Abstract Endochondral ossification (EO), the main form of bone formation, has been well studied, and collagen type X (Col X) is considered as an essential component for EO, and its expression has been detected in the hypertrophied chondrocytes in developing cartilage tissues. In this regard, deer antlers arguably fit this purpose the best. In this paper, an in vitro model was established using antler stem cell to investigate the role of Col X in EO through RNAi approach. High score shRNA sequences were designed and four (S1~S4) were selected after removing off the target ones based on general design rules. Then, oligo DNA comprising both sense and antisense strands of each shRNA was ligated into pLVTHM (lentiviral carrier plasmid using green fluorescent protein (GFP) as a maker) using T4 DNA ligase. Positive clones named as pLVTHM S1~S4 were selected by using PCR screening and then sequenced. The carrier plasmid containing shRNA (pLVTHM S1~S4), packaging plasmid (pCMV-dr8.91) and enveloping plasmid (pMD2.G) were co-transfected into a 293T cell line. The lentiviral particles produced from the co-transfected 293T cells were harvested and concentrated. Antlerogenic periosteum (AP), within which antler stem cells reside, was sampled from a 1-year-old male sika deer and enzymatically digested to release cells for in vitro culture. The resultant cells were subsequently infected by the lentiviral particles (S1~S4), or a negative control construct (PBS), and then seeded in a high-density (10 /mL) to establish micromass culture. Knock-down efficiency of Col X was examined using both qRT-PCR and Western blot analysis. The results showed that cell nodules were formed around 70 h after micromass seeding; These AP cell nodules strongly expressed GFP, implicating that shRNA that targets Col X gene had been expressed in these nodules, whereas negative control was completely dark under UV light. The qRT-PCR results showed that Col X gene was significantly silenced in micromass-cultured AP cells compared to the negative control. Among these shRNAs, S3 was the one that had most dramatic effects on down-regulating Col X gene expression reaching 94.6%. Western blot analysis further confirmed the results of qRT-PCR. Histological examination revealed that, in the absence of Col X, the matrix of the cellular nodules of pLVTHM-S3 infected group had little alcian blue staining with empty lacunas, whereas nodules of the negative control were darkly stained, which indicated that Col X plays a key role in the process of EO through maintaining the matrix of chondrocytes. Overall, this study successfully established an in vitro model using antler stem cells for the investigation of the role of Col X in EO through RNAi approach. Down regulation of Col X could promote the process of chondrocyte death in EO to occur, and result in empty lacunas. Further study shall use antler models to determine how bone replacement is affected after Col X silencing in vivo .

Abstract Annual full regeneration of deer (Cervidae) antlers has been proved to be a stem cell-based process, and antler stem cells reside in both antlerogenic periosteum (AP) and pedicle periosteum (PP). The extraordinary growth rates of antlers provide us a rare system in which rapid cell proliferation is elegantly regulated without becoming cancerous. Thus deer antlers are unique mammalian organs that have potential to be a valuable model for biomedical research, such as the areas of stem cell, organ regeneration, bone development and growth control. qRT-PCR is one of the most common-used techniques in molecule biology study of deer antler. Appropriate internal reference genes are prerequisites for the accurate analysis of gene expression in antler research by qRT-PCR. In order to perform accurate quantitative analysis for differentially expressed genes between antler growth center cells and antler stem cells. In this study, antler stem cell tissue samples including AP, PP and facial periosteum (FP) (as a perfect negative control in antler stem cells research) were collected using surgical operation in a relative sterile environment. Antler growing center tissue samples, including reserve mesenchymal (RM), precartilage (PC), transition zone (TZ) and cartilage (C) were isolated from growing antler tips. Primary culture of these cells were carried out after releasing cells from these tissue types using typeⅡcollagenase (150 U/mL) and cells were cultured in Dulbecco's modified eagle medium (DMEM) with 10% fetal calf serum (FBS) in 37 ℃ and 5% CO2. Stability of mRNA expression and levels of 6 candidate reference genes, including actin beta (ACTB), tubulin alpha1a (TUBA1A), TATA box binding protein (TBP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G6PDH) and beta-2-microglobulin (B2M) in antler growth center cells and antler stem cells were measured by through qRT-PCR method. Software, which are Delta-Ct method, geNorm (ver.3.5), Bestkeeper (ver.1.0), NormFinder (ver.0.953) and RefFinder, were used to analyze all the acquired data. The results showed that expressions of ACTB and TUBA1A genes were the most stable in antler stem cells; B2M and ACTB were the same in antler growth center cells. The results of comprehensive analysis using the software indicated that B2M, ACTB and TUBA1A genes were the most stable reference genes, and suitable for antler research. The present study provides a foundation for accurate quantification of differential gene expression for antler research and mechanistic study of this fascinating model for mammalian organ regeneration.

