pods of mature plants. However, the 3 genes showed their own specific expression pattern. The expression of MYB51 performed higher in ground tissue than in root tissue, while the MYB34 and MYB122 were mainly expressed in the roots. Hormone responsiveness analysis showed that MYB51 was induced by salicylic acid (SA) and MYB34 was induced by methyl jasmonate (MeJA). Compared with MYB34 and MYB51, MYB122 was not sensitive to hormone stimulation. Under the experimental conditions of this study, the expression level of MYB122 did not change significantly after several hormone treatments. The above research results suggested that the metabolism of indolic glucosinolates might be regulated by different signaling pathways in different tissues of the plant. This study preliminarily clarified the temporal and spatial expression characteristics of MYB34, MYB51 and MYB122, and provided basic data for further revealing the regulation mechanism of indolic glucosinolate biosynthesis.

Chrysanthemum morifolium originated from the ancient Huaiqingfu is one of the Four Famous Huai Medicines, which has high ornamental, edible and medicinal value. But it is rare reported about the research on its flower organ development. To study the function of PERIANTHIA (PAN) gene in flower development of Ch. morifolium, a 1 484 bp full-length cDNA sequence of homologous PAN gene was cloned by employing homology gene cloning and Rapid amplification of cDNA ends (RACE) from Ch. morifolium cv. Huaihuang. The cDNA sequence contained 64 bp 5' UTR, 106 bp 3' UTR and 1 314 bp CDS which encoded 437 amino acids. The protein of CmPAN had very high similarity with PANs from other plant species. C-terminal amino acids sequence of CmPAN included group D structural features with a well characterized family of plant bZIPs (basic leucine zipper motif) and 2 glutamine-rich regions. CmPAN was most close to AtPAN, so the cloned sequence was named CmPAN (GenBank No. KX380854). Putative protein molecular weight was 48.265 kD, the theoretical isoelectric point was 8.82, and it was non-secretory protein with nuclear localization sequence, its disordered degree was smaller than that of AtPAN. The qRT-PCR detection showed that CmPAN was higher expressed in flowers and stems than that in roots and leaves, and the CmPAN expession in floral development increased from bud emergence stage to visible color stage, then decreased at late phase. The CmPAN expession in floral organ ranked from high to low was petal of tubular flower, stamen of tubular flower, petal of ray floret, carpel of ray floret and receptacle. As a result, CmPAN might have important roles in early phase of floral development, and be closely related to the development of petals and stamens of tubular flower. The study provides some reference for further exploring the function of CmPAN gene.

Malate dehydrogenase plays an important role in plant growth and development. The objective of this study was to clone malate dehydrogenase (PpmMDH), a gene possibly related to fruit ripening and softening in peach (Prunus persica), investigate its sequence characteristics and analyze its expression on different peach tissues and fruits before and after ripening firm-fleshed and soft-fleshed peach varieties. The results indicated that the full-length cDNA of PpmMDH (GenBank No. KF017594) in peach was cloned. The sequence consisted of 1 239 bp with an ORF of 1 020 bp, encoding a polypeptide of 339 amino acids. Homology analysis showed that the deduced PpmMDH protein was highly homologous to other mMDH proteins from different species. Phylogenetic analysis also indicated that PpmMDH was very closely related to mMDH of plum blossom (Prunus mume)和 cherry (Pr. avium). qRT-PCR results showed that the PpmMDH expression was abundant in stamens, followed by petals and leaves, and lower in young fruits and pistil. During later stage of fruit ripening, the accumulation of PpmMDH was significantly higher in the firm-fleshed mutant 'Shuangjiuhong' than that in the soft-fleshed 'Kawanakajima Hakuto' (P<0.05). Abscisic acid (ABA), 1-naphthylacetic acid (NAA) and ethephon (ETH) treatments showed that the expression of PpmMDH was up-regulated by ABA, NAA in both cultivars, but the soft-fleshed peach variety 'Kawanakajima Hakuto' was induced in a short period of time, and the firm-fleshed peach variety 'Shuangjiuhong' was upregulated continuously. Different from ABA, NAA, the effect of ETH treatment on the induction of the gene in the soft-fleshed peach variety 'Kawanakajima Hakuto' was very weak, even negligible, but the gene was continuously upregulated in 'Shuangjiuhong', firm-fleshed peach variety. In the control group, the expression level of PpmMDH in 'Kawanakajima Hakuto' was significantly lower than that in 'Shuangjiuhong'. Infer from this, the expression of PpmMDH was antagonistic to ethylene during ripening and softening of soft-fleshed peach, the endogenous hormone dynamics of firm-fleshed peach and soft-fleshed each were different during fruit ripening and storage, and PpmMDH expression level was different from enzyme activity state. The above results indicated that PpmMDH was not necessarily the upstream gene to maintain fruit firmness, but showed the same enzyme activity difference as physiological difference between different materials of firm-fleshed and soft-fleshed peach. The present study laid a foundation for further exploring the role of PpmMDH in peach fruit ripening and softening. It also provides a scientific basis for the identification of the gene at the molecular level.

