Calcineurin B-like interacting protein kinase (CIPK) is a class of serine/threonine (Ser/Thr) protein kinases widely existing in plants that are involved in decoding calcium signals. CIPKs play important roles in abiotic and biotic stress signal transduction. In this study, BnaCIPK2 and BnaCIPK16 genes were successfully cloned by reverse transcription-polymerase chain reaction (RT-PCR) from oilseed rape (Brassica napus). Sequence analysis showed that the 2 proteins encoded by the 2 BnaCIPK genes contained conserved kinase domain and asparagine-alanine-phenylalanine (NAF) motif. The subcellular localization analysis showed that both BnaCIPK2 and BnaCIPK16 were localized in both cytoplasm and nuclei. qRT-PCR profiling at 3 time points revealed that jasmonic acid (JA), cold and polyethylene glycol (PEG8000) significantly induced the expression of BnaCIPK2. After methyl viologen (MV) treatment for 24 h, the expression of BnaCIPK2 was inhibited. Salicylic acid (SA), heat, cold, hydrogen peroxide (H₂O₂) and PEG8000 up-regulated the expression of BnaCIPK16, while JA and abscisic acid (ABA) treatments resulted in the repression of BnaCIPK16 expression. Yeast two-hybrid (Y2H) screening and bimolecular fluorescence complementation (BiFC) showed that BnaCIPK2 and BnaCIPK16 interacted with Brassica napus calcineurin B-like protein 4 (BnaCBL4) and BnaCBL3, respectively. The present study provides basic information for elucidating the functions and regulatory mechanisms of BnaCIPK2 and BnaCIPK16.

The EST-SSR markers obtained from transcriptome sequencing information have the advantages of good stability, high polymorphism, co-dominant inheritance, and simple operation, and are widely used in genetic diversity analysis of plant germplasm resources. In this study, high-throughput sequencing of the transcriptome of non-heading Chinese cabbage (Brassica rapa ssp. chinensis) was used to seek SSR loci and develop EST-SSR markers, and preliminary primer verification and polymorphism analysis were performed. A total of 17 009 SSR loci were found in the 48 817 unigene sequences of the transcriptional set of non-heading Chinese cabbage, with an occurrence frequency of 18.44% and a SSR distribution frequency of 34.84%. The SSR sites of non-heading Chinese cabbage contained 212 repeat elements whose SSR types were mainly concentrated on single, binary and trinucleotide repeats. Among them, single nucleotide repeats accounted for the most, accounting for 39.70% of the total, and the dominant repeating unit was T/A (26.83%). The EST-SSR primers designed by Primer Premier 3.0 soft were used to randomly selected for 40 pairs in batch design primers for synthesis and preliminary verification. Twenty-five pairs of primers could amplify the expected size bands in 92 non-heading Chinese cabbage germplasm resources. The average number of alleles was 3.72, and the effective number of alleles (Ne), Shannon's index (I), polymorphism information content (PIC) and gene diversity (GD) were 2.710 8, 0.999 1, 0.488 4 and 0.545 2, respectively. The obtained EST-SSR primers could provide effective molecular markers for genetic structure analysis, germplasm utilization and genetic map construction of non-heading Chinese cabbage and other Brassica plants.

The various degrees sterility fluctuation of CMS (cytoplasmic male sterility) lines directly affects the seeds purity of hybrid rice (Oryza sativa). For analyzing the fertility sensitivity by photoperiods and temperatures of duration of cultivation to CMS-WA (cytoplamic male sterility of wild abortion), 4 temperatures of duration of cultivation and 4 photoperiods were selected to study male fertility changes of 3 CMS-WA lines ('Zhenshan 97A', 'Longtepu A' and 'Jinfu 1A') which had different numbers of minor effect restoring gene. The dark pollen rate by I₂-IK (mixed solution of I₂ and IK) dyed and rate of florets with fertile anthers were analyzed by linear regression model. For constructing predictive analysis models, and exploring the genetic basis of dark pollen rate by I₂-IK dyed and rate of florets with fertile anthers caused by the interaction between temperatures of duration of cultivation and photoperiods and restoring gene with minor effect. The results showed that dark pollen rate by I₂-IK dyed and rate of florets with fertile anthers of male sterile lines were importantly affected by the interaction between photoperiods and temperatures of duration of cultivation. Generally, with temperature rising, dark pollen rate by I₂-IK dyed and rate of florets with fertile anthers of CMS lines were increasing. With photoperiod increasing, significant impact was observed in 'Longtepu A', but not in 'Jingfu 1A' and 'Zhenshan 97A'. So, 'Longtepu A' should be classified as photo-thermo-sensitive type, it meant that 'Longtepu A' had mutual promotion effect by photoperiods and temperatures of duration of cultivation, and early season propagation and early season production were priority adopted to ensure the purity of seeds. Breeding and purification of photo-thermo-sensitive type CMS lines, should be conducted at short photoperiods and high temperature of duration of cultivation. Thereby the unqualified individual plant or lines could be eliminated as soon as possible. 'Zhenshan 97A' and 'Jingfu 1A' belonged to thermo-sensitive type, and had no obvious photoperiod effect. Low temperature of duration of cultivation was large contribution to propagation and production seed, and high temperature was beneficial to breeding and purification of thermo-sensitive type CMS lines. The result could provide reference basis for breeding, purification, propagation and reproduction of male sterile lines.

