Heat shock factor (Hsf), a kind of transcription regulation gene family in plants, plays an important role in response to high temperature and other vital movement. In this study, Hsf were identified using tomato (Solanum lycopersicum) whole-genome sequencing results. Multiple sequence alignments were performed using the Cluster W and Bioedit program. A phylogenetic tree was created using the MEGA5 program. Chromosomes were located by MapDraw program. Expression pattern of Hsf gene family at different development stages and in response to high temperature were analyzed based on the existing microarray database and qRT-PCR. Results showed that the tomato Hsf gene family contained 25 members. Sequence alignment showed that these members had a DNA binding domain (DBD) of specific transcription factor. Transcription factors from Arabidopsis thaliana, rice(Oryza sativa) and cucumber(Cucumis sativus L.) were divided into three branches (A, B and C), and further subdivided into 15 categories (A1~A9, B1~B4 and C1~C2). Chromosomal localization found that the Hsf unevenly were distributed in the ten of twelve tomato chromosomes except Chr.1 and Chr.5. Most of the Hsf genes had differential expression pattern and response to high temperature at seedling stage. The results provide foundation for revealing function of Hsf genes in tomato.

Ursolic acid (UA) is an important active component in loquat (Eriobotrya japonica L.), and squalene epoxidase (SQE) is one of the key rate-limiting enzyme in pentacyclic triterpenoids biosynthesis, such as UA. To clone SQEs gene, find the suitable stress temperature in loquat suspension cultured cells, and further to find out the correlation between the different expression of SQEs and the content of UA under suitable stress temperature, cells of Eriobotrya japonica L. in the late logarithmic growth phase of cell suspension had been chose. Both the cell growth and the accumulation of UA were investigated at the low temperature of 15 ℃ and the high temperature of 35 ℃ treatments. At the same time, the homology-based cloning method was employed to determine the different transcription levels of two genes (ejSQE1 and ejSQE2) at 15 ℃ treatment. The amino acid sequence similarity between ejSQE1 (GenBank accession No. JQ294053.2) and ejSQE2 (GenBank accession No. JQ294054.2) was 69.89%, and the significant differences between sequences were in the heads and tails. The results indicated that the total UA production of the 48 h at 15 ℃ treatment was the highest of all. Moreover, the expression of ejSQE1 and ejSQE2 peaked in 48 h treatment, and expressions of both genes declined significantly in 24 h treatment at 15 ℃(P＜0.05). The results revealed that there was significantly positive correlation between expressions of ejSQE1 and ejSQE2, as well as expression of ejSQE2 and accumulation of UA with any treatment longer than 24 h at 15 ℃. This study reveals the gene sequences and expression characteristics of SQEs under stress temperature, and provides the foundations for the further researches on UA regulatory mechanisms in loquat cell suspension cultures.

MADS-BOX gene family is one kind of important transcription factors which regulates multiple developmental processes in plant, and AGAMOUS LIKE 6(AGL6) and FRUITFULL(FUL) genes belong to its subfamily. In this study, cDNA sequences of two Brachypodium distachyon MADS-BOX genes BdAGL6 and BdFUL1 were cloned. Furthermore, the alternative splicing and expression pattern of both genes were analyzed. As a result, three BdAGL6 transcripts were cloned. Further analysis showed three isoforms were generated by alternative splicing through intron retention mechanism. BdAGL6-3 was conservative with AGL6 transcription factors in rice (Oryza sativa) and maize (Zea maize). Compared with BdAGL6-3, additional 7-aa and 5-aa were inserted in the C domain of BdAGL6-1 and BdAGL6-2, respectively. BdAGL6 strongly expressed in flowers, and the expression strength of BdAGL6-3 was the strongest while the expression strength of BdAGL6-1 and BdAGL6-2 were weaker. Meanwhile, alternative splicing also occurred in BdFUL1 too, and two transcripts of BdFUL1-1 and BdFUL1-2 with similar expression pattern were generated, and both of them expressed in flowers, stems and root of seedlings. These findings provide data for further functional elucidation of these Brachypodium distachyon MADS-BOX gene family.

