Sucrose metabolism plays important roles in the development of plant sink organs, particularly in the process of fruit-setting and fruit development. Invertase (INV; EC 3.2.1.26) hydrolyzing sucrose into glucose and fructose is a key player in sucrose metabolism. Through exploring the correlation between INV enzyme activity, related genes expression characteristics and parthenocarpy, this study was to provide candidate genes and a theoretical basis for understanding the molecular mechanism of sucrose metabolism regulating plant fruit-setting. A parthenocarpic (CP28) and a non-parthenocarpic (CL26) cherry tomato (Solanum lycopersicum var. cerasiforme) inbred line were taken as materials to investigate the INV activities, sugar contents and INV genes expression patterns in buds, flowers and fruits under different developmental stages. The results indicated that cell wall invertase (CWIN) activities of buds and flowers were higher than those of fruits in both CP28 and CL26 lines, and moreover, the CWIN activity of CP28 was significantly higher than that of CL26 in middle bud stage (P＜0.05). The expression patterns of genes encoding tomato CWIN (Lin5, Lin6 and Lin7) and CWIN inhibitor (SlINVINH1) in buds and flowers of both CP28 and CL26 lines were analyzed by qRT-PCR. The results showed that the relative expression levels of Lin6 in buds and flowers of CP28 line were about 93~ 205 times of those of CL26 line, i.e. significantly higher than those of CL26 line (P＜0.01). Meanwhile, the expression patterns of Lin5, Lin7 and SlINVINH1 showed no significant difference between the 2 lines. Taken together, these results indicated that Lin6 was likely the key tomato CWIN gene taking part in the regulation of tomato fruit parthenocarpy through sucrose metabolism. This finding provides helpful information for further understanding the molecular mechanism of CWIN participating in the regulation of parthenocarpy in tomato fruit.

The study aimed to screen endophytic diazotrophs with high capability of phosphate solubilizing and potassium decomposing from Oryza officinalis. The endophytic diazotrophs with high capability of phosphate solubilizing and potassium decomposing properties potentially supplied the foundation for the future agricultural application. The endophytic diazotrophs isolated from the O. officinalis were evaluated by using 2 nitrogen-free selective media in combination with the acetylene reduction method, the capability of phosphate solubilizing and potassium decomposing. The isolated endophytic diazotrophs with high efficient capability of phosphate solubilizing and potassium decomposing were grouped by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the whole-cell protein electrophoresis. The representative strains were further studied by physiological and biochemical tests, and 16S rRNA gene sequencing analysis. The soluble phosphorus and decomposing potassium content in the fermentation liquid were determined by molybdenum antimony colorimetric method and 4 benzene boron sodium assay, respectively. The possible promotion of rice growth was also carried out. The nitrogenase activity of 4 isolated endophytic diazotrophs ranged from 8.78~8.88 μmol C2H4/(mL·h). The 4 strains were assigned to one group by analysis of SDS-PAGE patterns of the whole-cell protein electrophoresis. 16S rRNA gene sequencing analysis and physiological and biochemical tests of the representative strain yy01 showed that the strain was closely related to Burkholderia kururiensis. The inorganic phosphorus solubilizing ability (116.28 mg/L) of strain yy01 was 2.73 times as much as the reference strain Klebsiella variicola, and potassium decomposing activity (268.31 mg/L) was 4.67 times as much as the the reference strain K. variicola within 5 d fermentation in the liquid medium. In addition, the strain yy01 could also secrete indole-3-acetic acid. Strain yy01 inoculated with rice showed significant promotion rice growth, the length of rice leaf increased of 25.9%, the length of root increased of 42.30%, tiller number increased of 79.60%, fresh weight increased of 166.90%; The N, P and K content of rice increased of 61.54%, 41.18% and 54.05%, respectively. Based on above results, these 4 endophytic diazotrophs with high capability of phosphate solubilizing and potassium decomposing isolated from the O. officinalis have the potential application in agriculture production.