Anthocyanin synthase (ANS) is a key enzyme at the end of plant anthocyanin biosynthetic pathway,which catalyzes leucoanthocyanins into anthocyanins.In this study, the ANS gene of Camellia sinensis was cloned by reverse transcription-polymerase chain reaction (RT-PCR) based on tea full-length transcriptome sequencing data (GenBank No. AY830416).The full-length cDNA coding region was 1 068 bp and encoded 355 amino acids. The plant expression vector pSH-CsANS was constructed, and Agrobacterium tumefaciens was used to infect tobacco (Nicotiana tabacun) leaves for genetic transformation. Thirty-five transgenic plants were obtained by tissue culture. And after resistance selection and PCR identification, three PCR-positive tobacco lines TP-1, TP-2 and TP-4 were selected for gene expression and anthocyanin and proanthocyanidin content analysis. Quantitative real-time PCR (qRT-PCR) analysis showed that over-expression CsANS gene resulted in up-regulation of endogenous flavonoid biosynthesis pathway genes chalcone isomerase(CHI), (2S)-flavanone 3-hydroaylase (F3H) and dihydroflavonol4-reductase (DFR) and down-regulation of flavonol synthase (FLS) gene in tobacco. The results showed that over-expression of CsANS gene promoted the synthesis of tobacco anthocyanins, and the anthocyanin content was increased by about 45% compared with wild type. Moreover, over-expression of CsANS gene could increase the content of flavan-3-ol, a prerequisite material for the synthesis of proanthocyanidins, and increase the content of proanthocyanidins in transgenic plants by about 34% compared with wild type. This research has provided a foundation for future study on gene expression and functional analysis.

Retrotransposons, as important parts of Phyllostachys edulis genome, participate in the regulation of the growth and development of Ph. edulis through their own transposonsition, and it is of great significance to obtain active transposons for the molecular breeding of Ph. edulis. In this study,a complete LTR retrotransposons from the Ph. edulis genome that named PHRE9 was characterized. The PCR amplification and bioinformatics analysis of the full length retrotransposon of PHRE9 show that the structural characteristics and evolution patterns as well as the expression patterns under adversity was systematically analyzed. The length of PHRE9 is 6 370 bp, belonging to the Tork branch in Ty1-copia super-family. The transcriptional activity levels of genes in three domains of PHRE9 was measured by qRT-PCR under different treatment conditions, such as DNA methylation inhibitors, irradiation, high salt, high temperature and low temperature. The results showed that PHRE9 expression level was raised under the stress of DNA methylation inhibitors treatment, irradiation, high salt, high temperature (42 ℃), low temperature (4 ℃). The results showed that PHRE9 was a transcription-active transposon. This study could provide basis data for the study of adversity adaptation mechanism and molecular breeding of Ph. edulis.