Low temperature is one of the major abiotic stresses in northern China. In this study, the maize (Zea mays) inbred line 'He 344' was used as the experimental material, which were analyzed changes of antioxidant system, osmotic adjustment substances and related gene expression of root under low temperature at different time points (12 h, 24 h, 3 d and 7 d). The result suggested that compared with the control at low temperature for 7 d, the fresh and dry weights of maize seedling roots were reduced by 47.9% and 66.7%, respectively; Superoxide dismutase (SOD) and peroxidase (POD) activity gradually increased, compared with the control, which increased by 44.8% and 30.1%, respectively; Proline (Pro) and malondialdehyde (MDA) content increased by 77.6% and 83.1%, respectively, compared to the control. A total of 10 ZmSODs genes were screened from transcriptome data of maize seedlings (3 d low temperature treatment vs control). Most ZmSODs genes were up-regulated, among which ZmSOD1, ZmSOD7 and ZmSOD9 were found higher relative expression compared with other genes. A total of 12 up-regulated ZmPODs genes were screened, of which ZmPOD35, ZmPOD92, ZmPOD94 and ZmPOD108 were highly up-regulated. A total of 9 genes encoding lipoxygenase (LOX) enzyme were screened, among which ZmLOX3, ZmLOX5, ZmLOX6 and ZmLOX7 genes were up-regulated. A total of 3 genes related to proline synthesis (delta 1-pyrroline-5-carboxylate synthetase, P5CS) gene were identified, among which ZmP5CS1 and ZmP5CS4 were up-regulated. This study provides a reference for further study on physiological indicators and their related gene expression regulation in the root of maize seedlings under low temperature.

Aluminum-activated malate transporter 4 gene (ALMT4), aluminum-activated malate transporter 9 gene (ALMT9) and tonoplast dicarboxylate transporter gene (tDT) may be important genes involved in malate transportation and accumulation in plum (Prunus salicina) fruit, to analyze their codon bias is of great significance for subsequent research of gene function. This study used CodonW and CUSP programs to analyze the codon bias of ALMT4, ALMT9 and tDT genes in Prunus salicina 'Huangguan' and compared with the codon bias of the genome of the model species contained Escherichia coli, Saccharomyces cerevisiae, Nicotiana tabacum, Arabidopsis thaliana and Lycopersicon esculentum etc. The results showed that the effective number of codons (ENC) of ALMT4, ALMT9 and tDT genes in Prunus salicina ‘Huangguan’ were much higher than 35, close to 61, and the codon adaptation index (CAI) was much less than 1, close to 0, which indicated their codon bias were weak. ALMT4 and ALMT9 preferred to the codons ending with A or U(T), tDT preferred to the codons ending with U(T) or C. GCA, AGA, UGU, GGU, CCU, ACU, UAU, UUU, GAA, CAU, AAU, GAU were the high frequency codons of ALMT4, GCA, AGG, GGA, CAU, UUG, UCA, ACA, AAU, GAU, UUU, UAC were the high frequency codons of ALMT9, GCU, AGG, CGA, UGC, GAG, GGA, UUG, AGC, UCC, UAU, GAU, CAA, AAG were the high frequency codons of tDT. The gene tree was more able to reflect the botanical classification relationship of species than the cluster tree constructed based on relative synonymous codon usage (RSCU). Considering codon bias differences, cost, convenience and other factors, S. cerevisiae was an ideal heterologous protein expression system for ALMT4, ALMT9, and tDT genes in P. salicina 'Huangguan', and L. esculentum was an ideal heterologous plant expression system. Furthermore, S. cerevisiae and L. esculentum were used as examples to optimize the CDs sequences of ALMT4, ALMT9 and tDT genes in P. salicina 'Huangguan', the modified gene sequences had codon bias features of S. cerevisiae and L. esculentum genome. These results provide a theoretical basis for subsequent research of genes function.

