Abstract Acrossocheilus fasciatus is a type of stream fish having great economic value in China. Recently, resource of A. fasciatus has declined rapidly due to overfishing and habitat destruction. Sperm cryopreservation is an effective way to protect germ cells; therefore, research in germplasm resource protection and germ cell conservation are in urgent need. In this study, the sperm was collected from cultured mature male fish and was stored in 0.25 mL straws for the study of sperm cryopreservation. Different conditions were evaluated for sperm cryopreservation, including extenders (Kurokura-1, Ringer, Cortland, D-14, D-15, D-16, D-17, D-19, D-20 and D-21), dilution ratios (1∶1, 1∶2, 1∶3, 1∶4, 1∶5, 1∶7 and 1∶9), cryoprotectants (dimethyl sulfoxide (DMSO), methyl alcohol (MeOH), ethylene glycol (EG), and propylene glycol (PG)), equilibrium times (0, 10, 20, 30, 40, 50 and 60 min), cooling heights (using two-step cooling procedures) (2, 3.5, 5, 6.5 and 8 cm), and thawing temperatures (27, 32, 37, 40 and 42 ℃). In addition, we detected the quality of fresh and corresponding frozen-thawed sperm through their enzyme activities. The experimental data were expressed as X±SD, using SPSS 17.0 statistical software. The statistically significant differences in sperm activation rate and enzyme activities of each group were detected by one-way ANOVA. P＜0.05 were considered significant. The fresh sperm density of Acrossocheilus fasciatus was (9.32±2.03)×109 cells/mL. The optimum result was obtained with the cryoprotectant comprising D-15 diluted with 10% DMSO, which was mixed with sperm at a ratio of 1∶5, equilibrated for 30 min, swung at 3.5 cm above nitrogen for 5 min, and finally stored in nitrogen. After thawing in a water bath at 37 ℃, the activation rate of the frozen-thawed sperm was (35.33±2.52)%; meanwhile, the rate of fresh sperms was (87.67±3.06)%. Enzyme activities were examined using reagent kits. Compared to the fresh sperm, the activities of the total ATPase, succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) in optimized frozen-thawed sperm decreased from (9.31±0.17) U/mL, (30.33±5.69) U/mL, and (7 454.84±252.42) U/L to (7.23±1.08) U/mL, (17.67±6.03) U/mL, and (2 172.48±209.62) U/L, respectively. Thus, significant differences were observed in sperm activation rates and enzyme activities between the two groups. The decrease in enzyme activities indicated that cryopreservation caused damage to the sperm. With the given protocol, we got early success in sperm cryopreservation of A. fasciatus. This study provide a technical basis for the foundation of the cryobank for A. fasciatus; however, further studies on parametric optimization are warranted to improve the quality of frozen-thawed sperms.

Abstract Cucumber (Cucumis sativus), a worldwide distributed vegetable, is an economically important?crop in Chongqing. However, viruslike symptoms were observed in greenhouse- and field-grown in the major cucurbit cultivation regions in Chongqing from 2013. In this study, 11 common vegetable viruses specific primers were used to detect the pathogenic agents associated with 5 diseased cucumber plants exhibiting typical stunting, wrinkled, mosaic and distortion symptoms collected from Chongqing. The detection results showed that 5 cucumber plants were mix-infected with more than 4 viruses including Cucumber mosaic virus(CMV), Squash mosaic virus(SqMV), Turnip mosaic virus(TuMV), Watermelon mosaic virus(WMV) and Zucchini yellow mosaic virus(ZYMV). Using PCR, cloning, and sequencing techniques coat protein (CP) gene of WMV (WMV-H3) and ZYMV (ZYMV-H3) were identified for phylogenetic analysis. Compared with reported sequences of different geographic origins, the CP genes of WMV-H3 and ZYMV-H3 shared similarity of 92.3%~96.9%, 93.0%~97.7% at nucleotide level, respectively. WMV-H3 had the highest sequence identity with WMV-T-2-9 and ZYMV-H3 shared the highest sequence identity with ZYMV-Hefei. The phylogenetic trees constructed with CP genes showed that 44 WMV isolates were clustered into 5 groups and WMV-H3 belonged to group Ⅲ, the genetic variation of which was related to geographical distribution and host origins; 80 ZYMV isolates were divided into 6 groups and ZYMV-H3 belonged to groupⅢ, the distribution of which exhibited a distinct geographical differentiation. This is the first report of SqMV, WMV and ZYMV infecting cucumber in Chongqing. The results provide theoretical basis for further study on the distribution of the disease in cucurbit-growing regions and the disease management.