At present, the evolution mechanism of Lycoris radiata genome is unclear and retrotransposons serve as the main source of plant genome evolution. Ty1-copia retrotransposon reverse transcriptase gene (RT) fragments were cloned from genomic DNA of diploid (2n=2x=22) and triploid (2n=3x=33) L. radiata, respectively, using universal primers of Ty1-copia retrotransposon RT, and then analyzed by the relevant bioinformatics softwares in this study. Sixteen (2RA1~2RA16, GenBank accession: KP275626~KP275641) and 17 (3RA1~3RA17, GenBank accession: KP275642~KP275658) RT sequences were cloned from diploid and triploid L. radiata, respectively. The length of these sequences ranged from 248 to 266 bp. The similarity of RT nucleotide sequences from diploid L. radiata ranged from 49.05% to 87.83%, while that from triploid L. radiata ranged from 40.75% to 89.73%. And the similarity of RT inferred amino acid sequences from diploid L. radiata ranged from 18.60% to 91.95%, while that from triploid L. radiata ranged from 18.07% to 88.51%. The results showed that 33 RT sequences obtained from L. radiata had polymorphism and high heterogeneity, and the difference of RT sequences among triploid L. radiata was larger than that among diploid L. radiata. Phylogenetic trees, which were constructed by MEGA 4.0 using the Neighbor-Joining method, indicated that Ty1-copia retrotransposons in L. radiata happened not only horizontal transmission, but also vertical transmission. In addition, there was no direct correlation between classification and ploidy. Compared with other plants, it was found that they had higher homology with herbaceous plants: Volvox carteri from algae, Dicranum scoparium from bryophytes, Osmunda cinnamomea and Equisetum scirpoides from fern, and monocotyledonous plants (Zea mays, Triticum aestivum, Equisetum scirpoides and Dendrobium officinale), and dicotyledonous plants (Arabidopsis thaliana, Lycopersicon esculentum, Populus ciliate, Picea abies, Beta procumbens and Glycine max). Moreover, 2RA16 from diploid L. radiata was the most ancient Ty1-copia retrotransposon RT sequences. Diploid L. radiata was likely to be the primitive group in L. radiata complexes, and triploid L. radiata was proved to be homologous triploid. This study is helpful for understanding intra- specific evolution of L. radiata and the origin of triploid L. radiata, and it provides scientific evidences for further revealling karyotype evolutionary mechanism and theoretical foundation for polyploid breeding of L. radiata in the future.

Two peach (Prunus persica) cultivars Qinwang (with good storage property) and Shahong (with poor storage property) fruits harvested at commercial maturity were investigated. We analysed the difference of ethylene production through gas chromatography approach, and the genes expressions involed in ethylene biosynthesis and fruit softening between the two peach cultivars by quantitative Real-time PCR PCR (qRT-PCR). The results showed that Qinwang peach remained high firmness during storage accompanied by a quite low expression level of ACC synthase gene (1-aminocyclopropane-1-carboxylic acid synthase gene, PpACS1) and ethylene production rate. However, Shahong softened quikly, and the relative expression of PpACS1 was increased significantly, along with lot of ethylene produced. ACC oxidase gene (1-aminocyclopropane-1-carboxylic acid oxidase gene, PpACO1) expression did not increased in Qinwang while presented rising trend during Shahong softening. In Qinwnag, the expression of pectin methylesterase gene (PpPME) and polygalacturonase gene (PpPG) were extremely low, however, it increased continuously during Shahong softening. In addition, the expression level of expansin gene (PpEXP) was very low in Qinwang compared with Shahong. The gene expression of N-glycan processing enzymes α-mannosidase gene (Ppα-Man) and β-hexosaminidase gene (PpHex1 and PpHex2) expression level were increased in both of Shahong and Qinwang, but the expression in Shahong were significantly higher than that in Qinwang (P＜0.05). These results indicated that the delay of Qinwang fruit softening was due to lack of enough ethylene production by limited expression of PpACS1, which caused PpPME and PpPG expression and enzyme at lower level, unable to take the role in pectins degradation. However, Shahong softened rapidly as a result of production of ethylene, thus, PpEXP, PpPME and PpPG were induced and pectins degradated. Ppα-Man, PpHex1 and PpHex2 maybe not key factors acting on Qinwang softening. This research indicates mechanism of peach fruit ripening and softening, and provides the basic evidence for the long shelf-life peach cultivar breeding.