Muscle satellite cells are potential myogenic cells capable of self-renewal and with potential multi-directional differentiation. Under normal circumstances, these cells are in resting state, but they can proliferate, differentiate and integrate into myotubes and form muscle cells. However, in vivo, these mesoderm originated satellite cells can not trans-differentiate into endoderm-derived insulin-producing cells. In this study, a muscle satellite cell line was isolated, cultured, identified and established from a bovine (Bos taurus) fetal muscle tissues. The muscle satellite cells were then induced to differentiate into insulin-producing cells. During pancreatic induction process, the genes associated with pancreatic development such as pancreatic and duodenal homeobox 1 (PDX1), neurogenin 3 (NGN3), Amylase and Insulin (INS) were analyzed. PDX1, an important transcription factor in differentiation of pancreatic islets, started to express after induction 2 d, reached to the highest level after 3 d, and kept lowest level after 4 d. NGN3 was an important transcription factor in the formation of endocrine cells, and was a key gene that controlled insulin secretion to extracellular, which the mRNA started to express after induction 3 d, reached to the highest level after 4 d and then declined. As a pancreatic extracellular secretion associated gene, Amylase mRNA appeared after 6 d and reached to the maximum after 8 d. INS expressed from 1st day and reached to the maximum at 11~12 d. Enzyme-linked immunosorbent assay (ELISA) of the culture media showed that the concentration of insulin secreted by the cultured cells reached to the maximum at 11~12 d. The insulin secretion was significantly increased in the glucose added culture medium, and glucose concentration regulated insulin secretion in the cultured cells, which was similar to adult islet fuction. In conclusion, the bovine muscle satellite cells could be trans-differentiated into pancreatic cells, and make response to glucose stimulation. The study provides basic data for further research and clinical application.

Basic leucine zipper (bZIP) transcription factor family widely distributes in plant and involves in multiple stress response and developmental stages. In this study, we cloned a heat induced transcription factor gene TabZIP28 (GenBank accession No. KT753298.1) from wheat (Triticum aestivum), the length of ORF was 1 713 bp, encoding a protein of 570 amino acid residues. Sequence analysis indicated that TabZIP28 possessed a conserved bZIP domain, a transmembrane domain (TMD) and a canonical site 1 protease (S1P) cleavage site. The promoter region of TabZIP28 contained several stress related cis-element. The expression patterns of TabZIP28 were analyzed by qRT-PCR under stress conditions. The results showed that TabZIP28 was induced by heat and peaked at 1 h; the transcript level of TabZIP28 was increased to the highest level by 20% PEG treatment for 6 h and reduced rapidly after 12 h; TabZIP28 was found to be slightly up-regulated by treatment of 5 mmol/L H2O2 for 12 h and TabZIP28 could not be induced by DTT treatment. To investigate the function of TabZIP28, we overexpressed TabZIP28 in Arabidopsis thaliana and assessed the thermotolerance of the transgenic lines. The results showed that the survival rate and germination rate of transgenic lines after heat stress were statistically higher than the wild type. This indicated that TabZIP28 could contribute to heat stress tolerance and could be used as a candidate gene for breeding of thermotolerant cultivars.

Plant auxin receptor is the key point in auxin signaling pathways. We had cloned the auxin receptor homologous genes in cucumber (Cucumis sativus) in the previous work with the name of auxin signaling F box protein (CsAFB) and transport inhibitor response(CsTIR). In order to verify and further explore their function, we transformed them into both TIR1 loss-of-function mutant tir1-1 and wild-type Col-0 Arabidopsis thaliana and obtained homozygous transgenic plants, respectively. With the transformation of CsAFB and CsTIR into tir1-1 mutant, the abnormal phenotypes of root system development, exogenous auxin sensitivity and cell elongation response in tir1-1 could be rescued. Overexpression of CsAFB/CsTIR in wild-type A. thaliana, especially CsTIR, led to inhibited elongation of primary root, increased numbers of lateral roots, curled cotyledons, upward petioles, bending leaf margins towards abaxial surface and had obvious apical dominance. This work illustrated that CsAFB/TIR functioned similarly as AtTIR1 in Arabidopsis. These genes were probably auxin receptor homologous genes in cucumber. Overexpression of CsAFB/TIR could enhance the number of auxin receptors, amplify auxin signal and regulate plant growth and development in A. thaliana. This work will contribute to the further study of CsAFB/TIR functions and regulation mechanisms in cucumber.