Studies about angiotensin converting enzyme 2 (ACE2) have focused on rodents and humans (Homo sapiens), and there are few data on pigs (Sus scrofa domesticus). The good anti-inflammatory and anti-injury effects of ACE2 in different tissues and organs have been widely recognized, but the role of ACE2 in intestinal tract is still unclear. This study intends to use the CRISPR/Cas9 gene editing technology to establish ace2 gene knockout method in porcine intestinal epithelial cell (IPEC-J2), and to explore the effect of ace2 deletion on cellular inflammatory injury. First, the presence of ACE2 protein in selected IPEC-J2 cells was confirmed by Western blot and immunofluorescence staining. The ace2 gene was then selected as the knockout target, and 3 pairs of single guide RNA (sg RNA) were designed and synthesized, and inserted into the pX330 plasmid containing Cas9 backbone to construct the ace2 knockout targeting vector pX330-ace2-1, pX330-ace2-2 and pX330-ace2-3. The correct recombinant plasmid pX330-ace2-2 was transfected into IPEC-J2 cells. Western blot and immunofluorescence staining showed that ACE2 protein expression was absent. The inflammatory factors were detected by enzyme linked immunosorbent assay (ELISA). The results showed that lipopolysaccharide (LPS) treated ace2 knockout cells and the pro-inflammatory factors interleukin (IL)-1β and IL-8 were significantly increased (P<0.01), the anti-inflammatory factor IL-10 was significantly decreased (P<0.05), and the cellular inflammatory injury was aggravated which suggested that intestinal ACE2 might had certain anti-inflammatory effect. The present study established a method for knocking out ace2 gene in pig intestinal epithelial cells by CRISPR/Cas9 system, and preliminarily explored the anti-inflammatory effect of ACE2, which might provide basic information for further research on ACE2 related functions and mechanisms.

As one of the major factors for affecting the growth and meat quality of pigs (Sus scrofa), subcutaneous fat deposition has always been a hot issue in pig production. Catalase (CAT) is a key antioxidant enzyme in the oxidative metabolism process and plays vital roles in the fat deposition of animals. To investigate the role of CAT gene in the porcine subcutaneous preadipocytes differentiation, the expression patterns of CAT during the preadipocyte differentiation process and CAT mRNA levels in various tissues of Erhualian pigs were detected using qRT-PCR. Moreover, CAT gene was knockdowned by small interfering RNA (siRNA) fragments, and then, the ability of subcutaneous preadipocytes differentiation was assessed via oil red O staining. The expression levels of important candidates in siCAT treated adipocytes, including the key adipocyte differentiation-related genes such as peroxisome proliferator activated receptor γ gene (PPARγ), CCAAT enhancer binding protein α gene (C/EBPα), fatty acid binding protein 4 gene (FABP4), apolipoprotein E gene (ApoE), adiponectin, C1Q and collagen domain containing gene (ADIPOQ), perilipin 1 gene (PLIN1), and hormone-sensitive lipase gene (HSL), and the antioxidant-related genes such as superoxide dismutase 2 gene (SOD2), glutathione synthetase gene (GSS), and peroxidase 3 gene (PRDX3) were detected by qRT-PCR. The results indicated that CAT gene was mainly expressed in subcutaneous adipose, kidney, and liver tissues of pigs and its expression levels in subcutaneous adipose tissues were significantly higher than that in intramuscular adipose tissues (P<0.01). During the differentiation process, the expression levels of CAT gene increased significantly at different differentiation stages, with the highest expression levels on the 4th day of the preadipocytes differentiation. Silencing of CAT increased the contents of hydrogen peroxide (H₂O₂) (P<0.01) and also decreased the triglyceride levels of subcutaneous preadipocytes (P<0.05). Furthermore, interfering CAT expression reduced the expression levels of ADIPOQ (P<0.05) and PLIN1 (P<0.01) and simultaneously increased the levels of HSL gene (P<0.05), with non-significant effect on the expression levels of PPARγ, C/EBPα, FABP4, ApoE, SOD2, GSS, and PRDX3 genes (P>0.05). Taken together, this study demonstrated CAT gene knockdown could inhibit the differentiation and lipid deposition of subcutaneous preadipocytes of Erhualian pigs, which might result from downregulating lipogenesis-related genes of ADIPOQ and PLIN1 and simultaneously upregulating the expressions of lipolysis-related HSL gene. These results could provide new evidence and ideas for further revealing the mechanism of CAT gene on adipogenesis and reducing the subcutaneous fat deposition of pigs.