Lipoprotein lipase (LPL) has the function of regulating fat deposition in animals, but its molecular genetic mechanisms is seldomly studied in local mini-pig (Sus scrofa) breeds. In order to understand the function of gene LPL and reveal its effects on meat quality traits in Congjiang pigs. In this study, the CDS region of LPL gene was cloned by RT-PCR from Congjiang pigs, the physical and chemical properties as well as structure characteristics of LPL protein was predicted using bioinformatics softwares. The genotype of AfaⅠlocus of intron 5 in Congjiang pigs was tested by polymerase chain reaction-restriction fragment length polymorphism technique (PCR-RFLP). Meanwhile, the expression profile of LPL gene was investigated by qRT-PCR and their association with content of intramuscular fat and activities of LPL in dorsal muscles of different stages Congjiang pigs were analyzed. The results showed that the CDS of LPL gene was 1 437 bp encoding a stable, hydrophilic protein with 478 amino acids and a PLAT/LH2 domain. There was high amino acid homology of LPL among different breeds of pig, the amino acid sequence of LPL of Congjiang pigs had similarity of 99.8% with Norwegian Landrace and Luchuan pigs, the lowest homology with Guizhou baixiang pigs was 99.2%. PCR-RFLP results showed that the AfaⅠlocus of the LPL gene have 3 genotypes in Congjiang pigs, i.e. CC, CG and GG, and the CG was dominant genotype. The allele frequencies of C and G at AfaⅠlocus were 0.350 5 and 0.649 5, repectively. The Congjiang pig population investigated was at Hardy-Weinberg equilibrium in this polymorphic sites. The skin thickness of CC genotype was significantly higher than that of CG genotype at AfaⅠlocus in Congjiang pigs (P＜0.05). Furthermore, the backfat thickness, lion eye area and intramuscular fat content of CC were also higer than those of other genotypes (P＞0.05). qRT-PCR results suggested that the expression level of LPL gene in the dorsal muscles of Congjiang pigs at different stages was decreased first and then increased, the correlation analysis indicated that there was a significant correlation between LPL mRNA expression and intramuscular fat content (P＜0.05). In conclusion, the LPL gene might play an important role in the IMF deposition of Congjiang pigs, this study could provide a theoretical basis for the follow-up analysis of structure and function of LPL gene in Congjiang pigs.

Abstract Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear transcriptional factor, which plays an important role in regulating lipid metabolism. This study was carried out to clone the full-length cDNA of pigeon (Columba livia) PPARγ gene, analyze the protein structures, physical, chemical properties and their functional domain structures which encoded by PPARγ gene, and reveal its tissue expression pattern. In our experiment, rapid-amplification cDNA ends (RACE) was used to obtain pigeon PPARγ cDNA. The structure and function of PPARγ gene was analyzed by bioinformatics methods. Real-time PCR was performed to detect PPARγ mRNA expression levels in various tissues. The results showed that the full-length of pigeon PPARγ (GenBank No. KU166859.2) cDNA was of 1 846 bp including a 1 428 bp ORF, 63 bp 5'UTR and 355 bp 3'UTR. The ORF encoded a polypeptide of 475 amino acids, with a calculated relative molecular mass of 54.438 79 kD, pI of 6.19 and an average of hydropathicity -0.287. The predicted protein of pigeon PPARγ was composed of 7 661 atoms with the formula of C2436H3840N644O714S27. Structure prediction showed that there were 2 zinc finger structures and a ligand binding domain in the pigeon PPARγ protein. The deduced amino acid sequence of pigeon PPARγ shared 97%, 97%, 96%, 96%, 96%, 96%, 96%, 92%, 91%, 90% and 64% identities with those of Cuculus canorus, Anas platyrhynchos, Melopsittacus undulates, Taeniopygia guttata, Meleagris gallopavo, Gallus gallus, Coturnix japonica, Sus scrofa, Homo sapien, Mus musculus and Danio rerio, repectively. Phylogenetic relationship analysis indicated that all birds and mammals each formed a large branch, and zebrafish formed another independent branch. Columba livia was closely related with Cuculus canorus and Anas platyrhynchos, which was similar to the results of the biological classification. Real-time PCR analysis revealed that the mRNA level of pigeon PPARγ was highest in lung and abundant in kidney, liver, spleen and heart, while relatively low in muscle. The results showed the full-length cDNA sequence, molecular characteristics and tissue-specific distribution of pigeon PPARγ, which suggested that the PPARγ gene might have various effects on different tissues of pigeion, and also provide theoretical foundation for the further study of the structure and function of PPARγ.