The main function of establishment of sister chromatid cohesion N-acetyltransferase 2 (ESCO2) is to regulate the aggregation of sister chromatid, thus it plays an important role in the repair of DNA double strand breaks in cells. In this study, dairy goat (Capra hircus) ESCO2 was cloned by RT-PCR, and the sequence was analyzed by modern bioinformatics. Further, the expression pattern of the gene was analyzed through PCR, qRT-PCR, bioinformatics analysis, immunofluorescence staining and related experiments. Moreover, eukaryotic expression vector carrying ESCO2 (pESCO2-IRES2-AcGFP) was also constructed successfully in our work, which was transfected into male germline stem cells of Dairy Goat (mGSCs-I-SB) in the following study. The results showed that, the sequence of dairy goat ESCO2 gene had a length of 1 834 bp and encoded an amino acid sequence with 612 residues (GenBank accession No. KP341998.D), which was highly conservative in evolution, its homology between dairy goat and Bos taurus was up to 97.82%. While widely-expressed throughout the body organs of dairy goat, ESCO2 was expressed highest in heart and lung, followed by testis and liver. The ESCO2 gene began to express after the birth of dairy goat and reached its peak at the age of one-year old, then falled down with the age of the goat in the testis. After being transfected with pESCO2-IRES2-AcGFP, mGSCs-I-SB showed an up-regulation in the expression level of meiosis-related genes, indicating that ESCO2 had a significant effect on the meiosis of spermatogenesis of dairy goat. Our work set a molecular basis for further study of the meiosis and differentiation process of dairy goat's spermatogonium.

To analyze the genetic diversity of 4 cattle (Bos taurus) species respectively including Xizhen cattle, Chiya cattle, Yunba cattle and Xuanhan cattle in Qinling-Bashan Mountain area, the research chose Shuxuan breed cattle and local varieties of Jiaxian Red Cattle as the control, studied the genetic diversity of 289 cattle individuals from 6 cattle species by using 12 pairs of micro satellite primers to detect the genetic diversity, and analyze the allele and their frequency, the number of effective alleles, genetic heterozygosity, polymorphism information content and genetic distance among populations, and conducting cluster analysis. The results showed that 110 alleles were found at 12 microsatellite loci in 6 cattle breeds. The allele frequencies ranged from 0.001 6 to 0.517 3 and the average number of alleles per locus ranged from 7.132 6 to 2.787 7. The polymorphism information content of each point ranged from 0.519 2 to 0.895 3. The average heterozygosity was from 0.667 2 to 0.724 1. Eleven unique alleles were found among the populations and the gene frequency was from 0.008 1 to 0.381 6. The dominant allele was 19(P＞0.4) and the gene frequency was from 0.403 2 to 0.820 0. There were 41 common alleles, accounting for 37.27% of all alleles, and only 13 alleles were dominant (P＞0.4), accounting for 37.71% of all alleles. Among populations the genetic identity of Nei's was 0.596 5 to 0.840 8 and the standard genetic distance (Ds) was from 0.173 5 to 0.524 7. Cluster analysis showed that 6 cattle breeds were clustered into three categories. Yunba cattle and Xuanhan cattle first got together, at the same time with the xizhen cattle gathered in a same class. Next Chiya cattle and Jiaxian Red cattle gathered for second class. At last, Shuxuan cattle was a category alone. The results of research showed that 12 pairs of microsatellite markers could be used for the analysis of genetic diversity of cattle in the Qin-Ba mountain area. The genetic diversity of cattle breeds in the Qin-Ba mountain area was rich, and the degree of breeding was also different. Although the geographical distribution pattern and the natural environment of 4 cattle breeds were similar, their genetic relationship was not close. It was confirmed that 4 cattle breeds were different varieties from different sources. This study provides the scientific basis for researching on the characteristics of genetic co adaptation, predicting heterosis, and formulating breeding strategies.