Abstract Assignment of the human 14-3-3 epsilon isoform (YWHAE) gene encodes 14-3-3ε protein, which highly specifically expresses during antler development of sika deer(Cervus nippon). The study was to clone the YWHAE gene's cDNA coding domain sequence of sika deer and predict its structure, function and expression profiles. Using antlerogenic periosteum as materials, the sika deer's YWHAE CDS was cloned by RT-PCR and its protein structure and functions were predicted through several bioinformational tools as well. The results showed that the sika deer's YWHAE CDS consisted of 768 base pair (GenBank No. KY797653) that encoded 255 amino acids residues, and the amino acid homology of deer YWHAE shared 99.6% identity with bovine (Bos taurus). The analysis of amino acid sequence revealed that the sika deer YWHAE encoded 14-3-3 protein and its relative molecular weight was 29 163.86 D. Subcellular location of 14-3-3 protein was primarily in the cytoplasm, and it belonged to the water-soluble protein. It was predicted that the 14-3-3 protein contained 19 phosphorylation sites, and there was no glycosylation sites. The secondary structure of the protein mainly had helixs, while the tertiary structure of domain area showed a helical state. The study predicts the characteristics of the 14-3-3 protein structure and provides basis theory on exploring the 14-3-3 proteins interacting with other proteins. Furthermore, it provides a basis for further studying on the regulation mechanism of antler development.

Abstract Quantitative Real-time PCR is popularly used to study gene transcription levels in plants. Validation of candi-date reference genes is the key step to ensure accurate quantification. Nitrogen element assimilation and utilization play a critical role in sorghum (Sorghum bicolor) yield. The study was to select the most reliable reference genes for quantitative Real-time PCR (qRT-PCR) analysis of target sorghum genes under low nitrogen stress. We chose 7 commonly used housekeeping genes including Actin, ubiqutin conjugating enzyme (UBQ), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), UBQ10, eukaryotic translation initiation factor 2B (EIF2B), glyceraldehyde-3-phosphate dehydrogenase (sbGAPDH) and eukaryotic translation initiation factor 4a (EIF4a) to systematically assess their expression levels in leaves and roots of sorghum under low nitrogen stress by qRT-PCR. The results of primers specificity analysis showed that all the primers were in line with the requirements of the stability screening. The consequences of qRT- PCR indicated that the stability and expression level of candidate genes varied in the different tissues and under different treatments. The expression level of GAPDH was the highest in sorghum leaves, and that of UBQ10 was the highest in roots. We also analyzed and evaluated the stability of candidate reference genes by geNorm software. The results showed that GAPDH and EIF4a were the most suitable reference genes for normalizing target gene expression in sorghum leaves under low nitrogen stress, whereas sbGAPDH and EIF4a were recommended as the optimum combination in roots. In conclusion, this study has provided the basic data for expression analysis of target sorghum genes under low nitrogen stress and reference data for similar studies of other plants.