In order to analyse the situation of gene flow(Nm) and the degree of genetic differentiation, Hubei black-boned goat(Capra hircus) and other 3 geographical closely breeds of Youzhou black goat, Macheng black goat and Nanjiang yellow goat, as well as Boer goat as out group breed, were chosen in the preset research. Allele heterozygosity(h), Shannon-Weiner index(S) and the efficient allelic number(Ne) which can reflect population genetic diversity were calculated using 14 single nucleotide polymorphism (SNP) markers of the 5 goat breeds. Meanwhile, the situation of gene flow and genetic differentiation between Hubei black-boned goat and the other 3 local goat breeds were also analyzed, with the linear model relationship establishment between geographical distance and genetic differentiation. Moreover, the clustering figure of the 5 populations was built using the genetic distance (Ds) data. The results showed that Hubei black-boned goat had the highest value of both allele heterozygosity (h), Shannon-Weiner index (S) and the efficient allelic number (Ne) which suggested Hubei black-boned goat had the most abundant genetic variation. The also finding was that the genetic differentiation(Fst) was medium which ranged from 0.072 9 to 0.137 2 between Hubei black-boned goat and other 4 goat populations, but the gene flow(Nm) was large of which ranged from 1.571 6 to 3.178 7. Unweighted pair-group method with arithmetic means(UPGMA) based clustering figure showed that Hubei black-boned goat was put the outermost of the graph for large genetic differentiation. Otherwise, there was positive linear relationship between genetic differentiation and geographical distance, genetic distance(Ds) and geographical distance(D) also had correlation relationship (r=0.934). There were significant genetic differentiation between Hubei black-boned goat and the other 3 goat populations and Hubei black-boned goat had the most abundant genetic variation, but the large gene flow was adverse for this germplasm resources. So it is necessary to take some measures to prevent germplasm genetic resources from loss by hybridization and dilution. The result provides some clues for conservation and breeding of Hubei black-boned goat.

Piriformospora indica, isolated from Indian Thar desert, s identified as a plant root endophytic fungus which plays an important role in inducing resistance against biotic and abiotic stresses. Recently, P. indica has been regarded as a fungal model to study the interaction between endophytic fungus and plants. In order to study the influence of P.indica on black shank resistance in Nicotiana tobacum, we analyzed the differences of the incidence, antioxidant enzyme activities, including phenylalnine ammonialyase (PAL), peroxidase (POD) and polyphenol oxidasei(PPO), and the expression levels of disease resistance-related genes in P.indica-colonized and non-colonized plants after Phytophthora parasitica var. nicotianae inoculation. We also studied whether P.indica influenced the growth of P. parasitica var. nicotianae by antibiosis assay. The results showed that: (1)The disease severity of P. indica-colonized N. tobacum was significantly lower than that of non-colonized ones; (2) After P. parasitica var. nicotianae inoculation from 1 to 10 days, the activities of PAL, POD and PPO in P. indica-colonized tobacco were significantly higher than those in non-colonized plants. P. indica-colonized plants showed 3.99-fold greater PAL activity in comparison with controls after P.parasitica var. nicotianae inoculation for 1 day. Meanwhile, compared with controls, POD and PPO activity in P. indica-colonized plants were elevated by 1.5- and 2.26-fold after infestation with P. parasitica var. nicotianae for 2 and 4 days, respectively; (3) Real-time quantitative PCR (qRT-PCR) results showed that the expression levels of disease resistance-related genes (PR1b, P450-2, hrs203J, GST, NmIMSP and P450-1) were up-regulated in P. indica-colonized N. tobacum after P. parasitica var. nicotianae inoculation at 6 to 96 h, respectively. The expression levels of PR1b, P450-2, hrs203J were up-regulated by 3.8, 15.7 and 3.1 folds in P. indica-colonized N. tobacum compared with controls after P. parasitica var. nicotianae inoculation for 12 h, respectively. The NmIMSP expression reached a level of 2.6-fold higher in P. indica-colonized plants in contrast to the controls after P. parasitica var. nicotianae inoculation for 48 h. The P450-1 was 1.5 folds of the control ones after P. parasitica var. nicotianae inoculation for 72 h. (4) The antibiosis assay showed that P. indica didn’t inhibit P. parasitica var. nicotianae growth. Thus, the resistance induced by P.indica against P.parasitica var. nicotianae in N.tobacum was not dependent on the antibiotic secretion, but on the up-regulation of resistance-related genes and improved antioxidant enzyme activity in N. tobacum. This study reveals the role of P. indica conferring N. tobacum disease resistance against P. parasitica var. nicotianae and correlative mechanism, and provides a new way for the biological control of black shank.