Escherichia coli F18 (E. coli F18) is the major pathogen causing porcine edema disease (ED) and porcine post-weaning diarrhea (PWD), which leads to tremendous damage and loss to the pig (Sus scrofa) industry all around the world. Previous study pointed that glycosphingolipid biosynthesis-globo series pathway and alpha (1,2) fucose transferase 1 (FUT1) as one of 7 key glycosyl transferase genes of pathway might play an important regulatory role to anti E. coli F18 in piglets. Therefore, given the importance of gene promoter in the regulation of gene transcription, the aim of this study was to identify the transcription initiation site and promoter of FUT1 gene by using bioinformatics to mine the RNA-seq results which obtained early. Besides, we analyzed the promoter CpG islands of FUT1 through online software. Then, dual luciferase reporter gene technology and AliBaba software were used for the analysis of promoter CpG islands and prediction of putative transcription factor binding sites (TFBS), respectively. By comparing the gene sequences of human (Homo sapiens) and porcine information from GenBank database, we found that FUT1 transcription initiation region had 5 kinds of alternative splicing (AS-1, AS-2, AS-3, AS-4 and AS-5) and 2 promoter regions named as promoter 1 and promoter 2. Further research by dual luciferase reporter gene technology detected the transcriptional activity of the 2 promoters. The result showed that transcriptional activity of promoter 2 was extremely significantly higher than that of the promoter 1 (P＜0.01) and the activity of the promoter 2 was 2.75 times of the promoter 1, which illustrated that promoter 2 played leading role in the transcription process. Then, analyzing the CpG island showed that FUT1 promoter 1 contained a CpG island within 500 bp length and promoter 2 with 2 CpG island. Using Alibaba version 2, we analyzed the identified CpG island, and found 20 TFBSs in the FUT1 gene, and specificity protein 1 (Sp1) located in many TFBS region, which might play important roles in the regulation of FUT1. All the results provide a deeper insight into the methylation detection and regulatory mechanism of FUT1 gene in piglets.

Virus disease is one of the 3 major diseases in the production of Chinese cabbage (Brassica campestris ssp. pekinensis), it has a strong impaction to the production and quality of Chinese cabbage. The virus disease in Chinese cabbage was resulted by 3 viruses alone or compound, which were Turnip mosaic virus (TuMV), Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV). TuMV was the main pathogen. Therefore, to breed TuMV resistant varieties became the main target. Molecular marker assisted selection can greatly speed up the process of breeding. Because of the complexity of the genetic law for resistance to virus of Chinese Cabbage. At present, the location of the resistant gene and the screening of the linkage markers can not meet the needs of breeding. In order to make better molecular marker assisted selection for virus resistance of Chinese cabbage (Brassica campestris ssp. pekinensis), we screened closely linked molecular markers for resistance to TuMV in Chinese Cabbage. Based on the mapping of that gene before, BC1 segregation population, BSA method, SSR and SSP technology techniques were used to develop molecular markers. Among 13 SSP and 16 SSR primers, 8 showed polymorphic between the 2 parents, markers amplified using 5 primers linked with gene TuRBCS01, which were mBr4072, Bra025493-1, Bra025493-2, Bra025467-4 and Bra025467-5. Two markers were developed closely linked to gene TuRBCS01, which were SAAS_mBr4072_240 (1.5 cM) and Bra025493-1(1.0 cM), respectively. In addition, SSP markers SAAS_ Bra025493-2_749, SAAS_Bra025467-4_780 and SAAS_Bra025467-5_956 were found co-segregation with that gene TuRBCS01. The markers have enriched the number and types of molecular markers about Chinese cabbage's resistance to TuMV, and they are expected to be used as marker-assisted selection in Chinese cabbage's resistance to virus disease.