Abstract Pig (Sus scrofa) is a good animal model for human disease and stem cell study, also is one of the important domestic species in the husbandry. In recent years, more and more scientific research needs pig preimplantation embryos for experiment material, due to the development and application of pig embryo engineering and related scientific research on porcine. Because of immaturation of the pig semen and embryo cryopreservation techniques, most of pig preimplantation embryos used in laboratory are obtained by in vitro fertilization (IVF) with fresh semen or frozen-thawed semen. Collection and transportation of fresh semen is inconvenient, and IVF with frozen semen is inefficient. Polyspermy is also easily to happen, which can lead to result mistake. Therefore, the establishment of a fast and efficient technological system for producing high-quality porcine embryos in vitro needs urgent resolve for scientists in the related research fields. In this study, we integrated and optimized porcine semen cryopreservation, oocyte in vitro maturation and oocyte intracytoplasmic injection (ICSI) techniques to establish a highly efficient pig embryo in vitro production system. First, we established stable system of oocyte in vitro maturation, the cleavage rate of parthenogenetic activation and blastocyst developmental rates were 79.51% and 32.30% respectively. And then, we performed IVF with fresh semen and frozen semen, embryo cleavage rates were 49.89% and 28.30% respectively, blastocyst developmental rates were 31.44% and 10.55% respectively. Embryo cleavage rate and blastocyst developmental rate of IVF with fresh semen were significantly higher than those of freeze-thawing semen (P<0.05). In addition, further test was conducted by in vitro production of pig embryo using the method of oocyte ICSI with frozen semen, the cleavage rate and blastocyst developmental rates were 83.00% and 23.32% respectively, while the embryo cleavage rate and blastocyst rates were 49.89% and 31.44% respectively by IVF with fresh semen. Blastocyst developmental rate of ICSI was lower than that of IVF and parthenogenetic activation. But for total blastocyst yield rate, it was significantly higher by oocyte ICSI than that of fresh semen IVF method. This study sets up an effective, economical system to produce embryos for scientific research and animal reproduction.

Abstract Fish cells are important tools for studying virology, immunology, genetics, and so on. Turbot (Scophthalmus maximus) is a widely cultivated marine fish species in Europe and China. However, no brain cell line has been developed so far. S. maximus brain cell line (SMB) was established by tissue block method from turbot brain in this research. The cell line was maintained in DMEM/F12 which was supplemented with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), antibiotic (mycillin), fetal bovine serum (FBS), antibiotics, basic fibroblast growth factor (bFGF), and 2-mercaptoethanol (2-ME). The cultured SMB cells were composed of fibroblast-like cells and epithelial-like cells, and they had been subcultured for more than 40 passages. The cells grew well in the temperature 18~30 ℃, and the optimum growth temperature was 24 ℃. Chromosome analysis indicated that the passage 30 cells exhibited chromosomal aneuploidy, and 43% of the cells maintained a normal diploid chromosome number (2n=44t). When the SMB cells were successfully transfected with pEGFP-N3 plasmid, the transfection efficiency was up to about 30%. The SMB cell line will provide a new important experimental material to study the mechanisms of infective viruses and gene function analysis.

Abstract The plant lateral buds are important organs which are involved into morphogenesis. Recently many progresses has been made on research of the development of lateral buds. This review systematically summed up the main advances in model plant lateral buds (including Arabidopsis thaliana and Oryza sativa) focusing on the plant hormone and gene expression regulations during the growth and development of lateral buds, and drew the involved gene expression regulating networks. Bamboo (Bambusoideae) shoot bud is a kind of plant lateral buds. Due to bamboo shoot bud differentiation and development have great influence on production of bamboo shoots and bamboo wood, many bamboo researchers focus on the research of bamboo shoot buds and have achieved many interesting results in recent years. So, we also summarized the research progress of bamboo shoot buds based on the research advance of model plant lateral buds. In this paper, the key candidate genes involved into the growth and development of bamboo shoot bud were emphasized the possible breakthrough for further research on the mechanism of the differentiation and development of bamboo shoot bud were also suggested.