Baicalin has many beneficial effects clinically such as heat-clearing bacteriostasis and anti-inflammatory. Its effects on improving the heat resistance of bovine(Bos taurus) sertoli cells(SCs) were studied by measuring expressions of glial cell line-derived neurotrophic factor(GDNF) and stem cell factor(SCF) of SCs treated with different concentrations of baicalin. Collagenase and typsin were sequentially used to separate SCs from calf testis, and then the differential adhesion method was used to purify the SCs(4 h). The obtained SCs were cultured in vitro and their GDNF and SCF mRNA expressions and satellite karyosomes were identified by RT-PCR and Feulgen staining, respectively. The SCs of logarithmic phase growth were selected and divided into different groups including control group(37 ℃), 42 ℃ heat stress group and 42 ℃ heat stress plus baicalin(0.1, 1, 10 and 20 μg/mL) treated group. The cell survival rate was observed with methyl thiazoletetrazolium(MTT) assay. The Real-time quantitative(qRT-PCR) and enzyme linked immunosorbent assay(ELISA) results showed that both marker genes of GDNF and SCF and special satellite karyosomes in SCs were all detected. The cell survival rate(P＜0.01) and the expressions of GDNF and SCF mRNA(P＜0.01) and protein(P＜0.05) reduced in the SCs in 42 ℃ heat stress group compared with the control group(37 ℃). However, compared with 42 ℃ heat stress group, the cell survival rates in the heat stress group treated with 1 μg/mL(P＜0.05) and 10 μg/mL(P＜0.01) of baicalin increased; the expressions of GDNF and SCF mRNA in the 42 ℃ heat group treated with 0.1~20 μg/mL baicalin very significantly(P＜0.01) increased, and so did the protein expressions of SCF(P＜0.01) and GDNF(P＜0.05) with 1 μg/mL baicalin, respectively. The results indicated that baicalin increased SCs survival rate under heat stress and improved mRNA and protein expressions of both SCF and GDNF. The present research revealed some heat-resistance mechanism of baicalin on SCs under heat stress, which provides theoretical basis for improving the cell resistance to heat shock in practice.

Arming at screening mesophilic and high temperature resistant microbes of highly efficient cellulose-degradation and constructing composite strains for application as compost inoculant, the microbes were isolated using cellulose Congo red selective medium under room and high temperatures from humus soil and compost samples. Detections of carboxymethyl cellulase(CMCase) activity, filter paper degradation and degradation ability of rice straw and radix(Sophorae flavescens Ait.) residues under liquid and solid state fermentation conditions of isolated microbes were performed, and highly efficient microbes which were used to construct the composite strains were selected. The finally constructed lignocellulose degradation composite strains of high efficiency, which was determined by antagonism and single strain contribution among the composite strains analysis, contained 3 bacteria(Beijerinckia fluminensis, Microbacterium sp. and Bacillus sp.), 1 Streptomyces sp. and 1 Chaetomium sp.. Results showed that the degradation effects on rice straw and radix residues were stronger by the tested microbes under liquid state fermentation than in the solid state fermentation. The rice straw was more likely to be degraded by most of the tested strains than the radix residues, and the fungi species showed greater effects among the tested microbes in the rice straw and radix residues degradation. The Beijerinckia fluminensis strain XM-3 degraded the filter paper stripe completely within 48 h. The composite strains degraded 31.4% and 63.1% of the radix residues and rice straw after 20 d of solid state fermentation. This study provides reference for the development of compost inoculants.