Alveolar epithelial typeⅡcells (AECⅡ) are the first infected target cells by tuberculosis pathogens inhaled from respiratory tract. The activation of toll-liking receptors (TLRs) signal pathway and expression of pro-inflammatory cytokines were analyzed by the different virulence Mycobacterium tuberculosis (Mtb) infected alveolar epithelial TypeⅡcells (AECⅡ) in different time, such as H37Rv and Bacillus Calmette-Guérin (BCG) Mycobacterium tuberculosis strains, and which was used for finding the molecular mechanisms and immune response of AECⅡcells infected with Mtb. The AECⅡ cell lines of A549 were infected by H37Rv and BCG strains respectively. The expression of key molecules and proinflammatories related to TLRs signal pathway was detected by qRT-PCR and Western-blot in the infected cell at different time points (6, 12 and 24 h). The result showed that, the mRNA expression of TLR2, TLR4, NF-κB and MyD88 etc. were up-regulated significantly in the presence of H37Rv compared with BCG infected group at 6 h time point. TLR2/4 was increased in both H37Rv and BCG infected groups and there was no statistically significant difference between the 2 infected groups at 24 h time point. The expression of NF-κB was up-regulated in H37Rv infected groups at 24 h time point. Furthermore, the protein expression of TLRs signal molecules was analyzed. The expression of interleukin-1 receptor-associated kinase 4 (IRAK4) and TNF receptor-associated factor 3 (TRAF3) in the infected A549 cells with BCG were significantly higher than with H37Rv infection. The expression of IRAK4 was up-regulated at the time of 6 and 12 h of BCG infection, but suppressed at 24 h. However, the expression of IRAK4 was all suppressed at those 3 time points of H37Rv infection. When the A549 cells infected with H37Rv, the expression of MyD88 and phosphorylated NF-κB were significantly increased at 6 h and down-regulated at 12 and 24 h. But in BCG infection, both of them were no significant difference. The expression of pro-inflammatory cytokines IL- 1a, IL-6, IL-12a, IL- 8, TNF-α and CSF2 were increased with the infection time, and the expression of IL-1a, IL-6 IL-8, TNF-α and CSF2 in H37Rv infection were higher than BCG infection significantly and there was no significant difference in IL- 12a expression. The results indicated that the TLRs signaling pathways apparently suppressed in AECⅡ cells. The inflammation response was increased when infected by virulence Mtb and decreased when infected by attenuated strain of Mtb. This study will help to understand the AECⅡ cell immune regulatory mechanism in different virulent M. tuberculosis infection and take a reference for the studies of clinical tuberculosis pathogenesis.

Southern rice black-streaked dwarf virus (SRBSDV), the genus Fijivirus in the family Reoviridae, is transmitted mainly by white backed planthopper (Sogatella furcifera, WBPH) in a persistent-propagative manner. The epithelial cells of the midgut is the initially infected and replicated tissue for SRBSDV during the whole infection route in its insect vector. The effective multiplication of the virus is the key factor to determine whether the virus can be transmitted. In this study, to investigate the interaction between SRBSDV and its insect vector, and the mechanism of midgut protein regulating viral proliferation to transmit virus successfully. The yeast two-hybrid cDNA library for the midgut of SRBSDV-infected WBPH was constructed through SMART technique. The midgut of WBPH was dissected firstly. The mRNA was isolated and purified from the midguts of viruliferous populations, and used as the template to synthesize the double-strand cDNAs. The cDNAs digested with SfiⅠ were ligated into the library vector pGADT7, and then were transformed into the Escherichia coli DH10B cells to amplify the cDNA library. The cDNA library was calculated the number of transformants per fraction from 1 μL of ligation reaction and the average insert size from the population of 32 released inserts. The results showed the titer of the amplified library was 1.5×106 cfu /mL. The average size of inserts was 1.0~2.0 kb in the cDNA library. The yeast two-hybrid cDNA library derived from the viruliferous WBPH midgut will be useful for the future study on the interaction between insect vector and SRBSDV.