Moso bamboo (Phyllostachys edulis) is the most important economic bamboo species in China. Drought, salinity and other stress not only affected the yield of bamboo, but also limited its distribution. NAC (NAM, ATAF1/2 and CUC2) is one of the largest transcription factor families in plants, which plays an important role in the plant growth, development and responding to stress. In this study, in order to explore the molecular mechanism in the abiotic stress, this study cloned the PeNAC047-1 (GenBankNo. MN013397) and PeNAC047-2 (GenBankNo. MN013398) which were the homologous gene of ANAC047. The phylogenetic tree of PeNAC047-1 and PeNAC047-2 proteins were constructed by MEGA7.0 software. Multiple sequence alignment of PeNAC047-1 and PeNAC047-2 proteins were analyzed by DNAMan. The gene structure was analyzed with NCBI Conserved Domains Database (CDD) and the acting elements in upstream regulatory sequence were detected using PlantPan 2.0 online software, respectively. Transcription factor characteristics were verified by subcellular localization and transcriptional activation experiments. The expression pattern of PeNAC047-1 and PeNAC047-2 in different tissue and abiotic stress including abscisic acid (ABA), methyl jasmonate (MeJA), gibberellin acid 3 (GA₃), drought and NaCl were analyzed by qRT-PCR. The result showed that the coding regions of the PeNAC047-1 and PeNAC047-2 were 879 bp and 1 095 bp, encoding 292 and 364 amino acids, respectively. The regulatory sequence upstream of PeNAC047-1 and PeNAC047-2 were 2 000 bp, which were including many kinds of responsive elements such as GA responsive element, MeJA responsive element, ABA responsive element etc. It's indicated that PeNAC047-1 and PeNAC047-2 might be regulated by hormones and abiotic stress. Based on subcellular localization and transcriptional activation experiment assay PeNAC047-1 and PeNAC047-2 was shown to be the nuclear protein and transcriptional activator. Tissue specific expression indicated that PeNAC047-1 and PeNAC047-2 transcripts gene were expressed in all tissues of bamboo, among which the highest level of PeNAC047-1 and PeNAC047-2 was found in root. Besides, PeNAC047-1 and PeNAC047-2 was induced by high salt. Under drought stress, the expression of PeNAC047-1 and PeNAC047-2 were inhibited, but the degree of inhibition of PeNAC047-1 was weakened, and PeNAC047-2 was inhibited continuously. The expression of PeNAC047-1 and PeNAC047-2 was induced by high temperature and the expression pattern was consistent basically. Similarly, the expression of PeNAC047-1 and PeNAC047-2 was induced by MeJA and the expression pattern was consistent basically. After ABA treatment, the expression pattern of PeNAC047-1 and PeNAC047-2 were different, PeNAC047-1 was induced by ABA, and PeNAC047-2 was inhibited. In addition, under the GA₃ treatment, the expression patterns of PeNAC047-1 and PeNAC047-2 were also different. The response of PeNAC047-1 to GA₃ was earlier (3~6 h), and the response of PeNAC047-2 to GA₃ was later (48~72 h). In conclusion, PeNAC047-1 and PeNAC047-2 had the highest expression levels in root and respond to abiotic stress. And then the expression patterns of high salt, drought, high temperature and MeJA treatment were similar. Specially, the response of PeNAC047-1 and PeNAC047-2 with ABA and GA₃ were inconsistent, so, it was speculated that it might be respond to abiotic stress by different pathways. This study explored the response pattern of PeNAC047-1 and PeNAC047-2 with abiotic stress preliminarily, which provides some basis data for the molecular mechanism of Moso bamboo in the abiotic stress.