miRNA (micoRNA) is a very important post transcriptional regulatory factor, and have been identified in many species, whereas miRNA related studies were poor in horse (Equus caballus). In this study, we carried out small RNA sequencing on mixed sample of 8 tissues of Mongolian horse with Solexa sequencing technology and analysed expression abundance of conserved and candidate miRNA, and predicted their target genes with relevant bioinformatic analysis. 5 922 735 clean reads were obtained after trimming low quality reads and adapter sequences from 9 478 925 raw reads which were generated from Illumina Miseq platform. Among them, 5 199 802 reads (15~30 nt) were selected from clean reads for subsequent analysis. After length distribution analysis, we found that total sequences were abundant in 20~24 nt that mainly based on 22 nt, which similar with length distribution of unique sequences. For small RNA annotation, total sequences distributed most in known miRNAs (69%), while unique sequences distributed most in rRNA (49%). Then we compared unique sequences with known miRNAs in miRBase and identified 239 conserved miRNAs and 243 miRNA precursors. In addition, we predicted 510 miRNAs and 375 miRNA precursors with all sequences using Mireap software, in which 238 miRNAs could be matched with horse known miRNAs and 272 non-matched miRNAs might be horse candidate novel miRNAs. The minimal folding free-energy (MFEs) of miRNA precursors reached 18.2~62.8 kcal/mol, which accordant with the requirement of its secondary structural stability. 23 037 target genes and 1 974 153 target sites for all predicted miRNAs were predicted by using miRanda software. These results enrich the horse miRNA database and can provide useful information for further related researches.

Germplasm resources are foundation for superior cultivar breeding, and essential for improve the economic efficiency for Cornus officinalis, a special herb tree species in China. The current study select C. officinalis core collection in the samples from all over the country by inter-simple sequence repeat (ISSR) markers, for the collection, evaluation, preservation and usage of germplasm. One hundred and twenty nine samples were collected from 11 provinces all over China. Total DNA were extracted by modified cetrimonium trimethyl ammonium bromide (CTAB) method and amplified by 11 ISSR markers. To select the core construction, cluster sampling adopts 2 strategies: Random sampling and site sampling, while genetic distances were estimated in 3 ways: Similar matching (SM) genetic similarity coefficient, Jaccard genetic similarity coefficient or Nei & Li genetic similarity coefficient. T test was carried out on the genetic parameters of the original collection, core collection and germplasm collection, to evaluate the representative and heterogeneity. The heterogeneity of the original germplasm and core collection was tested to confirm the representative of the core collection. Eleven primers amplified 87 bands from 129 samples, all of which were polymorphic, and the average loci number of each primer was 7.91. For the whole 129 samples, the number of alleles (Na) was 2.000 0, the average effective number of alleles (Ne) was 1.438 8, average Nei's genetic diversity index (H) was 0.259 3, the average Shannon's information index (I) was 0.400 6. These parameters showed the samples had high genetic diversity at the DNA level. Cluster analysis showed that geographical isolation and genetic differentiation existed in the populations from different areas. Cluster sampling results under 2 sampling strategies and 3 types of genetic distance were compared, the S1D3 group selected 34 samples by site preferred sampling strategy and Nei & Li genetic distance clustering showed the highest genetic diversity parameters. The number of effective alleles, Nei 's genetic diversity index and Shannon information index of the S1D3 group were significantly higher than the initial collection in the 0.01 level of probability, and the genetic diversity was more abundant than the reserve germplasm. Thirteen phenotypic indicators of the original germplasm and the core collection were compared, except for leaf width, petiole length and dried fruit weight differed at the 0.05 level, other 10 traits had no significant difference with the original germplasm. The phenotypic indicators of the selected core germplasm could represent the original germplasm resources. A C. officinalis core collection including 34 samples were selected from 129 germplasms collected from 11 provinces all over China, contained 26.36% of the original germplasm. By comparing the genetic diversity parameters of the core collection，original germplasm and the reserve germplasm, along with the confirmation by 13 phenotypic traits data, the core collection can represent the genetic diversity of the original germplasm. The result can be used in C. officinalis germplasm resource conservation and superior cultivar breeding.