As a natural insecticidal tetranortriterpenoid compound extracted from the seeds of the neem tree (Azadirachta indica), azadirachtin is currently widely used in agricultural products and vegetables for pest control. A large fraction of azadirachtin is lost and likely affects the environment and domestic animals by straw feeds exploitation. It has been reported that the intra-vas administration of neem oil resulted in the block of spermatogenesis in male rats. It is still unclear whether there is a role of azadirachtin on the function of pig testis. In order to gain insight into the importance of azadirachtin in male pig reproduction, this study was undertaken to investigate the effect of azadirachtin on testis in vitro by using pig testicular ST cell line. The status of ST cell growth was tracked under optical microscope and the proliferation or viability of cells were measured by 3- (4, 5-dimethyl thiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) assay. Flow cytometry with annexin V FITC/ PI double staining was used to detect apoptosis of ST cell before and after azadirachtin treatment. The expression levels of four genes were determined by using quantitative real-time polymerase chain reaction (qRT-PCR) method, in which included MMP15 (matrix metallopeptidase 15), PICK1 (protein interacting with Cα kinase), BMP6 (bone morphogenetic protein 6), and MSH4 (MutS homolog 4). Using different dose of azadirachtin from 1 to 9 mg/L, the number of adherent cells was reduced, with cells rounded and suspended and appeared to be clustered under microscope. Based on determination of MTT assay, azadirachtin exhibited cytotoxic effects on the viability of ST cells in a dose and time-dependent manner with IC₅₀ value of 7.646 mg/L at 24 h. Azadirachtin induced the apoptosis of ST cells in a dose- and time-dependent manner with a higher rate of early apoptotic cells at concentrations of 8 mg / L than that of 5 mg / L for 24 h of incubation (P<0.05). As the treatment concentration of azadirachtin changed from 5 mg/L to 8 mg/ L, the expression of all genes except MSH4 were rapidly decreased after dealting with azadirachtin at a dose-dependent way. The above findings demonstrated that azadirachtin had a cytotoxic impact on pig testis cell, and the induction of apoptosis might be one of the causes. It made a reasonable speculation that the function of pig testis was influenced by azadirachtin because the several gene expressions related with reproduction ability were disturbed intensely. It suggested that azadirachtin might inhibit the fertility of boar and need to have a mention on the detection and amount of azadirachtin residue in plant feedstuff in pig industry. The results could provide a basis for the residue control of azadirachtin in plant feedstuff.

As an excellent domestic breed with fecundity, Small Tail Han sheep (Ovis aries) could get high ovulation rate and little size as a result of A476G mutation in bone morphogenetic protein receptor 1B (BMPR1B) gene (FecB mutation), under which the mechanism remains to poorly understand. The objective of this study was to investigate the effects on the number and diameter of follicle and the expression patterns of anti-Müllerian hormone (AMH) gene and its receptor Ⅱ (anti-Müllerian hormone receptor Ⅱ, AMHR2) gene in Small Tail Han sheep with or without FecB mutation (mutant homozygous BB and wide type ++, respectively). Based on TaqMan probe genotyping and estrus synchronization, the number and diameter of follicle over 3 mm from ++ (n=21) and BB ewes (n=16) were recorded. qRT-PCR technology was applied to detect the expression of AMH and AMHR2 genes in 14 tissues (hypothalamus, pituitary, ovary, heart, liver, spleen, lung, kidney, brain, adrenal gland, small intestine, oviduct, uterus, thyroid) from 3 BB ewes during follicular phase and 3 tissues (hypothalamus, pituitary and ovary) from BB and ++ ewes during luteal phase and follicular phase (n=3, 3, 3, 3, respectively). The results revealed that FecB mutation had a highly significant increase in number of follicle (P<0.01) and a highly remarkable decrease in follicular diameter (P<0.01). Two genes were expressed in 14 tissues of BB ewes during follicular phase, wherein both genes expressed highly in hypothalamus-pituitary-ovary gonad axis as well as AMH gene expressed highest in ovary and AMHR2 gene in adrenal gland. The expression of AMH gene in hypothalamus-pituitary-ovary gonad axis between BB and ++ animals during different phases of follicular development were not significantly different (P>0.05). There was no significant difference for the expression of AMHR2 gene in ovary between 2 categories of animals during different phases of follicular development (P>0.05). The expression of AMHR2 gene in pituitary of BB ewes was higher than ++ contemporaries (P<0.01). Stage of follicular development and FecB genotype had a highly significant effect on expression of AMHR2 gene in hypothalamus (P<0.01). A conclusion can be drew that FecB mutation caused the change of follicular development and expression of AMHR2 gene in time and space in Small Tail Han sheep and the special expression of AMHR2 gene in hypothalamus-pituitary-ovary gonad axis system may be involved in the regulation that FecB mutation alters follicular development, which would give novel theoretical basis to multiparous meat sheep breeding in our country.