As a main aquatic vegetable in southern China, Zizania latifolia could be affected by temperature in the field. The two double-cropping cultivars of Z. latifolia were comparatively analyzed by the method of two-dimensional electrophoresis (2-DE), including the chilling tolerance cultivar of Longjiao 2 and the main cultivar of Zhejiao 911. Among the 822 to 955 protein spots of leaves, differential expression protein spots were selected and analyzed. It was found that the cultivar of Longjiao 2 contained more up-regulated spots and fewer down-regulated spots than that of Zhejiao 911 under the treatments of low temperate and recovery, respectively. Six differential expression chilling tolerance protein spots were successfully identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF-TOF MS), including IAA auxin amido hydrolase (IAA-AH), putative formate-tetra-hydrofolate ligase (FSH), malate dehydrogenase (MDH), mannose-6-phosphate reductase (M6PR), ATP synthase CF1 alpha chain (ATPase) and the molecular chaperon cpn60-1, which were related to the plant hormone metabolism, nucleic acid synthesis, energy metabolism, photosynthesis and protein folding, respectively. The expression trend of IAA-AH, MDH and the molecular chaperon of cpn60-1 were similar in the two cultivators of Longjiao 2 and Zhejiao 911 with increasing expression in the treatments of low temperature and decreasing expression in the following treatments of recovery, while the other three were expressed differently between the two cultivators. Especially in the treatments with the temperate of 5 degrees, there were more steady changes of expression and better recovery in the cultivator of Longjiao 2 than that in Zhejiao 911. Based on transcriptome sequencing of Z. latifolia, parts of the coding sequences of 6 differential expression proteins were cloned and the changes of expression were analyzed by Real-time PCR. In this research, the proteins related to chilling tolerant of Longjiao 2 were identified and discussed, which could be contributed to the illumination of the special features of Longjiao 2 and chilling tolerance. The results would provide theoretical basis to the breeding of Z. latifolia cultivars which could be harvested in different times.

miR-148a is a member of the miR-148/152 family, which participate in the regulation of many biological processes, such as cell proliferation and differentiation, cancer formation, and so on. In the present study, the expression of miR-148a was detected in Jinhua pig (Sus scrofa) liver at various developmental stages by qRT-PCR. The result showed that its expression was abundant in embryonic period and became lower as the days increased after birth. To understand its biological functions and processes, GO (Gene ontology) annotation and KEGG (kyoto encyclopedia of genes and genomes) pathway analyses were subsequently performed on miR-148a. Bioinformatics comprehensive analysis indicated that miR-148a participated in the regulation of liver growth and development, especially in cell proliferation and differentiation. To confirm the putative miR-148a target genes, the expression changes of ten predicted genes were quantitatively analyzed at mRNA in over-expressed miR-148a mouse (Mus musculus) embryonic hepatocytes. The results revealed that over-expression of miR-148a could significantly reduced the mRNA expression of CCAAT-enhancer binding protein-alpha gene (Cebpa) (P＜0.05). Moreover, the expression of miR-148a and Cebpa also showed an inverse correlation in Jinhua pig liver at various developmental stages. These results suggested that miR-148a might reduce the expression of Cebpa to regulate the balance between cell proliferation and differentiation in liver development.

Porcine (Sus scrofa) SARM1 (sterile-α and TIR motif containing protein 1) plays an important role in innate immune response during Porcine reproductive and respiratory syndrome virus (PRRSV) infection. Establishment of a MARC-145 cell line expressing Porcine (Sus scrofa) SARM1 is an important step to study function of porcine SARM1 gene. In the present study, the recombinant expression vector (pEGFP-N1-pSARM1) was successfully constructed, and transfected into MARC-145 cells using LipofectamineTM 2000. Fluorescence imaging using confocal microscope demonstrated localization of porcine SARM1-GFP protein in mitochondrion of MARC-145 cells. The stably transfected MARC-145 cell line was screened with 800 μg/mL of G418 for 6 d. The monoclonal cell was acquired by limiting dilution from the polyclonal mass of stably transfected cells. The stably transfected MARC-145 cell line had normal morphology and exhibited strong green fluorescence. After continuous passage culture, green fluorescence in stably transfected cell line could be observed. Porcine SARM1 and SARM1 protein stably expressed in stable transfected cell line by reverse transcription PCR and western blot, respectively. 0.1 multiplicity of infection (MOI) of PRRSV was used to infect stably transfected cell line to test whether this cell line could support PRRSV replication in vitro. Result of qRT-PCR showed that PRRSV ORF7 expressed in stably transfected cell line at 12 hour post infection (hpi), and the expression of ORF7 at 12, 24 and 48 hpi was decreased extremely significantly compared with that in normal MARC-145(P＜0.01). 50% tissue culture infective dose(TCID50) assay also showed viral load of PRRSV in stable transfected cell line was lower. The study provides foundation for investigating role of porcine SARM1 in immune regulation with PRRSV infection in future.