Placenta is the bond between the mother and the fetus during pregnancy, During pregnancy, it undergoes a dynamic process from formation, development to maturity and degradation, and its abnormal function may lead to abnormal fetal development or even abortion. Apoptosis is an important way for placental tissue to maintain homeostasis and function properly. Studying the dynamic changes and molecular mechanism of apoptosis in placenta tissue at various stages of yak (Bos grunniens)'s pregnancy. In this study, the placental tissue of calves at various stages of pregnancy were collected to detect the expression of apoptosis-related proteins Fas (factor associated suicide)/Fasl (factor associated suicide ligand), Bcl-2 (b-cell lymphoma-2)/Bax (b-cell lymphoma-2 assaciated X protein) and Caspase3 (cysteinyl aspartate specific proteinase3)/Caspase9 (cysteinyl aspartate specific proteinase 9) by immunohistochemistry. It was shown that Fas and Fasl protein were highly expressed in the placenta and early pregnancy, and showed a downward trend as the pregnancy progresses. Bcl-2 protein was mainly expressed in fetal villus epithelium and gradually decreasing with prolonged expression during pregnancy. Bax was highly expressed in fetal villus epithelium and early trophoblasts, it had a marked increase in late pregnancy. Caspase3 was mainly expressed in the endometrium, while Caspase9 was clearly expressed in the fetal trophoblast. Evidently apoptosis-related proteins Fas/Fasl, Bcl-2/Bax and Caspase3/Caspase9 had extensive cell-specific and time-specific expression patterns in placental tissues of yak during pregnancy. It was speculated that apoptosis-associated protein-mediated apoptosis would play a important role in the normal function of placental tissue during the dynamic changes of placental tissue formation, development, maturation and degeneration. This experiment provides a theoretical basis for studying and solving the problems in the production process of yak, such as abortion, fetal growth restriction and other anomalies.

The keratinocyte-associated protein 3 gene (KRTCAP3) affects the growth of skin, nails and hair by regulating keratin. In order to uncover the genetic variation in the KRTCAP3 gene and its genetic roles in wool production and growth performance in nangan (South African Mutton Merino (♂)×Gansu alpinefine-wool (♀)) and nannangan (South African Mutton Merino (♂)×nangan (♀)) crossbred sheep breed. DNA pool sequencing was used to scan the potential SNPs within the full-length DNA of KRTCAP3 gene in AF×MF population. The correlation between SNP locus and hair growth and growth traits of hybrid sheep was also analyzed. Six hundred crossbred sheep were individually genotyped by SNPscan^TM method. Then, the association analysis between SNP locus and wool production and growth performance was implemented. The results showed that one SNP (g.34287851C>G) was identified in intron2. Three genotypes, CC, CG and GG, in this locus were found in AF×MF crossbreed sheep population. The genotypic frequencies of CC, CG and GG were 0.35, 0.52 and 0.13, respectively. The allelic frequencies of C and G were 0.61 and 0.39, respectively. The population genetic parameters analyses showed that the homozygosity (Ho), heterozygosity (He) and effective allele number (Ne) were 0.53, 0.47 and 1.90, respectively. The polymorphism information contents (PIC) was 0.36 and suggested that this SNP was moderate diversity (0.25<PIC<0.5). The χ² test indicated that this locus was not in Hardy-Weinberg equilibrium (P<0.05). The association analysis of polymorphisms in KRTCAP3 gene with wool traits revealed that g.34287851C>G was significantly associated with the wool length of shoulder and side , wool length of thigh and notum, crimpness and crimp recovery (P<0.05 or P<0.01). The association analysis of polymorphisms in KRTCAP3 gene with growth performance revealed that g.34287851C>G was significantly associated with tube circumference and chest girth (P<0.05 or P<0.01). In summary, the g.34287851C>G site in KRTCAP3 gene could be used as a useful molecular marker for selection wool traits and growth performance. This study provides a molecular basis for utilization of fine wool resources and accelerates the breeding progress.

miR-486 is expressed in animal muscle at a high level and plays a key role in growth and development of myoblast in different tissues. To illuminate the polymorphism and expression pattern of miR-486 in Bashby sheep (Ovis aries), the stem-loop qRT-PCR was used to detect the expression levels in different tissues, and in skeletal muscle tissues of 8 fetal and postnatal periods of Bashby sheep. Then the mutation of miR-486 sequence in Bashby sheep group and its effect on cutting process of precursor miR-486 (pre-miR-486) in skeletal muscle cell were also researched. The result showed that the miR-486 was expressed at a high level in myocardium and skeletal muscle of the 100 d of fetal period of Bashby sheep, moderately expressed in stomach and intestines, and lowly expressed in liver, spleen, lung, skin and brain tissues; The expression levels of miR-486 in skeletal muscle of the 40~100 d of fetal periods were increased gradually, to a peak level on the 100 d of fetal periods then decreased gradually, and to a minimum level on the 120 d of fetal periods then increased continually until the 90 d of postnatal period; A G>C mutation was detected in pre-miR-486 sequence, which located at the first base of upstream of miR-486 mature sequence, the GG, GC and CC genotype could be detected in Bashby sheep group, but 91% individual of them were GC genotype, the further investigation indicated that the CC genotype could significantly up regulate the level of miR-486 in skeletal muscle cell (P<0.05). The present research could provide basic data for further uncovering the regulation mechanism of miR-486 in development of Bashby sheep skeletal muscle and relationship between its expression and excellent meat quality traits of Bashby sheep.