Recently, the use of biological control agents has received great attention because of their ability of suppressing different plant diseases. Some species of the genus Bacillus are the most promising candidates for bacterial biocontrol agents, as the species are well known as safe micro-organisms in the environment, versatility to protect plants effectively against plant pathogens. Bacillus species also have an outstanding ability to sporulation, which assures their prevalence in the environment. Bacillus strains often produce a range of antimicrobial cyclic lipopeptides, including iturins, fengycins and surfactines antibiotics in the family iturin, especially iturin A, is most famous for biocontrol activity. Iturin A is heptapeptide with a β-amino fatty acid that exhibits strong antifungal activity, and shows a strong antibiotic activity with a broad antifungal spectrum. A Bacillus strain FJAT-28592 kept in Microbiological Culture Collection Center of Fujian Academy of Agriculture Sciences was selected to search for naturally occurring biological control agents. The Bacillus strain FJAT-28592 identified as Bacillus siamensis by 16S rRNA analysis had antifungal activity against fungal pathogens Fusarium oxysporum. FJAT-28592 growed at 25~50 ℃, pH 5.0~9.0 and in the presence of 0~6% (W/V) NaCl. The optimal culture conditions was 35 ℃, pH 6.0~8.0 and salt concentration of 2%. Colonies grown on LB agar for 2 d were white, dry and flat. Cell morphology was observed with transmission electron microscopy, B. siamensis FJAT-28592 was motile rods, 0.76~0.94 μm in diameter and 1.76~2.94 μm in length after 2 d culture on LB agar. By blood agar plate and oil spreading test，the strain was confirmed to produce biosurfactant. The crude extract was gained by acid precipitation. Menthol extraction was compared with standard iturin A by high performance liquid chromatography (HPLC). Iturin A synthesis related malonyl-CoA-transacylase gene (ituD) and 4 '- phosphate generic acyl cysteamine transferase gene (lpa-14) were cloned by PCR. The chemical and molecular genetic evidence thus indicated that the biosurfactant from FJAT-28592 was a kind of iturin A. The fermentation crude extract containing iturin A possessed significantly inhibitory activities against F. oxysporum. B. siamensis FJAT-28592 was an ideal potential biological control agent with the aim of reducing the use of chemical pesticides in agriculture. This study provides basic data on the developing biocontrol Bacillus and exploring bacterial biocontrol substances.

Tomato (Lycopersicon esculentum) has emerged as an emblematic model plants for tomato fruit ripening and senescence, and tomato fruit ripening is a complex and highly coordinated developmental process which is companied by color, texture, flavor, aroma and other metabolic physiological and biochemical changes. In recent years, small RNAs are new post-transcriptional regulatory factors which received more and more attentions and take part in almost all physiological and biochemical processes in plants and animals. In plants, small RNAs participate in development, fruit ripening, and biological and abiotic stress process. This paper systematically summarized the current situation of small RNAs and the small RNAs related to ethylene pathway, fruit color, fruit flavor, fruit softening and other metabolic process. It also emphasized the new perspectives now possible in the small RNAs regulation research in tomato fruit ripening and senescence.