Abstract With the development of the new genome editing technology, a large number of genome editing pigs (Sus scrofa) with excellent traits and their products spring up. The analysis of literatures and patents about transgenic pigs and genome editing pigs shows that China is in the leading position in this field. This review summarizes research and development status of domestic and foreign genome editing pigs, predicting their development directions in the future. According to the safety management regulations of genetically modified organisms and the attitudes for genome editing organisms in different countries, we also offer some suggestions regarding safety management of genome editing animals and their products in order to promote the industrialized application in China.

Abstract Myostatin (MSTN) is the negative regulator in the muscular development. MSTN gene has great potential application values in genetic breeding. Acrossocheilus fasciatus is a kind of economic fish which has great prospects for the development, but its growth is slow. In order to clarify the gene characteristic and seek molecular markers associated with growth traits, the MSTN gene of A. fasciatus was cloned, detected its expression level in different tissues and screened single nucleotide poly morphism (SNP) sites. The full length of MSTN cDNA sequence was 2 163 bp, including a 1 128 bp open reading frame (ORF) that encoded 375 amino acids. The putative MSTN protein of A. fasciatus had the essential characteristic of vertebrate MSTN, such as the proteolytic site RIRR and nine conserved cysteines in the C-terminal. Sequence alignment showed that homology of MSTNs were greater than 80% among different teleost, and fish had low homology (approximately 65%) with other vertebrates. A. fasciatus MSTN was highly homologous with other Cyprinidae fish (＞96.5%). The phylogenetic tree based on MSTN amino acids showed all the fish clustered together, which was seperated from other vertebrates. A. fasciatus was located in the cluster of Cyprinidae. Semi-quantitative RT-PCR showed that there were high level of MSTN mRNA expression in brain and muscle, weak expression in liver and intestine, and no expression was detected in spleen, kidney, gill, fin and heart. The full-length of MSTN gene DNA sequence was 5 828 bp, which contained 3 exons and 2 introns. Ten SNPs sites were detected in the second intron, but there was no significant correlation (P＞0.05) between these SNP and growth traits (body weight and body length). These results provide reference for the function research of MSTN gene and seeking molecular markers associated with growth traits in A. fasciatus.

Abstract Iron-regulated transporter 1 gene (IRT1) has been proved to be involved in plant iron absorption as the main transporter when plants are under iron deficiency stress. In this study, for cloning the IRT1 gene sequence and investigating its expression characteristics of Pyrus betulaefolia, the full length cDNA of iron-regulated transporter gene named PbIRT1 (GenBank accession No. KX355331) was cloned with reverse transcription PCR (RT-PCR) and RACE by roots of Py. betulaefolia seedings. The full length cDNA of PbIRT1 was 1 369 bp, 5'-UTR was 269 bp, 3'-UTR was 490 bp and contained 6 bp PolyA tail, including an 1 095 bp ORF. The protein consisted of 364 amino acids encoded by the PbIRT1, and it had 9 transmembrane domains located in 10~28, 55~74, 89~109, 128~153, 212~232, 242~266 272~290, 303~323, 346~364 of amino acid sequence, and a complete ZRT/IRT-like protein (ZIP) transport family domain architecture which could transport divalent metal ions, whose protein molecular weight was 39.17 kD and isoelectric point was 8.84. Hydrophobicity analysis speculated that PbIRT1 was a hydrophobic protein and there were 10 serine phosphorylation sites and 5 threonine phosphorylation sites in coding region. The secondary structure prediction of Py. betulaefolia IRT1 protein showed that 37.64% α-helix, 29.67% extended strand and 24.45% random coil might be formed. The amino acid identity of PbIRT1 protein among the Py. betulaefolia, Py. bretschneider, Malus xiaojinensis, Prunus mume and Pr. persica were 100%, 97.8%, 87.9% and 87.4%. In addition, subcellular localization prediction analysis indicated that the protein was most likely targeted to the plasma membrane. It was found that the protein sequence of PbIRT1 was the closest with MxIRT1 (M. xiaojinensis) (GenBank accession No. AAO17059.1) and PbrIRT1 (Py. bretschneideri) (GenBank accession No. XP_009357272.1) in genetic relationship by the evolutionary trees analysis of amino acids. RT-PCR and qRT-PCR analysis of PbIRT1 showed that it expressed mainly in the roots of Py. betulaefolia seedings but almost no expression was found in the leaf or the stem, which suggested a typical expression of root specific. The expression levels in the roots of Fe-deficiency treatment were significantly higher than that of Fe-sufficient treatment. Moreover, the expression levels of PbIRT1 in roots decreased firstly (6 h) and then increased over time under the Fe-deficiency stress and reached the highest at 5 d, and then decreased but remained at relatively high level from 5 d to 15 d, being 3.5~5.5 times as much as in the Fe-sufficient treatment, whereas its expression levels were rapidly inhibited when iron resupplied which reduced by 43.1% compared with 1 d and 63.4% compared with 15 d. The expression levels under the Fe-sufficient treatment remained stable throughout. It could be speculated that the PbIRT1 gene could be induced by iron deficiency stress and was involved in the process of iron uptake and transport. This study can contribute to further investigation for the function of IRT1 and the mechanism of Py. betulaefolia iron-deficiency.