Autophagy is one of the basic researches of viral infection and tumor development. In order to understand the characteristics of autophagosomes, the determination of autophagosomes and the ultrastructure of cells, this experiment used Porcine reproductive and respiratory syndrome virus (PRRSV) to induce autophagy in African green monkey (Cercopithecus aethiops) embryonic kidney cells Marc-145. The cells and autophagosomes were observed by transmission electron microscopy and fluorescence microscope. The occurrence of autophagy was initially determined, and changes in structures such as lysosomes, endoplasmic reticulum, mitochondria and nucleus were observed. Marc-145 cells changed from a typical fusiform to a round shape after PRRSV infection, and there were significant autophagosomes in the infected cells. Ultrastructural changes such as mitochondrial ridge dissolution, endoplasmic reticulum degranulation, lysosomal activity enhancement, and nuclear abnormalities were observed during ultrastructural observation. The results showed that PRRSV induced autophagy in Marc-145 cells and the degree of autophagy were closely related to ultrastructure. The ultrastructural state could be used as an important reference for determining the development of autophagy. The results showed that PRRSV induced autophagy in Marc-145 cells, the degree of autophagy was closely related to the ultrastructure, and the state of the ultrastructure could be used as an important reference for judging the development of autophagy. This study could provide important reference for the study of autophagy, cell ultrastructure and viral infection.

Bisphenol A (BPA) is a ubiquitous environmental endocrine disruptor. This study was conducted to investigate the effects of BPA exposure to the dams during gestation and lactation on ovarian development in offspring. In this experiment, 8-week-old female mice (Mus musculus) at pregnant 0 d were randomly divided into 7 groups with 20 each group, and distilled water and 0.05, 0.5, 5, 10, 20, 50 mg / kg BPA were given through drinking water from 0 d pregnant to 21 d weaned. In this test, the offspring mice did not directly contact BPA, but indirectly contacted BPA through placental transmission and lactation during breastfeeding. And the mice gave birth at 20 d of pregnancy, so the indirect contact time of BPA was 41 d. The period of exposure was from gestation day 0 until the weaning of the F₁ mice for 41 d. The results showed that the BPA content in the serum and ovary of the offspring mice was significantly increased when the dose of BPA≥20 mg/kg (P<0.05). Organ index showed that the dose of BPA≥10 mg/kg resulted in a significant increase in the ovarian index of the pups (P<0.05). The observation of hematoxylin-eosin staining (HE) pathological tissue sections showed that low doses of BPA (≥0.05 mg/kg) could damage the ovaries of the offspring and cause irregular architecture in follicular granule cell layers. And the pathological changes of the ovary also increase as the dose increases. The results of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) showed that the ovarian granulosa cells were apoptotic when the BPA exposure was at or over 20 mg/kg (P<0.05). The expression of estrogen receptor α (ERα) in the ovary significantly increased when BPA exposed at no less than 20 mg/kg (P<0.05). And the dose of BPA of 50 mg/kg had significant affect on ERβ expression in ovarian progeny 21-day-old mice. The results of transcriptome sequencing showed that there were 81 significant differentially expressed genes in the ovary of the offspring compared with the control group, 38 of which were up-regulated and 43 genes were down-regulated. Differentially expressed genes were enriched in transcriptional functions and ribosomal metabolic pathways, in which the ribosomal genes ribosomal protein L3 (Rpl3), Rpl21 and Rpsa were significantly down-regulated, and the Rps2、Rps28、Rpl26、Rpl32 and Rpl10a were significantly up-regulated. These genes are associated with developmental arrest of the reproductive system of the embryo, apoptosis and ovarian precocity. The expression levels of Rps2, Rpl21 and Rpsa in all 7 groups of ovarian were detected by real-time PCR, which was consistent with the results of transcriptome, but the effect of BPA on Rps2 varied with doses. When BPA dose was 0.05 mg/kg to 20 mg/kg, the expression of Rps2 was lower than that of the control group, but the expression level was higher than that of the control group at the dose of 50 mg/kg. These results indicated that BPA could be indirectly transmitted to progeny mice through the placenta and milk of maternal mice. Ingestion of BPA could cause lesions in the ovaries of offspring mice, affected the development and function of the ovaries. Further studies at the genetic level revealed that abnormalities in ribosomal functional protein synthesis were associated with reproductive toxicity of BPA, and that low-dose BPA inhibited ribosomal protein expression while high-dose BPA promoted ribosomal protein expression, demonstrating the duality of BPA in ovarian development. The difference in doses used may be the reason for the current contradiction in the results of the BPA study. In this study, the offspring mice did not directly contact BPA, but the ovarian development of the pups was still affected by BPA, and it was further confirmed that BPA disrupted the normal ovarian development by affecting ovarian ribosomal function. The results of this study imply that the use of BPA-containing plastic products in pregnant livestock may affect offspring ovarian development, Which means that avoiding the use of BPA-containing plastic products in livestock breeding can help ensure the reproductive health of offspring, improve the breeding efficiency of livestock.