Spinosad, produced by Saccharopolyspora spinosa, is a novel natural macrolide antibiotic with the properties of both safety of biopesticide and the efficiency of chemical pesticide. With an unique mechanism of action, spinosad has a weak toxicity towards mammalian, natural enemies of insects and environment. Thus, it has obtained permission and wildly used as a new biopesticide in different fields, such as pesticide, veterinary drug, sanitary field, etc. At present, the research of spinosad in our country is still in the laboratory scale due to its low yield. Industrial production of spinosad still has not been implemented for the lack of breeding of high-yield strains of spinosad. The mutation breeding systems and rational screening of Saccharopolyspora spinosa for high-yield spinosad were summarized in this paper. The common mutation types, including ultraviolet mutagenesis, ion beam implantation, γ ratial and atmospheric and room temperature plasma, have the features of high safety for the operators and environment, easy operation, rapid mutation capability, high mutation rate and genetic stability of targeted mutants. The optimization of the media, including seed and fermentation medium were reviewed. The effects of breeding temperature, dissolved oxygen, inoculum level, seeding age, medium volume, the initial pH and various precursors on the spinosad fermentation were also studied in the shaking flasks. Several precursors, such as short-chain alcohols, amino acids and methylating agents, could enhance biosynthesis of spinosad. The biosynthesis, separation and detection methods for this kind of compounds were also summarized. It is helpful for encouraging the industrial production of spinosad to develop high performance and accurate analysis methods. The domestic and foreign research status was analyzed from 4 aspects. Meanwhile, the trends in the development of spinosad were also discussed.

Banana bunchy top virus (BBTV) is an important pathogen of banana (Musa paradisiaca), which seriously harm banana production worldwide. The purpose of this study was to develop serological methods for detecting BBTV using prepared monoclonal antibodies (MAbs) against BBTV, and to provide technical support for the diagnosis and scientific prevention and control of BBTV in fields. BBTV-infected banana plants were identified by PCR and nucleotide sequencing. A 513 bp coat protein gene (CP) was amplified from infected banana plants, which had 99% nucleotide sequence identities with Chinese isolates of BBTV in GenBank. BBTV particles with a diameter of 18 nm were obtained from BBTV-infected plants by an improved purification method. Electron microscopic observation showed that concentration of purified BBTV particles was high enough to prepare MAbs against BBTV. After cell selecting and cloning, a hybridoma cell line (22E3) which secreted a MAb against BBTV was produced by fusing mouse (Mus musculus) myeloma cells (SP2/0) with spleen cells from BALB/c immunized by purified BBTV particles. The hybridoma was injected into pristane-primed BALB/c mice to prepare the ascetic fluid, which contained the MAb. The titer of this MAb in ascites determined by an indirect-enzyme-linked immunosorbent assay (in-ELISA) is up to 10-7. Isotype and subclass of this MAb belonged to IgG1, κ light chain. The IgG yield of this MAb in the ascetic fluid was 8.32 mg/mL. Western blot analysis indicated that this MAb could specifically react with the 19.3 kD CP of BBTV in BBTV-infected leaf crude extracts, had a negative reaction with healthy banana leaf crude extracts. Using the MAb, dot-ELISA for detecting BBTV was established. Results of phalanx tests showed that the dilutions of 22E3 at 1∶5 000 and goat anti-mouse IgG conjugated with alkaline phosphatase at 1∶8 000 were suitable in dot-ELISA for BBTV detection in field samples. Specificity analysis of the dot-ELISA demonstrated that this method could strongly react with BBTV-infected banana plant tissues, not with the healthy banana plant tissues. The detection sensitivity of the dot-ELISA based on 22E3 was up to 1∶640 (W/V, g/mL). The field banana samples, collected from Guangdong, Yunnan and Hainan Provinces, were tested the presence of BBTV by developed dot-ELISA, and the detection results demonstrated that the assay could accurately and reliably detect BBTV in field banana plant samples. The anti-BBTV MAb and the developed serological dot-ELISA provide material and technology for diagnosis, production of BBTV-free seedlings and scientific prevention and control of banana bunchy top disease.