Dopamine receptor D1(DRD1) can promote or inhibit the secretion of prolactin in the birds' brain. It is associated with the reproductive system and highly expressed in the hypothalamus and pituitary. The experiment was designed to study of polymorphism of 5' flanking regulating of DRD1 gene on sexual maturity traits in cock (Gallus gallus), 140 W lines breeding from Poultry Institute, Chinese Academy of Agricultural Sciences were used in the experiment. The SNPs was detected by PCR-RFLP (cleaved amplification polymorphism sequence-tagged sites) and analyzed the association of polymorphisms sites with body weight, comb height 1, comb height 2 and comb length of cock at 77, 84 and 91 days of age. The results indicated that one mutation site was detected in 5' flanking regulating of DRD1 gene. Three genotypes (AA, AG and GG) were found at G-570A mutation locus at the distance of CDS-570 locus in 140 cocks by digesting the PCR product with MspⅠ. The relationship between DRD1 gene and the sexual maturity traits was analyzed by least squares linear model. The individuals with GG genotype had significant effect on the body weight (P＜0.05), and had no significant effect on the comb height 1, comb height 2 and comb length of cock at 77 days of age. At 84 days of age, the individuals with different genotype of body weight comb height 1, comb height 2 and comb length were not significant. The individuals with GG genotype had significant effect on comb height 1 and comb length (P＜0.05). The DRD1/883 promoter of DRD1 gene was amplified using PCR technology, inserted into the dual-luciferase reporter vector (pGL3-Basic) to construct pGL3-DRD1/883/promoter. 293T cell were co-transfected with a plasmid containing the pGL3-DRD1/883/promoter, pGL3-Basic and PRL-CMV, respectively. The activity of luciferase gene in 293T cell was testing using the Dual-Luciferase Reporter Assay kit. The results showed that the intensity ratio of firefly to sea renal luciferase of DRD1/883G prompter enhance 13.3 times than DRD1/883A promoter, showed high expression (P＜0.01). Thus it can be seen, GG was the most favorable genotype for early maturity traits in chicken. Individuals with DRD1/883G had higher levels of transcription. These results imply that DRD1 gene may have a certain effect on early maturity on chicken and provide a theoretical basis for the expression and function of DRD1 gene and breeding of high quality broiler.

Kernel ratio in maize (Zea mays) is a typical quantitative trait, which is affected by the minor-gene and is susceptible to environmental effects, thus limiting the ability of genetic improvement in breeding. Multi-environment experiment was conducted at 3 locations for 2 years in order to explore QTLs which controlled kernel ratio traits in maize stably inheriting under different environments, and analyze the interaction effects between environments and QTLs. In this study, 150 recombinant inbred lines (RIL) derived from Xu178×K12 were used for the experimental materials. QTL analysis was conducted through single environment analysis, joint analysis and epistatic analysis, respectively. Results showed that 13 QTLs were detected through single environmental QTL analysis, which distributed on Chr.1, Chr.3, Chr.5, Chr.6, Chr.7, Chr.8 and Chr.9, respectively. The phenotypic variance explained by these QTLs ranged from 6.74% to 22.18%. Four QTLs were detected by using best linear unbiased prediction (BLUP) data, all of which were also detected through single environment. The phenotypic variance explained by interaction of additive effect for single QTL ranged from 0.78% to 2.31%, Eight QTLs were identified through joint analysis, the phenotypic variance explained by interaction of additive×environment for single QTL ranged from 0.21% to 1.96%. Fifteen pairs of QTLs with epistatic effect were detected through epistatic analysis in total, which distributed on all chromosomes. Four key areas, including Bin1.06~1.07, Bin6.01~6.02, Bin8.07 and Bin9.03~9.05 controlling kernel ratio were filtered through multi-environments QTL analysis. Besides, our results showed that the interaction effect between kernel ratio trait and environments was complicated. Epistatic effect was also an important genetic basis for the performance of kernel ratio in maize except for additive effect. The results of this study will be instructive for improvement of germplasm resources and molecular marker assisted selection (MAS) in further study.

Sucrose which involved in the plant growth and development processes, the signal transduction pathway and other physiological activities is an important carbon source in plants. Sucrose transporter genes (SUT) play a key role in the long distance transport activities of sucrose in plants. In order to understand the transport mechanism of the SUT in Paeonia suffruticosa, P. suffruticosa 'Luoyanghong' was used as experimental materials in this study. The sucrose transporter gene named PsSUT2 was separated and cloned from the leaf by reverse transcription-PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE). The full length cDNA of PsSUT2 is 2 347 bp, and the ORF encoding a protein of 601 amino acids, was 1 803 bp. The accession number is KC542396 in GenBank. Its molecular mass was 64.6 kD and pI was 6.14, The homologous amino acid sequence of PsSUT2 compared with that of SUT gene from Vitis vinifera was 75.27%. PsSUT2 protein contained 12 transmembrane helical structures, which belongs to the major facilitator superfamily (MFS). The relative expression of PsSUT2 gene and the soluble content in different organs were determined with qRT-PCR and chromatography technology at the different developmental stage of tree peony. The main results showed that the PsSUT2 gene expressed abundantly in the petals at the flowering stage, and the relative expression was 11.18, which was significantly different from other organs of the same stage even every organ of other stages. Meanwhile, the PsSUT2 gene expressed in every organ such as the root, the perennial stem, the leaf, the bulbil, the petal at different developmental stage, but the relative expression were low, for example, the relative expression of petals in the flowering stage was 4.26 times as much as that of roots in the same stage, and 4.40 times as much as that of roots in the little bell stage, and 10.48 times as much as that of petals in the peach stage, and 30.55 times as much as that of bulbil in the bulbil differentiation stage. So, it indicated that the PsSUT2 gene took part in the sucrose unloading activities in the sink organs probably. The contents of sucrose, glucose and fructose of every organ were significantly different at different developmental stage of tree peony, and there was no sucrose almost but much more glucose and fructose in the bulbil or petal before the arrival of flowering. So, the petal of P. suffruticosa 'Luoyanghong' accumulated hexose (glucose and fructose) mainly and the accumulation of hexose was benifit to the flower opening. However, the differences were significantly negative between the sucrose content of leaf, the glucose content of root and the relative expression level of PsSUT2 by the correlative analysis method. Therefore, The expression of the PsSUT2 gene may be inhibited by the sucrose in the leaves and the glucose accumulation in the roots. The results provide a theoretical basis for further understanding of the function of the PsSUT2 gene, and the next step, the gene can be used to carry out molecular breeding and genetic improvement work on the tree peony.