Abstract The aim of the study was to clone the CDS of retinol binding protein 4 gene (RBP4), and to determine its expression in different months old testis of sheep (Ovis aires), which might provide basis for its functional research on testis development. Sheep testis was used as material in this study. The sequence of RBP4 gene was cloned by RT-PCR, and structure of RBP4 protein was analyzed with relevant bioinformatics softwares. The mRNA and protein expression of RBP4 was determined using qRT-PCR and immunohistochemistry, respectively. The results showed that the CDS length of RBP4 gene was 606 bp, and it encoded goat (Capra hircus), cow (Bos taurus), pig (Sus scrofa), human (Homo sapiens) and mouse (Mus musculus), respectively. The sheep RBP4 gene was the closest with that of goat, followed by cow, pig, human and mouse. RBP4 mRNA expressed in all different months old and the expression was firstly decreased and then increased with month increasing. The difference between the adjacent two groups were extremely significant (P＜0.01). RBP4 was mainly localized in the primary spermatocytes, spermatid, spermatozoon and seminiferous tubule. The positive reaction integrated optical density showed a trend of first increasing and then decreasing with month increasing, which was opposite to the expression of RBP4 mRNA. The study result indicated that the RBP4 gene may participate in the control of sheep testis development, which provides reference to further exploration of the influence of RBP4 in sheep reproductive process.

Abstract Swine (Sus scrofa) leukocyte antigen (SLA) gene is one of the genetic factors that cause xenotransplantation cell immune rejection. To enrich the classical experimental animal model and solve the problem of the lack of heterotopic transplantation model, blood samples were obtained from specific pathogen free (SPF) Yorkshire and Landrace for isolation of peripheral blood mononuclear cells, and SLA-1 gene was amplified by PCR after reverse transcription. SLA-1 gene sequences were obtained by analyzing the direct and cloning results. The polymorphism of alleles was analyzed using the bioinformatics software. A total of 16 alleles was identified at SLA-1 gene, containing a full-length 1 086 bp open reading frame. Nucleotide diversity (Pi) was 0.045 59, haplotype diversity (Hd) was 0.000 49, the average nucleotide difference (K) was 49.508, and the content of G+C reached 60.9%. Among 150 nucleotide mutations, the number of nucleotide parsimony informative sites (NPi) were higher than those of the nucleotide singleton variable sites (NS), and 82 of the 361 amino acids were mutated and the numbers of non-synonymous sites (NSS) were obviously higher than those of synonymous sites (SS). The polymorphism of SLA-1 allele was mainly concentrated in exon 2 and 3. Nine of 33 key amino acids were completely conserved, and eight residues showed complete consistence with the corresponding amino acids of human leukocyte antigen (HLA) allele. Eleven of the 19 amino acids, which bind β2-microglobulin (β2m) to HLA-A, showed complete consistency among the alleles identified in this study. The key amino acid binding sites of CD8 to major histocompatibility complex (MHC) were highly consistent in human (Homo sapiens) and SPF Yorkshire and Landrace, except for 225 (Thr/Ser) and 228 (Thr/Met) sites. The result of phylogenetic relationship showed that there was a close relationship among SPF Yorkshire and Landrace pigs, Korean native pigs and Meishan pigs. This study can provide genetic basis for establishment of animal models antigen presentation and immune response.