Appropriate reducing agent is very crucial for complete dissolution of protein and better results in two-dimensional gel electrophoresis (2-DE). Tributyl phosphine (TBP) is considered to be a more effective reducing agent than DL-Dithiothreitol (DTT) in general. In this study, we detailly compared DTT with TBP in term of 2-DE map quality and protein spot distribution of total proteins and sarcoplasmic proteins from Qinchuan cattle (Bos taurus) longissimus muscle tissue for the first time. The result implied that the reducing agent of DTT produced a higher resolution 2-DE gels with a clearer background than TBP. For the total proteins, DTT extracted more proteins with molecular weight ＞20 kD and protein volume ＜1 000 than TBP. For the sarcoplasmic proteins DTT promoted extraction of proteins with molecular weight＞50 kD and protein volume＜5 000 compared with TBP. DTT was more appropriate to 2-DE of Qinchuan cattle longissimus muscle tissue than TBP and this result wasn't consistent with traditional view. Further more, we increased TBP concentration and gave up the use of thiourea and the result wasn't improved. Through centrifugation protein precipitations appeared at room temperature. The short half-life of TBP might result in the poor effect in 2-DE. This study offers technical support for sample preparation and the proteomics research of Qinchuan cattle muscle tissue.

Canine distemper virus (CDV), which causes fever, diarrhea, even death of Canidae and other animals, is a pathogen hazards of infectious diseases. It is important to diagnosis and control diseases using rapid and sensitive method of detecting CDV. In the work, M gene of CDV was cloned by RT-PCR from cDNA of clininal samples. The plasmid of pET-28(a)-CDV-M was constucted and identified by PCR and restricttion endonucleases digestion. The recombinant protein CDV-M were expressed in Escherichia coli by IPTG induction. The antigenicity of CDV-M were identified by Western blot. The purified recombinant protein his-CDV-M were coated as antigen, the optimal protein coating concentration was determined by ELISA square tests. The indirect ELISA method for CDV-M antibody was established and applified to detection of clinical canine serum samples. The specificity and sensitivity were completed by comparison tests of this test method and other kits. The CDV-M were expressed in E. coli BL21 (DE3). The purified protein concentration was 2.840 mg/mL. Square test showed that the optimum coated concentration of antigen was 8.88 μg/mL and the optimum dilution of antibody was 1∶160. Using this method, 223 clininal samples was detected, and the result showed that coincidence rate was 96% compared with import diagnostic kit. This study established the indirect ELISA based on M protein and can provide tools for CDV diagnosis and M protein function research.

As the aggravation of the greenhouse effect, global warming has become a serious change for the modern animal husbandry and caused billions of dollars of losses annually around the world. Elevated temperature has become the major abiotic stress affecting animal growth. During heat shock, organisms experience orchestration of a multitude of biochemical and physiological changes to acquire heat resistance, and most of these depend on changes of heat shock proteins (Hsps). Therefore, this paper aimed to state the mechanism of the heat shock signal transduction that the Ca2+-CaM involved in. The concentration of intracellular free Ca2+ is increased by heat stress, and CaM is activated by the increased Ca2+, then CaM function on the 2 downstream target proteins that effect the transcriptional activity of HSF1 to regulate the expression of Hsp70. There are 2 specific pathways participating in the expression of Hsps: ①The transcriptional activity of HSF1 is increased by the phosphorylation of HSF1 (Ser230), which was activated by CaMKⅡ that one of the downstream target proteins regulated by Ca2+-CaM; ② During heat shock, HSF1 is released, and activated by the compound of Hsp70-CaM for that Hsp70 is another downstream target proteins regulated by Ca2+-CaM, HSF1 is converted from a transcriptional inactive monomer to active trimmer that is capable of binding to HSE and exhibits transcriptional activity, and finally induced the expression of Hsp70. The regulation activity of the HSF1 is the central mechanism of transcriptional regulation for Hsps gene expression. This paper reviewed the mechanism of the Ca2+-CaM-HSF1-Hsp70 signal transduction system in reception, transduction and response to heat stress.