Calcium-dependent protein kinase (CDPK) is a kind of protein kinase that can respond to abiotic stresses. Over the past few years, many CDPK genes have been isolated from Arabidopsis thaliana, Oryza sativa and Populus trichocarpa. To study the structure, function and expression patterns of CDPK genes in Dendrobium officinale, DoCDPK6 (GenBank No. KY682702) and its promoter were cloned from D. officinale, sequence characteristics, structure and evolutionary relationships were analysed by bioinformatics, which showed that the length of cDNA sequence of DoCDPK6 were 1 976 bp, with 1 725 bp open reading frame which encoded 574 amino acids. The molecular formula was C2845H4500N766U858S24 and the molecular weight is 63.9 kD. Structural analysis showed that DoCDPK6 gene had 4 typical conserved structure of CDPK genes: variable domain, protein kinase domain, autoinhibitory domain and calmodulin-like region. The phylogenetic analysis showed that it is most relative to Phalaenopsis amabilis, and in accordance with botanical classification status, which illustrated that DoCDPK6 gene was conservative in evolution. The core promoter elements of TAAT-box and CAAT-box were found in the 1 500 bp upstream sequence of DoCDPK6 gene, and adverse signal related response elements like light, temperature, salicylic acid, abscisic acid (ABA), auxin, gibberellic acid (GA) and so on, which was speculated that it could participate in the regulation of DoCDPK6 response to stress. By qRT-PCR the expression of DoCDPK6 could be detected in roots, stems, leaves of D. officinale, the most in leaves, followed by stems and roots, which showed the expression of DoCDPK6 has tissue specificity. It was found that the expression of DoCDPK6 were up to varying degrees, through the low temperature of 4 ℃, ABA and NaCl treatment. Under 4 ℃ treatment the expression of DoCDPK6 increased and reached the peak at 4 h, and had 3.5 times as much as 0 h. Then as time went on it decreased, the expression at 24 h returned to the level as much as 0 h, that low temperature induces the expression of DoCDPK6 can inferred .Under the ABA treatment the expression of DoCDPK6 was up-regulated in all times periods and reached the peak at 12 h and was 4 times as much as the level at 0 h, which showed the expression was regulated by ABA of high concentration. Under NaCl treatment the expression of DoCDPK6 increased to the highest level at 4 h, 4.9 times as much as 0 h, after that the expression decreased but also significantly higher than 0 h, which indicated that the expression was regulated by NaCl of high concentration. CDPK is a kind of Ser/Thr protein kinase response to the second messenger of Ca2+ and plays an important role in plant growth, adversity stresses and cell signaling. The study indeed proves DoCDPK6 can response to stresses such as low temperature, NaCl and ABA, it is speculated that DoCDPK6 plays a role in stresses, which provides information to the regulatory mechanism and pathway of signal transduction of DoCDPK6 gene and its promoter in abiotic stress in the next study.