Abstract S-adenosylmethionine decarboxylase (SAMDC) is a crucial enzyme related to biosynthesis of polyamines (PAs). In this study, the full-length 1 083 bp cDNA sequence of Sesamum indicum (SmSAMDC) (GenBank No. GU983041.1) was amplified from total mRNA of S. miltiorrhiza by RT-PCR and designated as SmSAMDC. Phylogenetic analysis of this deduced protein inferred using the unweighted pair-group method with arithmetic means (UPGMA) method indicated that SmSAMDC was quite similar to SAMDC from other plants, especially S. indicum up to 82%. The SmSAMDC protein showed an obvious homology with other species of plants. To reveal the role of polyamines against drought, the ampli?ed coding sequence of SmSAMDC was overexpressed in tobacco (Nicotiana tabacum). To screen the positive plants, PCR analysis was performed and DNA of the transgenic lines (T0 and T1 plants) was used as templates. RT-PCR analysis was performed using gene-specific primers to screen 5 transgenic plants, and an amplicon of 500 bp was showed. These results clearly demonstrated the overexpression of the SmSAMDC gene in the genome of the transgenic plants. During drought, the growth rates of the wild type and transgenic tobacco significantly decreased, and their development process was also inhibited. However, after 18 d drought treatment, obvious wilting was observed in wild type, meanwhile, much fewer wilting was detected in T1 generation plants of transgenic tobacco from lines S-7, S-8 and S-14 until 18. After rewatering, much more transgenic tobacco plants survived and recovered comparing with the wild type. This results showed that overexpression of SmSAMDC greatly increased the survival rate under drought condition. The physiological study indicated plant lines with SmSAMDC showed a higher relative water content (RWC) and stronger activities of antioxidant enzymes, with lesser contents of malondialdehyde (MDA) and superoxide radical anions (O2·-) compared with the wild type lines under drought condition. To reveal the role of SmSAMDC in preventing membrane injury caused by drought stress, the activities of several antioxidant enzymes, comprising of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were analyzed. And the activities of these enzymes were almost the same between the transgenic lines and wild type in normal growth. However, after 18 d of drought treatment, the activities of SOD and CAT were slightly higher in the transgenic lines than that in wild type, on the other hand, the activities of POD dropped a lot. To further explore the role of SmSAMDC in ROS reduction, the accumulation of O2·- and H2O2 were examined in transgenic lines under drought stress using nitroblue tetrazolium (NBT) and diaminobenzidine (DAB) staining, separately. The staining results indicated that overexpression of SmSAMDC decreased the accumulation of O2·- (NBT staining) and increased H2O2 (DAB staining) under drought stress for 18 d comparing to wild type. Furthermore, the results showed that overexpression of SmSAMDC signi?cantly increased the accumulation of hydrogen peroxide (H2O2) in tobacco under drought stress. These results indicated that overexpression of SmSAMDC increased drought tolerance in tobacco. Furthermore, this is the foundation that SmSAMDC could be applied in other species.