Intramuscular fat content is a key indicator of animal breeding. It is an important task to study the regulation mechanism of lipid metabolism by molecular biology. The present study aims to clone the carnitine palmityl transferase 1 B gene (CPT1B) and determine its expression in various tissues and intramuscular precursor adipose cells of goats (Capra hircus), which will lay the foundation for further study of CPT1B gene in regulating goat fatty acid metabolism. Seven healthy Jianzhou Big-eared goats aged 1 year old were selected, and the tissue samples of heart, liver, spleen, lung, kidney, back longissimus dorsi, biceps femoris, triceps, subcutaneous fat and abdominal fat were quickly collected and stored in liquid nitrogen. The intramuscular fat contents of back longissimus dorsi, biceps femoris, triceps were detected by Soxhlet extraction method. The total RNA of the above-mentioned tissue samples was extracted using Trizol reagent and the sequence of CPT1B was cloned by RT-PCR following by the sequence bioinformatics analysis. The expression of CPT1B in various muscle tissues were determined by qRT-PCR method, which were then used for correlation analysis with the intramuscular fat content. The results showed that the goat sequence was 2 619 bp (GenBank No. MH340532), including 2 313 bp CDS, 52 bp 5'UTR and 254 bp 3' UTR, encoding 771 amino acid residues. Tissue quantification results showed that CPT1B was expressed higher in heart, longissimus dorsi, biceps femoris and tricep, and expressed lower in liver, spleen, lung, kidney, subcutaneous fat and abdominal fat of Jianzhou Big-eared goat. The qRT-PCR results of intramuscular precursor adipose cells showed that the expression of CPT1B during differentiation of preadipocytes was significantly higher than that before and after differentiation (P<0.01). The expression of CPT1B gene was significantly positively correlated with the intramuscular fat content in all the 3 muscle tissues (P<0.05). In summary, CPT1B might have a regulatory role in goat intramuscular fat deposition, which also lays a foundation for the application of CPT1B gene in meat goat breeding.

Secreted frizzled-related protein 5 (SFRP5) is a crucial anti-inflammatory adipokine secreted from white adipose tissue. It can regulate lipid metabolism, reduce insulin resistance, and keep inflammation balance by Wnt singal pathway. To investigate the relationship between SFRP5 gene polymorphism and muscle quality in Changshun Blue-eggshell chicken (Gallus gallus domesticus), and find molecular markers related to muscle quality. In this study, the single nucleotide polymorphisms (SNPs) of SFRP5 gene were detected by direct sequencing, and its correlation with the meat quality of chest muscle in Changshun Blue-eggshell chicken was analyzed. The results showed that 2 SNPs (g.23253254G>A and g.23253269T>G) were firstly found in SFRP5 gene exon 3. The dominant genotype and dominant allele of the g.23253254G>A locus was GG genotype and G allele, and its frequencies were 0.569 and 0.756, respectively. The g.23253269T>G locus was TT genotype and T allele with frequencies were 0.681 and 0.812, respectively. Two mutation sites of SFRP5 gene were synonymous mutations. Polymorphic information content (PIC) of the 2 loci were between 0.25 and 0.5, and belong to moderate polymorphic loci. The chi-square test indicated that the genotypes distribution of 2 SNPs (g.23253254G>A and g.23253269T>G) were agree with the Hardy-Weinberg equilibrium (P>0.05). There was no strong linkage disequilibrium (LD) between each SNP. Among the flock of Changshun Blue-eggshell chicken, 3 haplotypes and 6 diplotypes were formed on SFRP5 gene. The frequency of 3 haplotypes H1, H2 and H3 were 0.569, 0.187 and 0.244 respectively, and then the frequency of 6 diplotypes H1H1, H1H2, H1H3, H2H2, H2H3 and H3H3 were 0.313, 0.200, 0.313, 0.056, 0.063 and 0.056, respectively. Genetic correlation analysis demonstated that g.23253254 G>A locus was significantly correlated with crude fat of chest muscle (P<0.05), and further the diplotype H3H3 was obviously higher crude fat content than other 5 diplotypes (P<0.05). The present research revealed that SFRP5 gene had significant genotype effects on chicken meat quality and also suggested that both g.23253254 G>A and g.23253269 T>G variant sites of SFRP5 gene may be used as molecular markers to improve meat quality in Changshun Blue-eggshell chicken. This study provide theoretical basis for further exploring the molecular mechanism of SFRP5 gene variation in poultry muscle quality.