Atrina pectinata and Crassostrea rivularis are important marine economic shellfish, but their output is declining sharply due to overfishing, disorderly use and environmental deterioration. The establishment of sperm cryobank and cryopreservation technology was significant to their germplasm resources preservation and genetic breeding. In this study, in order to establish a technology of sperm cryopreservation suitable for Atrina pectinata and Crassostrea rivularis, the series of selecting experiments for type and concentration of cryoprotectant, freezing procedure, balance time, sample volume, sperm dilution method and freezing time were designed and compared. The sperm survival rate was detected by both direct microscopic examination and eosin Y staining detection. The data indicated that the protection of dimethyl sulfoxide (DMSO) to sperm was obviously superior to glycerol (Gly). The sperm survival rates of both Atrina pectinata and Crassostrea rivularis were up to 60% with the protection of DMSO, while only 40% or so with the protection of Gly. The terminal concentration of DMSO was 8%, which was suitable for Atrina pectinata, and 10% suitable for Crassostrea rivularis. The cryoprotectant was divided into 3 parts and added to sperm by 3 times, interval 8 min every time. Using this dilution method, the sperm survival rate was enhanced about 30% than that using once-mix method. The precooling of samples for 15~25 min at 4 ℃ was necessary and could increase sperm survival rate by about 50% compared with direct freezing method. The optimum freezing procedure was firstly to prefreeze for 5 min at 15 cm above liquid nitrogen level, then to prefreeze for 10 min at 5 cm above liquid nitrogen level, and to freeze in liquid nitrogen at last. The sperm survival rate was more than 60% with this freezing procedure, while near to zero without any freezing procedure. Under this freezing procedure, the volume of sample between 1.0 to 1.4 mL could make the sperm survival rate of 60% or so, and less than 1.0 mL or more than 1.8 mL would reduce the sperm survival rate and were unsuitable for sperm cryopreservation of Atrina pectinata and Crassostrea rivularis. After cryopreservation, the sperm survival rate decreased sharply to 60% or so from 90% of fresh sperm. However, there was no significant difference in survival rate of sperm, which was frozen less than 180 d. The sperm survival rate was further decreased to about 56% after 180 d. Therefore, combined with the optimum condition above, we obtained the optimum sperm cryopreservation project for Atrina pectinata and Crassostrea rivularis. This study on the technology of sperm cryopreservation of Atrina pectinata and Crassostrea rivularis provides the theoretical and technical basis for germplasm resources preservation and genetic breeding in shellfish.

Genetically modified organism (GMO) ingredients detection is an important part of GMO safety management and GMO labeling, GMO detection reference material (RM) is guaranteed to get accurate, reliable, comparable GMO ingredients detection results. GM soybean (Glycine max) MON89788 is a GM plant that has been approved to import and use as in-process raw material in China. Therefore, preparation of GM soybean MON89788 RM is essential for its safety management. In this study, GM soybean MON89788 and traditional soybean seeds A3244 were milled using a cryo-grinding vibrating mill separately, then the GM soybean MON89788 matrix RM was prepared by weighting and mixing of GM and Non-GM powder. The value of MON89788 matrix RM was 49.3 g/kg in mass fraction. The mixture was further dispensed into brown bottles. Analysis of variance (F test) was performed to test homogeneity of RMs. The calculated F-statistics is 1.618, less than the critical value F (0.05, 11, 24). The results indicated that there was no significant difference between bottles and within bottles, and the RMs were homogeneous. Stability study results indicated the RMs could be stored under ambient conditions for 4 weeks, and stored at - 20 ℃ for 30 months or more. The certified value was assigned based on mass fraction of GMO powder and non-GMO powder with the units of g/kg, and mass fraction of GMO DNA and non-GMO DNA with the units of ng/μg. The certified value was expressed as (49.3±2.0) g/kg, and (49.8±5.9) ng/μg. This batch of RMs was 1.0 g/bottle, and the recommended minimum sample intake was 100 mg. The RMs could be used in qualitative and quantitative detection of GM soybean MON89788, as well as in standardization of GMO detection method and laboratory quality control. This work provides theoretical basis and technical support for the development of GM detection matrix RMs.