Thinopyrum ponticum (2n=10x=70), which contains excellent resistance and stress tolerance genes to against biotic and abiotic stress, is an important germplasm for wheat (Triticum aestivum) improvement. The development of novel Tr. aestivum (common wheat)-Th. ponticum derivatives will provide valuable germplasm to wheat resistance breeding programs. CH1115-B15-1-5-1-1 (CH1115-B15), a novel disomic addition line from the cross of 7182 (Tr. aestivum)/Th. ponticum//87-1-9 (Tr. aestivum), was characterized by cytological identification, morphological analysis, salt tolerance analysis, genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) analysis, simple sequence repeat (SSR), expressed sequence tag (EST) and PCR based landmark unique gene (PLUG) markers analysis. Cytological observations showed that CH1115-B15 contained 44 chromosomes and formed 22 bivalents at meiotic metaphase Ⅰ and the addition line was stable in morphology and cytology. GISH and FISH analysis suggested that, of 44 line chromosomes, 42 chromosomes were common with wheat chromosomes, and 2 chromosomes came from Th. ponticum chromosomes. In addition, the pair of alien chromosomes carried on similar Oligo-pTa535 signals. GISH analysis showed that the pair of alien chromosomes belonged to the genome J(E). The polymorphic analysis with 2 SSR markers, 4 EST markers, and 4 PLUG markers showed that the pair of alien chromosome belonged to the fifth homeologous group. Salt tolerance analysis at the germination stage and evaluation of adult stripe rust resistance showed that line had high resistance to wheat stripe rust (IT=1) at the adult stage and excellent salt tolerance at the germination stage. Therefore, CH1115-B15 is Tr. aestivum- Th. ponticum 5J(E) disomic addition line with excellent stripe rust resistance and salt tolerance, and it could be exploited as a promising germplasm in wheat breeding and chromosome engineering.

Abstract Milk fat is an important part of milk. The main feature of yak (Bos grunniens) milk is high fat rate. Diacylglycerol acyltransferase 2 (DGAT2) plays an important role in triacylglyceride synthesis for the milk and is an important candidate gene for milk fat traits. In this study, the polymorphism of DGAT2 gene in Gannan yak, Tianzhu white yak and Datong yak were detected with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and the effects of mutations on milk quality traits were analyzed in Gannan yak. The results showed that 4 SNPs were identified, including c.613-214 A＞G and c.613-337 C＞A in intron5 and c.787＋616 G＞A and c.787＋691 T＞G in intron6 which led to six alleles (A1, B1, C1 in intron5 and A2, B2, C2 in intron6) and two regions were moderate polymorphism in yak populations. Seven haplotypes were found and 4 SNPs were linked but closed to linkage equilibrium between intron5 and intron6 of yak DGAT2 gene. In intron5 of Gannan yak DGAT2 gene, individuals possessing the allele B1 had higher milk fat rate than those no- possessing in second parity yaks (P＜0.05). In intron6 of Gannan yak DGAT2 gene, individuals with alleles A2 showed higher milk fat rate and total solid content than those no-possessing and significant effects were also found in second parity yak (P＜0.05), which indicated that selecting those yaks with allele A2 had the tendency of increasing the milk fat rate and total solid content in offspring groups. The second parity yaks with genotype A2C2 showed higher protein rate than those with genotypes A2C2 and B2B2 (P＜0.05). Gannan yak with haplotype combinations H1H3 had the higher milk fat quality traits than others. The mutations in intron5 and intron6 might be used as genetic markers to improve milk quality traits in Gannan yak. This study has accumulated candidate genes of molecular genetic research data for Yak milk traits.