Parkinson's disease (PD), the second most common neurodegenerative disease. It includes the major motor symptoms of akinesia, rigidity and tremor. A lot of study indicates that apoptosis is involved in the pathogenesis of PD. The present study is to investigate the mRNA expression of tumor suppressor gene P53, cysteinyl aspartate-specific proteinase-3 (Caspase-3), B cell lymphoma-2 (Bcl-2) and BCL-2-associated X protein (Bax) in peripheral blood before and after treatment in monkey (Macaca mulatta) with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Six healthy young rhesus monkeys were respectively daily injected a small dose (0.2 mg/(kg·d)) of MPTP. The behavioral manifestations of all monkeys were evaluated before and after the injection for 30 min, respectively. The forearm venous blood of rhesus monkeys were took at 0 week(non-injected MPTP) and 4, 8, 12, 16, 20, 24 week after injection MPTP. qRT-PCR detected P53, Bax, Bcl-2 and Caspase-3 mRNA expression in peripheral blood. The correlations between mRNA expression of P53, Caspase-3, Bcl-2 and Bax and the severity of behavior scores were analyzed. Behavioral observation indicated that from the 4 week, the experimental animals showed moderate bradykinesia, and then the symptoms gradually worsened, exercise capacity continued to decline, experimental animals showed a significant reduction in activity at 12 week, posture stiff and severe movement obstacle. After the 13 week of clinical symptoms was basically stable, the experimental animals showed slow movement, abnormal posture, muscle rigidity and resting tremor. qRT-PCR detected of apoptosis regulatory factors, compared with the 0 week, the expression of P53 and Caspase-3 significantly increased at the 4, 8, 12 week (P＜0.05), but the difference was not significantly at the 16, 20, 24 week; compared with the 0 week, Bcl-2/Bax ratio at the 4, 8, 12 week significantly decreased (P＜0.05), and the difference was not significantly at 16, 20 and 24 week. The mRNA expression of P53 and Caspase-3 were positively correlated with behavior scores in 0~12 week (P＜0.05). The above results suggested that when P53 and Casapse-3 significantly increased, the clinical symptoms of experimental animals also continued to intensify with the same period. The mRNA expression of P53 and Caspae-3 in peripheral blood can be used as one of the indicators for diagnosing PD, assessing the severity of PD.

The trigeneric hybridization is mainly produced by recombination or structural rearrangement among chromosomes of different species, and they are the individuals with excellent gene set. Through screening, identification and breeding, the wheat traits were obtained, as well as genes, resistance to germplasm or direct breeding of new varieties. So far, the domestic and foreign scholars have been a large number of polygeneric hybrids. Its descendants were analyzed and the results showed that the offspring differentiation of the hybridization was extremely complicated, which generated some new germplasm for the improvement of wheat genetics. In this study, polygeneric hybrid was produced by an octoploid trititrigia (wheatgrass 8, AABBDDEE, 2n=56) and a hexaploid triticale (Hashi 209, AABBRR, 2n=42). Wheatgrass 8 of the male parent was obtained from the hybridization of Chinese Spring and Thinopyrum intermedia. Hashi 209 of the female parent was final election in 1996 and called "Nongqing 2" by Heilongjiang Provincial Variety Approval Committee, which was cultivated in 1990 using the hybridization between "triticale 2" (octoploid triticale, bred by Academician Bao Wenkui from Chinese Academy of Agricultural Sciences) and "Rosner"(a hexaploid triticale). Then, a part of hybrid F6 generations of polygeneric hybrid were analyzed and identified by morphological and molecular cytogenetic methods, which was aimed at discussing the cytogenetics' rule of polygeneric hybrid and breeding hybrids of Triticum-Thinopyrum-Rye with the important agronomic traits, such as a large spike, more flowers and grains, and resistance to disease. It will provide the basic material for the improvement of Triticale and Triticum. The morphological results showed that the significant differences of morphological characteristics were divided into triticale type, normal wheat type and intermediate type. There were 5 lines of triticale type, which were lodging resistance and large spikelets. Their pollen mother cell meiosis was observed with the univalent, and the thousand kernel weight was more than 40 g in 4 lines. The results of molecular marker and genome in situ hybridization showed that 3 lines (14-4, 3-29-3, and h-29) were hexaploid triticale with R and E chromosomes. The kernel of line 14-4 was full and the thousand-grain weight was 47.6 g, which was better than its parental Hashi 209. There were 8 lines of normal wheat types. Their maturity was normal and the chromosome number of root tip cell was 42. There were 5 lines with the genetic components of R group chromosome and line 14-22 carried the chromosomal translocation of R genome, additionally, line w-16 carried the chromosomal translocation and substitution of E genome. There were 7 lines of intermediate type and all lines had the genetic components of R genome. The plants were height (94~120 cm), more tillers (5~15) and grains. The spikelet number was from 16 to 36. The kernel number of main spike in line h-7 was 122 and the threshing was difficult. Line 11-26 carried the chromosomal translocation of R genome. Besides of carrying the genetic components of R group chromosome, line 11-21-2 carried E chromosomes, but the chromosome number of root tip cell was 52. It was less the genetic instability. Line14-4, 3-29-3 and h-29 that obtained from the research were new germplasms of Triticum-Thinopyrum-Rye, which provides theoretical and material bases for breeding triticale and creating wheat polygeneric hybrids.