Autophagy and endocytosis are important life activities in the process of cell growth and development, which can regulate the growth state of cells to cope with the threat of harsh environment. To investigate the effect of epidermal growth factor (EGF) on autophagy and endocytosis of mouse (Mus musculus) cumulus cells, the concentrations 0, 50, 100, 150, 200 ng/mL of EGF and appropriate concentration (40 μg/mL) epidermal growth factor receptor (EGFR) inhibitor Gefitinib were applied to mouse cumulus cells, and cellular total RNA and total protein were extracted. The relative expression levels of autophagy related gene 5 (Atg5), Bcl-2 homologous domain protein (Beclin1), microtubule-associated protein light chain 3 (LC3), caveolin 1 (Cav1) and caveolin 2 (Cav2) mRNA at various concentrations were determined by qRT-PCR. The relative expression levels of Atg5, Beclin1, LC3, Cav1 and Cav2 proteins in every concentration were determined by Western-blot. The protein expressions were localized by immunofluorescence technique. The results showed that Atg5, Beclin1, LC3, Cav1 and Cav2 proteins were expressed in the normally growing mouse cumulus cells, and the main expression site was cytoplasm. After EGF treatments, gene expression levels of Atg5, LC3, and Cav1 increased significantly and were highest at 150 ng / mL (P<0.05), while the relative expression levels of Beclin1 and Cav2 decreased significantly and got lowest at 150 and 200 ng/mL (P<0.05). Protein expression levels of Atg5, Beclin1, and LC3 all increased significantly (P<0.05) and were highest at 150~200 ng/mL (P<0.05). In addition, LC3Ⅰ and LC3Ⅱ proteins were highest at 200 ng/mL and LC3Ⅱ/LC3Ⅰ was highest at 100 ng / mL. And Cav1 and Cav2 proteins were significantly increased at 50 ng/mL (P<0.05), while they were significantly decreased at 200 ng/mL (P<0.05). After inhibiting EGFR, the relative expression levels of Atg5, LC3, and Cav1 genes were significantly increased, while the expression levels of Beclin1 and Cav2 genes were significantly reduced (P<0.05). Protein expression levels of Atg5, Beclin1, LC3, LC3Ⅱ/LC3Ⅰ, and Cav1 were significantly increased (P<0.05, P<0.01), while the expression of Cav2 protein was significantly decreased (P<0.01). The results showed that EGF factor played an important role in the autophagy and endocytosis of mouse cumulus cells. The levels of autophagy and endocytosis were dose-dependent with the concentration of EGF. This study provides theoretical basis for further study of the regulation mechanism of EGF factor on autophagy and endocytosis of mouse cumulus cells.

On the lysosome surface mammalian target of rapamycin (mTOR) can phosphorylate transcription factor EB (TFEB) to cause nuclear translocation and thus plays a biological role in lysosomal biosynthesis, autophagy and lipid metabolism. In this study, The Northeast Agricultural University High and Low Fat (NEAUHLF) broiler (Gallus gallus) lines were used to detect the expression of TFEB in abdominal fat tissue and adipocytes by qRT-PCR. In addition, TFEB protein was localized in broiler adipocytes by immunofluorescence, and the changes of lysosome number during the differentiation of broiler adipocytes were analyzed. The results showed that the expression of TFEB in abdominal fat of lean line was higher than that of fat line at 1, 4 and 7 weeks of age, and the expression of TFEB in abdominal fat of 2 lines was significantly different at 4 weeks of age. During the differentiation the expression of TFEB in primary preadipocytes of 2 lines showed an opposite trend. To be precise, the expression of TFEB decreased during the differentiation of fat line broilers and increased in that of lean lines. At the same time, the expression of TFEB decreased during the differentiation of primary preadipocytes of arbor acres (AA) broilers, which was significantly different between 72 h and 0 h. Moreover, TFEB protein was detected in the cytoplasm and nucleus of adipocytes of broilers and the number of lysosomes decreased during adipocyte differentiation. The results would lay the foundation for further investigating the molecular mechanisms of how TFEB regulates lysosome biogenesis and affects the fat metabolism in broilers.