Abstract Longping206 (Zea mays), a spreading hybrid in south areas of the Yellow River, Huai and Hai Rivers, with conspicuous growth heterosis and higher yield production, provides us an opportunity to explore the potential molecular mechanism for heterosis. In this study, the new technology Illumina Hiseq2500 was applied to explore the transcription level in sixth leaves stage between Longping206 and its parents (L7221 and L239). Gene expression level and function prediction were analysed with a lot of bioinformatic methods. From the comparison of phenogram, the hybrid was stronger than its paternal line in sixth leaves stage. The measurement of biomass had the same results with the phenogram. After the RNA-Seq sequencing, approximately 12.52 Gb clean data was generated after removing low-quality read. At least 69% read could be mapped into the reference genome of maize B73 with the software Tophat2. With the module Cuffdiff of Cufflinks, gene expression quantity was obtained. Then EBSeq software was used to calculate the differential expression. A total of 3 005 differentially expressed genes (DEGs) was discovered between Longping206 and its parental lines. All 1 788 DEGs were up-regulated in hybrid with 925 DEGs were down-regulated. To calculate the gene expression level in heterosis, traditional quantitative genetic parameters were used as the quantitative traits. To classify the function of DEGs, Gene Ontology (GO) analysis was used. In total, 2 399 of 3 005 DGHP (different expression genes in hybrid and parents) were assigned to at least one term in GO database including molecular function, biological process and cellular component. Most of important biological processes were enriched in hybrid such as hormone signal conditioning, energy metabolism, materials transport, stress threaten etc. To identify the metabolic pathways which the DGHP were involved and enriched, Kyoto Encyclopedia of Genes and Genomes (KEGG) database was performed to analyze the pathway. The results showed that at least 255 DGHP were classified into one functional category. 5.88% and 8.24% of these DEGs were involved in the carbon metabolism and plant signal transduction pathways, respectively. Interestingly, most of the DEGs were up-regulated, which revealed that the two ways were important to the formation of heterosis. qRT-PCR was performed to validate the consistence of transcriptome analysis with 10 random different expression genes. The extensive transcriptome data which was obtained from high-throughput sequencing given the comprehensive overview of the leaf transcriptomes at Longping206 and its parents in a heterotic maize cross. This study provides a useful resource for molecular mechanisms of the maize heterosis research.

Abstract Polyphenol oxidase (PPO) is a class of copper-containing oxidoreductase, and it is resistant to phytophagous insects or pathogens. In order to study the insect resistance of GhPPO1 gene in cotton (Gossypium arboreum), the gene silencing recombinant vector pTRV2-GhPPO1 was constructed, and was transformed into Agrobaoterium tumefaciens GV3101 by virus-mediated gene silencing (VIGS). The cotton plants were fed to 2rd instar larvae of cotton bollworm (Helicoverpa armigera), and the expression of GhPPO1 gene in cotton plants after GhPPO1 gene silencing was obtained by qRT-PCR. The results showed that the expression of GhPPO1 was inhibited by Tobacco rattle virus (TRV) system, and the expression of GhPPO1 gene was only 20% compared with the control group (empty vector pTRV0) (P＜0.05), and the GhPPO1 gene in cotton had a significant silence effect. Compared with cotton bollworm which was fed with common cotton leaf, the weight of the cotton bollworm fed with cotton leaf which was treated by gene silencing increased by 86% after 72 h, which indicated that GhPPO1 was a kind of defense mechanism gene in cotton. The expression of GhPPO1 in the directly induced leaves with cotton bollworm was higher than that of the indirect induced leaves. The results showed that the ability of cotton plants to direct defenses was weakened, and weight of the cotton bollworm increased. It was speculated that GhPPO1 gene might be involved in cotton direct defense response. This study has important significance for the role of GhPPO1 in cotton herbivorous insects, and lays the foundation for the relationship between cotton bollworm and cotton induced defense.

Abstract Chrysanthemum (Chrysanthemum morifolium) has been cultivated for a long history in China as an important ornamental plant, as well as plants of culinary and medicinal. Chrysanthemum tea has become one of the first choice of tea for health and leisure due to its beautiful shape and multiple medical effects, such as heat-clearing, detoxicating, calming the liver, improving eyesight and other efficacy. Chrysanthemum cultivar C. morifolium 'Zhongnong Xiazhuang' was bred by China Agricultural University, and can be used as courtyard landscaping and colorful tea variety. It was selected by the conventional hybridization method from the cross of male parent C. morifolium 'Fenling' and female parent C. morifolium 'Zhongnong Hongxiu'. This cultivar was suitable for application in most areas of China, with high plant, beautiful flowers, bright color, and strong comprehensive stress tolerance. The breeding process, main characters, cultivation techniques and application value of the colour tea cultivar of chrysanthemum ‘Zhongnong Xiazhuang’ were described in this paper.