Primordial germ cells (PGCs) are kind of cells which have the potential to differentiate into germ cells. To investigate the effect of activin A on the survival and proliferation of chicken (Gallus gallus) embryonic blood PGCs cultured in vitro, PGCs were isolated from stage 13~15 chicken embryonic blood, and then the cultured PGCs were identified using PGCs marker antibodies and marker genes. After addition of activin A (0 and 100 ng/mL) into complete medium for 48 h, the cell number, morphology and cell cycle were detected. To study the regulative mechanism of activin A in PGCs proliferation, the DNA methylation level of deleted in azoospermia-like (Dazl) gene was measured. The protein expression and phosphorylation of transforming growth factor-β (TGF-β) and SMAD2/3 were tested by Western blot. The results showed that PGCs isolated from chicken embryonic blood was distinct from blood cells with big nuclear and rich in glycogen particles. The purity of PGCs rapidly rised by treatment with red blood cell lysis buffer (ACK), from (0.026±0.005)% to (69.2±4.6)%. The high purity and excellent growing trend of PGCs was obtained by subculture. After subculture for 3 passages, PGCs was positive with stage specific embryonic antigen 1 (SSEA-1), chicken vasa homologue (CVH) and periodic acid Schiff reaction (PAS). The expression of pluripotent related genes, PouV, Nanog and sex determining region Y (SRY)-box 2 (Sox2), suggested that the biology characterist theics were well maintained in PGCs cultured in vitro. After treated with activin A, numbers of PGCs were significantly increased and the morphology of PGCs was better than that of control group, with clear boundaries and cubic morphology. And the mRNA expression of cyclin-dependent kinases (CDK2)/Cyclin D1 and CDK6/Cyclin E were obviously up-regulated. Flow cytometry analysis confirmed that PGCs populations treated with activin A displayed a significant increase in the proportion of S and G2 phase cells, which meant that there were more PGCs which entered into mitosis process than that of control group. The survival and proliferation ability of PGCs was enhanced by addition of 100 ng/mL activin A. Moreover, the ratio of Dazl gene mythelation was decreased from 94.7% in control group to 52.6% in activin A treated group. However, the mRNA expression of Dazl was significantly increased. The results indicated that activin A promoted PGCs proliferation by reducing the mythelation of Dazl gene. The protein expressions of TGF-β and SMAD2/3 were up-regulated and the phosphorylation of SMAD2/3 was activated when PGCs were treated with activin A. Collectively, these results suggested that activin A significantly promoted the survival and proliferation of PGCs cultured in vitro via TGF-β/SMAD signaling pathway and Dazl mythelation. These data provides basic information for the mechanism of PGCs proliferation regulated by activin A, and provides theoretical foundation to regulate germ cell development by using PGCs culture model.

Myo-inositol-1-phosphate synthase (MIPS) is a key rate-limiting enzyme in inositol biosynthetic, and plays important roles in plant growth regulation and abiotic stress response. To investigate fuction of MIPS in grape (Vitis vinifera), the full-length cDNA of VvMIPS was cloned from cultivar Zuoyouhong. Sequence analysis showed that the size of VvMIPS was 1 533 bp (GenBank No. 1984676), encoding a protein of 510 amino acids with molecular weight of 56.3 kD, and isoelectric point of 5.37. VvMIPS presented high similarity with MIPS proteins from Glycine max, Medicago sativa and Trifolium pratense. qRT-PCR analysis showed the expression of VvMIPS was the highest in flower buds, stems, and the lowest in root. In addition, VvMIPS was highly induced by low and high temperature stress, and induced by H2S, abscisic acid (ABA) and H2O2. The recombinant plasmid pET28a-VvMIPS was transformed into Escherichia coli BL21, and expression of VvMIPS protein was tested. The results showed that VvMIPS protein could enhance the resistance of E.coli to low and high temperature stress. This study provides the genetic resources for grape cultivars improvement with lower or higher temperature stress tolerance.

Melanocortin-1 receptor (MC1R) gene may play important roles in the regulation process of mammalian coat color, but the effects of exogenous MC1R gene on the micropht-halmiaassocitated transcription factor (MITF) and tyrosinase (TYR) genes in alpaca (Lama pacos) are less studied. To explore effect of MC1R gene on the expression of MITF and TYR, eukaryotic expression vector of MC1R gene pcDNA3.1(+)-MC1R was constructed and transfected into alpaca hair follicle stem cells (HFSCs). The relative expression of MC1R, MITF and TYR mRNA were detected by qRT-PCR, and MC1R protein was located by immunocytochemistry. qRT-PCR results showed that the relative expression level of MC1R was significantly increased (P＜0.05) in the group transfected with pcDNA3.1(+)-MC1R (P＜0.05). There were 2.15 and 2.26 times than that in the group of pcDNA3.1(+) and the control group. MITF and TYR gene also had higher expression, but not significant (P＞0.05). Meanwhile immunocytochemistry results showed that MC1R protein had a positive expression in HFSCs. The results showed that the MC1R gene excessively expressed in alpaca HFSCs, and indirectly up-regulated the expression of TYR and MITF. This study provides scientific basis for further study on the expression mechanism of MC1R in the alpaca HFSCs.