Southern rice black-streaked dwarf virus (SRBSDV) is transmitted by white-backed planthopper (Sogatella furcifera) in a persistent-propagative manner. During SRBSDV infection in its insect vector, viroplasm, putative sites of viral replication, contained the nonstructural viral protein P5, P6, P9-1 and insect vector proteins. The P6 protein is initially expressed, and interacts with P5, P9-1 and insect proteins in insect vector, respectively, which demonstrate that P6 protein is essential for viroplasm formation and viral replication. In order to investigate which insect proteins play an important role during viral replication, yeast two-hybrid experiments was be used to identify insect proteins interacting with SRBSDV P6 protein in the white-backed planthopper midgut. Firstly, SRBSDV P6 gene was constructed into the expression vector (pGBKT7-P6) and transformed into Y2HGold cell. Western blot showed P6 protein successfully expressed in yeast cell, which was non-toxicity and non-autonomous activation to cells. Then cDNA library plasmids were transformed into Y2HGold containing bait plasmid pGBKT7-P6. Transformants were selected from high stringency medium SD/-Leu/-Trp/-His/-Ade, and then replicated to SD/-Leu/-Trp/-His/-Ade/X-α-gal medium for further analysis. Colony growth and blue color indicated an interaction between the two-hybrid proteins. Plasmid DNA was isolated from yeast and then transformed into Escherichia coli DH5α. Lastly, cDNA inserts were sequenced to verify the preference of an open reading frame (ORF) and compared the sequence in Blast of NCBI.. The results showed 5 insect proteins, interacting with P6, which were involved in gene transcription, translation, post-translational modification and protein systhesis. Results of this study will be useful in revealing the replication mechanisms of SRBSDV in its insect vector, which will facilitate the development of effective control strategies against SRBSDV disease.

Carotenoids are one of the most important pigments in plants, apocarotenoids from carotenoids via carotenoids cleavage dioxygenases (CCDs) or non-enzyme pathway and their derivatives play an important role in plants as pigment, plant hormone, volatiles and signals. CCD family consists of two subfamilies: CCD and NCED. To date, five members in CCD subfamily including CCD1, CCD2, CCD4, CCD7 and CCD8 have been cloned from higher plants. It has been proved that CCD1 and CCD4 play important role in color and volatiles (eg, α-ionone and β-ionone) formation of flowers and fruits in plants. CCD2 was only found in Crocus plants, which was involved in the formation of aroma and pigments (eg, crocetin). CCD7 and CCD8 were involved in formation of strigolactone. In this paper, in order to provide references for the functional research and future application of CCD subfamily genes, the structure, expression patterns and functions of various members of CCD subfamily genes in higher plants are summarized, and further research directions and keynote are prospected.

Gonadotropin releasing hormone 3 (GnRH3) is a member of the gonadotropin releasing hormone family. GnRH3 has been derived from an earlier genome duplication in an ancestral vertebrate, followed by its loss in the tetrapod lineage,so it has been a unique GnRH type in fish. GnRH3 of fish has a classical decapeptide structure, which plays a crucial regulatory role in maturation of gonads and sensory regulation of fish by binding to gonadotropin releasing hormone receptors. This paper discusses from the following four parts. 1. Structure of GnRH3: It is composed of 3 introns and 4 exons. Non-coding regions and introns are different from vertebrates, but the coding regions are highly conserved; 2. Source of GnRH3: It was generated in two rounds of genome replication events and ultimately reserved in fish; 3. Production and migration of GnRH3: GnRH3 neurons in the olfactory area and the ectoderm of the embryo eventually migrated to the preoptic area and the ventral brain; 4. Research progress of GnRH3. In this paper, the research progress of GnRH3 was reviewed, and the future development prospect was prospected, so as to provide a theoretical basis for the regulation of fish reproduction.