Heart-type fatty acid-binding protein (FABP-3) is a member of the lipid-binding protein superfamily, and its main functions are uptaking and transportation of fatty acids. In order to understand the structure of FABP-3 and its potential function in nutrition metabolism and immune regulation of Acipenser dabryanus, the full-length cDNA of A. dabryanus FABP-3 was cloned from the unigenes database by full-length transcriptome sequencing of A. dabryanus. The expression profiles of FABP-3 in different tissues, starvation stress and bacterial infection were analyzed. The full length of FABP-3 was 641 bp (GenBank No. MN064725) which contained 89 bp 5' untranslated region (UTR), 150 bp 3' UTR and 402 bp open reading frame (ORF). The ORF of FABP-3 encoded 133-residue peptide. FABP-3 was predicted to have a lipocalin domain from the 6 to 120 amino acids by analysis of the deduced protein sequence using the SMART program. The results also showed the three-dimensional protein structure model predicted by SWISS-MODEL contained 10 antiparallel β-strands and 2 α-helixes in the lipocalin domain, and the 3 conserved amino acid residues (Arg 107, Arg 127, Tyr129) were also present. The tissue distribution and expression level during starvation stress were further analyzed. Results showed that FABP-3 was ubiquitously expressed in all tested tissues, and was mostly expressed in muscle (P≤0.05). During starvation, the expression level of FABP-3 in the brain, liver and stomach increased first and then decreased, while the expression of FABP-3 in the intestine and muscle declined significantly compared to the control group (P≤0.05). Bacterial challenge in vivo revealed significant changes in the gene expression of FABP-3 at immune-related sites. Striking upregulation of FABP-3 was observed in the hindgut at 12 h post-challenge, with increases by more than 219-fold compared to the control, and the expression of FABP-3 mRNA was decreased at first and remarkably elevated in the gill and spleen at 48 h post-challenge. The results indicated that FABP-3 was involved in biological pathways such as nutrition metabolism and immune response in A. dabryanus and would provide basic information for future studies on nutritional and immunomodulatory molecular mechanisms of the A. dabryanus.

Wheat (Triticum aestivum) leaf rust is an airborne fungal disease caused by Puccinia triticina, which occurs worldwide and causes yield loss up to 15%~40% or even more in wheat production. Utilization of resistance cultivars is the most effective, safe and economic method. However, the pathogenicity of Puccinia triticina is diversified and the toxicity is frequently mutated. Therefore, the wheat leaf rust resistance is continuously lost. To explore the molecular mechanism of wheat leaf rust pathogenesis is particularly important for the effective control of wheat leaf rust. In this study, based on a total of 9 samples transcriptome data of P. triticinia monospora 08-5-9-2 (KHTT), 13-5-28-1 (JHKT) and 13-5-72-1 (THSN) in dormant spore, germinating summer spore and haustorium formation period, effector proteins were initially screened by SignalP 4.1、TargetP 1.1, TMHMM 2.0 and EffectorP 2.0. Effector proteins of 357 candidates were obtained, and 20 candidate effector proteins were screened for their gradually up-regulated expression level during the interaction between Puccinia triticina and susceptible material Thatcher. The 20 candidate effector proteins were further screened by Nicotiana benthamiana and Bax and INF1 which could cause allergic necrosis, and results showed that the 20 candidates were all effector proteins. The above results showed that the heterologous expression system was a powerful tool for screening the effector protein of specific parasite, which could provide reference for further research on the pathogenicity of effector proteins.

Only one ubiquitin-like protein SUMO (small ubiquitin-related modifier) coupling enzyme E2 has been found in organisms, that is ubiquitin-conjugating enzyme 9 (Ubc-9). In all known organisms, Ubc-9 is highly conserved. In this study, the transcription level of Ubc-9 in mouse (Mus musculus) macrophages infected with Brucella ovis 16M at different time points were detected by qRT-PCR. Then the mouse macrophage cell line model with Ubc-9 gene silencing was constructed by lentivirus packaging technique. Finally, the effect of Ubc-9 gene silencing Brucella on apoptosis of mouse macrophages was analyzed by flow cytometry, and the intracellular bacterial load was detected.The results showed that the intracellular Ubc-9 transcription level in mouse macrophages infected with Brucella for 8 h was significantly higher than that in the control group (P<0.05). In Ubc-9 gene silenced mouse macrophages constructed by lentivirus packaging system, the transcription of Ubc-9 gene was significantly inhibited, and the apoptosis rate induced by Brucella infection was significantly decreased, and the intracellular bacteria load was higher than that in the control group (P<0.05). This study provides basic data for further exploration of the survival mechanism of Brucella in macrophages.