Abstract WRKY transcription factors are one of the largest families of transcription regulators in plants, and it can bind W-box in promoters of the target genes to regulate many specific transcriptional programs. This article summarized the recent advances on WRKY transcription factor functions and regulation mechanisms on biotic and abiotic stress responses in Arabidopsis and rice (Oryza sativa). The expressions of WRKY genes can be rapidly induced by many environmental signals. Many WRKY genes exhibit extensive autoregulation and cross-regulation. A single WRKY transcription factor can regulate several seemingly disparate processes, and several WRKY proteins can also work together in the same process to make the signal transduction pathway to be exactly regulated. WRKY proteins via cooperation with a diverse array of protein partners realize their functions, and they are usually phosphorylated by mitogen-activated protein kinase to modulate their activities in the nucleus. WRKY transcription factors in Group Ⅰ and Ⅱc can interact with VQ proteins that contain a short VQ motif and act as cofactors, and those in group Ⅱd contain C motif that can bind with calmodulin. In addition, the WRKY domain in RRS1-R represents an effector target 'decoy' which betrays the defense-suppressing abilities of PopP2 and AvrRps4 on their operational virulence targets, the defensive WRKY transcription factors. WRKY transcription factors play pivotal roles in regulating many stress reactions, but only a few mechanisms on signaling and transcriptional regulations are characterized more clearly. This article has revealed a corner of the complex regulatory network, and may help researchers to further understand the regulatory mechanisms of WRKY transcription factors.

Abstract Transgenic animal as bioreactors is a novel expression system capable of producing foreign proteins in live animal tissues and body fluids at an industrial scale level, such as the milk, blood, urine, seminal plasma and egg white. The use of the transgenic animal as bioreactors offers attractive advantages, residing on the low production costs allied to high productivity and quality and easy purification of the recombinant proteins. Since recombinant human antithrombin Ⅲ (ATryn), purified from the milk of transgenic goats (Capra hircus), was first approved by the European Medicines Agency (EMA) for marketing in European Union countries on February 6, 2009, this novel protein production system revolutionized the landscape of scientific and marketing possibilities of biopharmaceutical. Human lysozyme (hLZ), as a natural non-specific immune protein, is an important component of human breast milk. It hydrolyzes the glycosidic β-(1-4) linkage between Nacetylmuramic acid and N-acetylglucosamine of the peptidoglycan polymer in the bacterial cell wall and plays an important role in killing bacteria, modulating the inflammatory response, influencing the composition of intestinal microbiota populations. Compared to hen egg white lysozyme, hLZ is a safe and non-allergenic protein, which has more lytic activity. hLZ could be widely used in food preservation, infant milk powder, antibacterial anti-inflammatory drugs and antibiotic substitution. However, its commercial use is very limited due to the poor source of human breast milk. In this paper, we firstly summarized the progresses of recombinant human lysozyem expressed in the milk of transgenic mouse, cows, goats and pigs, and in the egg whites of transgenic hens. Then we talked about the physicochemical properties, purification process and biological function of recombinant human lysozyme, and biosafety evaluation of transgenic animals. Finally, we analyzed the existing problems and the future development of the industrialization of human lysozyme transgenic animal. Overall, the review aimed at providing better understanding of hLZ transgenic animal and industrialization to researchers.

Abstract Relaxin, as a polypeptide hormone of insulin superfamily, plays importantly regulatory roles in the reproductive and non-reproductive physiology processes. Besides, relaxin appeared in peripheral blood, which had been seen as a sign of pregnancy in canine (Canis lupus) and other some animal. To prepare a polyclonal antibody against canine prorelaxin, the cDNA fragment of canine prorelaxin gene was amplified from the recombined plasmid pMD19-T-Cpreprorelaxin1 containing cDNA fragment of canine preprorelaxin gene by PCR, and cloned into the prokaryotic expression plasmid pET28a. Secondly, the recombined expression plasmid of canine prorelaxin was transformed into Escherichia coli BL21 (DE3) and induced by isopropyl-β-d- thiogalactoside (IPTG), to harvest the fusion protein which were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Finally, the polyclonal antibodies were harvested after injecting the rabbits (Oryctolagus cuniculus) with the purified fusion protein of canine prorelaxin emulsified with the Freund's complete adjuvant as the immune antigens, and analyzed by enzyme linked immunosorbent assay (ELISA) and Western blot. The results demonstrated that the fusion protein with the consistent molecular weight of 22 kD indicated by SDS-PAGE, was highly expressed as the form of inclusion bodies and positively reacted with the His-labeled antibody by Western Blot. The harvested anti-serum with more than 1∶80 000 titered by ELISA, reacted specifically with the purified fusion protein of canine prorelaxin. This study would lay a potential foundation for further study and clinical application of canine prorelaxin.