Peanut (Arachis hypogaea) pod rot, a worldwide fungal disease, has become one of the important diseases that directly affect the production and quality of peanut. Pod rot, as a kind of soil-borne disease, is difficult to be completely controlled with chemicals, cultivation methods or even biological techniques. Disease resistance breeding is an economical and effective way to control the pod rot. The screening and utilization of resistance related molecular markers will be an important means to improve the selection efficiency and speed up the breeding process. According to the results of the previous study, 12 pairs of SSR markers related to peanut disease resistance in references and 48 pairs of insertion-deletion (InDel) markers developed based on the result of stress resistance in this lab were selected to detect 77 American peanut germplasm resources, and the correlation between pod rot resistance and markers were analyzed. Pearson correlation analysis and multiple linear stepwise regression analysis were conducted based on the evaluation criteria of peanut pod rot resistance established in this laboratory. And the associated SSR markers of pod rot were validated with a following F₂ segregation-population. The results revealed that there were abundant of genetic diversity and disease resistance in American germplasm resources, and inter-annual environmental factors had a significant impact on disease resistance of peanut germplasm. The results of SSR polymorphism showed that there were 103 polymorphic bands and the polymorphic rate of 20% to 100% with an average of 79.23% among 77 peanut germplasm. The results of correlation and multiple linear stepwise regression analysis between injury index and SSR markers revealed that four markers including 3A8, GM1760, PM163 and 14H6 were significantly associated with peanut pod rot resistance. These 4 markers were considered could be well used in further study on the development of molecular markers related to peanut pod rot resistance. The validation results in F₂ population showed that SSR marker 14H6 had a close linkage with peanut pod rot which considered being the practical application value. 48 pairs of InDel markers were also used to screen American germplasm resources and 15 of 48 InDel markers showed polymorphism among the germplasms with the average polymorphic rate of 90.67%. The results of Pearson correlation analysis and stepwise multiple linear regression analysis showed that only 2 InDel (InDel-004, InDel-020) markers were found to be significantly correlated with peanut pod rot resistance. And the proportions of phenotypic variance explained by InDel-004 and InDel-020 were 5.1% and 5.9%, respectively, for what these two markers were not validated by F₂ population due to low practical application value expectation. This study not only confirmed the resistance of peanut germplasm from different sources but also provided excellent materials forgenetic improvement of peanut disease resistance by rational utilization of germplasm. And furthermore, the acquisition of molecular markers related to peanut pod rot resistance could provide an important reference for molecular assisted selection breeding and the excavation of disease-related genes.

Bamboo mosaic virus (BaMV) is the only RNA virus that has been found to infect bamboo so far, seriously affects the economic value of bamboo.Therefore, development of a rapid and accurate detection method for the BaMV is of great significance for the prevention and control of BaMV. In this study, two pairs of specific primers according to the coat protein gene (CP) sequence of BaMV were designed . One sequence of BaMV CP gene were obtained from Phyllostachys bambusoides with mosaic symptoms by RT-qPCR.And the full-length sequence (GenBank accession number: KP256071) was 528 bp encoding 175 putative amino acid residues. The sequences shared more than 98% similarity at nucleotides level with those of Phyllostachys nigra BaMV strains.The recombinant plasmid was used as template for SYBR Green Ⅱ real-time PCR to generate standard and melting curves. The standard curve cycle threshold (Ct) had a good linear relationship with the logarithm of template concentration. Melting curve analysis indicated no primer-dimers and non-specific products in the assay. The amplification efficiency and correlation coefficient were 100% and 0.99, respectively. Repeat ability tests indicated that inter-assay variability of the Ct values was 1.5%. The purpose strips could be successfully amplified with the primers BaMV-F/BaMV-R only in the bamboo infected mosaic sample and the healthy young leaves without purpose strips, which indicated that the primers is highly specific. The sensitivity of RT-qPCR was 100 times higher than that of regular RT-PCR. The minimum detectable concentration of BaMV plasmid standard in RT-qPCR assay was 8.5×10¹ copies/uL.The presence of BaMV in 8 bamboo species were firstly detected by this method. The results show that the SYBR Green Ⅱ real-time fluorescent quantitative PCR method for detecting BaMV is sensitive, specific, and reproducible, and could be used for rapid detection of BaMV.