Canine parvovirus (CPV) non-structural protein 1 (NS1) is an important functional protein involved in CPV replication and transcription. NS1 is also a pathogenic protein to the host. In order to explore the biological functions of NS1, recombinant NS1 proteins are needed. To prepare high-purity NS1 protein, the human (Homo sapiens) CD5 signal peptide sequence was used to mediate NS1 secretory expression in eukaryotic cells. First, based on human CD5 signal peptide sequence (GenBank No. NM_014207.4), a pair of complementary full-length CD5 signal peptide sequences (2 oligos) with restriction sites at both ends were designed and synthesized. The CD5 signal peptide gene was then produced by annealing the 2 oligos. The resulting CD5 signaling peptide gene was inserted into a NS1 eukaryotic expression vector pcDNA3.1-NS1/H at the 5' end of the NS1 gene by the restriction sites and fused with the NS1 gene to generate the NS1 secretory eukaryotic expression vector pcDNA3.1-CD5-NS1/H. The NS1 vector was transfected into human embryonic kidney (HEK) 293T cells, and the expressed NS1 protein was purified by affinity chromatography with Ni-NTA agarose beads from the cell culture medium. The detection results of SDS-PAGE and Western blot showed that the high-purity NS1 protein was obtained (>95%) and it could specifically react with anti-His antibody, indicating that the constructed NS1 vector could mediate NS1 secretory expression in eukaryotic cells. In order to achieve a high level of expression, the effects of the vector doses, transfection time and expression time on NS1 expressions were analyzed and optimal conditions for NS1 secretory expression were preliminarily determined as vector dosage 30 μg in T75 cell culture flask, transfection time 5 h, expression time 48 h. This study could provide basic materials for further investigation on NS1 biological functions.

Anthocyanins, belonging to flavonoid compounds, are a kind of water-soluble natural pigments. Under natural conditions, anthocyanins always exist in the form of glycosides combined with glycosylation. The type, content and distribution of anthocyanins in plants contributed to the color of organs, such as plant flowers, stems, leaves and fruits. In addition, anthocyanins also play important roles in enhancing the tolerance and resistance of abiotic stresses such as drought, low temperature, salt stress, low nitrogen, and biotic stresses such as Erwinia amylovora, Pectobacterium carotovorum, and Verticillium dahliae. In this paper, we mainly reviewed the biosynthesis and metabolism of plant anthocyanins, the molecular mechanism of plant anthocyanins in the response to the biotic and abiotic stress, and proposed the direction for the future studies. This paper would be helpful for understanding and applying the molecular mechanism of plant anthocyanins in the response to the biotic and abiotic stress, and provides references for the cultivation of anthocyanin-mediated high tolerance and resistance plants.

The pandemic of avian influenza poses a serious threat to the breeding industry and public health which could cause great losses in national economy. Currently, vaccination against avian influenza is the most effective preventive measure. There are many problems in the traditional chicken embryo technology, which is gradually replaced by cell technology. In order to evaluate the application of suspension culture technology in the production of inactivated cell-derived Avian influenza virus (AIV) vaccine, a well-defined adherent canine kidney cells (MDCK) was selected and acclimated to the suspension condition by gradually reducing serum. The AIV H9 subtype was inoculated into the adherent and suspended MDCK for adaptation culture. The cell-derived and chicken embryo-derived virus were prepared into inactivated vaccine. The specific pathogen free (SPF) chickens (Gallus gallus) were immunized and the immune effect of cell-derived and chicken embryo-derived AI inactivated vaccine was evaluated by serological method. The results showed that the MDCK cell density proliferated to 3.0×10⁶ cells/mL after 48 h of suspension culture, and reached to 5.0×10⁶ cells/mL after 72 h with good morphology, high viability and single suspension in culture medium. The data and cell status of shaking flask and 7.5 L bioreactor culture showed that the cell strain was suitable for suspension culture in a bioreactor. The AIV H9 subtype proliferated by suspension MDCK cells was used to prepare inactivated vaccine. After 21 d of immunization with SPF chickens, the geometric mean value of Hemagglutinatio inhibion (HI) in serum was more than 6(log₂), which was equivalent to the immune effect of chicken embryo-derived vaccine. The above experimental results suggest that the selected suspension culture MDCK cell line had the characteristics of rapid growth and stable proliferation, and could be used to proliferate AIV H9 subtype with high hemagglutination (HA) titer. The inactivated vaccine had good immunogenicity and could stimulate the body to produce antibodies with high titers. This study provides basic cytological data for large-scale production of AI and other viral vaccines.