Cucurbita ficifolia genome contains a large number of beneficial genes of great agronomic value, which confers multiple traits including strong stress tolerance for low temperature, drought, poor soil and fusarium wilt of Cucurbits. However, it is difficult to transform these beneficial genes into other cultivated cucurbit crops by the conventional breeding program, while the transformation by TAC vector library may serve as an alternative. To facilitate the mapping and cloning of these beneficial genes, a genomic transformation-competent artificial chromosome (TAC) library of Cucurbita ficifolia was constructed by using the vector of pYLTAC747NH/sacB in this study．The library consisted of 57 560 individual clones, which was stored in one hundred fifty 384-well plates with an average DNA inserted size of 45 kb ranging from 20~100 kb(more than 40% of the clones were above 40 kb); and the whole library covers 7× genome equivalents. All 15 selected clones carried inserted fragments, and the restriction digestion of the plasmids also indicated that TAC clone could be stably replicated and retained in Escherichia coli DH10B after sub-culturing 30, 60 and 100 generations， thus demonstrating that high quality TAC library was created. To conclude, our study suggest that TAC library can provide valuable information for the isolation of genomic clones containing stress tolerance genes, functional validation, the construction of a physical map and the genome sequencing.

In the past two decades, many countries have been paying more attention to safety of genetically modified (GM) product, and thus, qualitative and quantitative test of GM products become more and more important. The test result can be more comparable and reliable by using GM reference material. This paper reviews the research progress of GM reference material and related techniques: it introduces the variety of GM reference material and techniques of value assignment for GM reference material, and also evaluates the key procedures in developing GM reference material. More importantly, it is concluded that how to assign the reference value for the plasmid reference material including the commutability between the plasmid and genomic DNA. Additionally, it summarizes the current international GM certified reference material. This aims to provide some valuable reference for developing GM reference material in China.

Mycoplasma bovis is an important pathogen in cattle. In this research we developed a new and rapid test method for Mycoplasma bovis with loop-mediated isothermal amplification(LAMP). With the 5 specific primers targeting to a total of six distinct sequences on the conservative gene uvrC, the new method could be completed in 60 min under the isothermal condition of 58℃. With the LAMP method, we could detect 20 pg DNA of M. bovis, which was 100 times lower than using the method of PCR. In addition, using M. bovirhinis, M. agalactiae, M. arginini, Bovine parainfluenza virus(BPIV), Bovine adenoviruses(BADV) and Infectious Bovine rhinotracheitis virus(IBRV) to determine the specificity of LAMP and PCR methods, both the methods of LAMP and PCR displayed the high specificity results. The research had also applied the calcein, a fluorescence visualization reagent, and Mn2+ in LAMP, so as to visualize the result of the test. Additionally, clinical samples after boiling 10 min which was the best compared with DNA extraction and FTA Card could be directly used for LAMP. In the test for 167 clinical samples of nasal swabs, the positive rate using LAMP (26.95%) was higher than the one using PCR (19.16%), which proved that LAMP method, in comparison with PCR method, showed a more efficient ability in testing the clinic samples. In conclusion, LAMP method, featuring simplicity, high-efficiency, rapidity, sensitivity, specificity, and more economical, etc., possesses a potential for base laboratory.

Seedless fruit has strong market competitiveness and the considerable economic value, seedless trees can effectively reduce the pollution of the environment, seedless transgenic plants can help prevent foreign gene dispersal. Barnase gene comes from Bacillus amyloliquefaciens, encoding a 12 kD small molecule extracellular ribonuclease (RNAase). In the absence of its natural inhibitor Barstar, the expression of Barnase gene usually causes the death of its host cell. The T-DNA vector i. e. pBIloxP2 PNap TAibarnase carrying Barnase gene driven by Brassica napus seed-specific promoter Napin was constructed and transformed into tobacco(Nicotiana benthamiana) with the leaf disc transformation method, and 55 tobacco transformants were obtained. In which, 10 transformants were randomly selected for floral morphology analysis. Comparison of floral organs indicated that no obvious morphological difference was observed at different developmental stages between the transgenic and non-transgenic plants before pollination. Whereas, the floral organs of transgenic plants were gradually shrinking and dry, and failing to produce seeds after pollination; the petals of non-transgenic tobaccos were normally withered and can produce seeds within torus after pollination. In order to understand the mechanism of seed abortion of transgenic tobaccos, the cross between transgenic and non-transgenic plants were performed. However, no seed was obtained with the transgenic plants either as female parent or male parent. The hybrid results revealed that the seed abortion of transgenic tobacco was not caused by the self-incompatibility. To analyze the pollen activity, Alexander solution was used to stain the pollens of transgenic and non-transgenic tobaccos, then we observed the size, shape and color of pollens from transgenic and non-transgenic tobaccos with microscope, no difference was found. Therefore both the transgenic and non-transgenic tobaccos could generate active pollens. It is concluded that the seed specific promoter Napin used in this study only drives the Barnase gene expression in the seed, and no leakage expression takes place in pollens. We successfully obtain seedless transgenic tobaccos through expressing the Barnase gene in the seed tissue. The present study creates a novel model of producing seedless plants via genetic transformation, especially for vegetatively propagated crops.

As a medicinal and edible plant, tartary buckwheat (Fagopyrum tataricum) is rich in flavonoids especially rutin. During germination, it has a higher concentration of ruin, but the molecular mechanism is still unclear. In this study, the total flavonoids in cotyledon and hypocotyl of F. tataricum during 6~10 days after germination were extracted by the methanol extraction method, and were determined by aluminum chloride spectrophotometry. Semi-quantitative RT-PCR was used to determine the expression level of the phenylalnine ammonialyase gene (Pal), chalcone isomeerase gene (Chi), flavonol synthase gene (Fls) and MYB transcription factor genes (FtMyb1, FtMyb2 and FtMyb3) in the cotyledon and hypocotyl during the seed germination, the relation among them analysed by statistics method. The result showed that: in cotyledons, the total flavonoids content was significantly and positively associated with FtMyb3 (0.9625), and negatively with FtMyb2 (－0.8572); Chi was significantly and positively associated with FtMyb2 (0.8468), and negatively with FtMyb3 (－0.8010); the three key enzyme genes were significantly and positively correlated to each other with 0.9119, 0.8920 and 0.7584, respectively. In hypocotyls, the total flavonoids content was significantly and negatively associated with Chi (－0.8989), while Fls was significantly negative associated with Chi (－0.7498). Visiblely, the regulation of flavonoid biosynthesis is rather complex during germination of F. tataricum, but some gene's expression is still significantly associated with each other.

In order to find the potential influence of Citrus tristeza virus (CTV) isolates in Jianyang Tangelo on Gannan navel orange, the CTV isolates were characterized at the molecular level. Jianyang Tangelo (Citrus paradisi × C. reticulata) is a new citrus hybrid cultivar with excellent quality. CTV isolate JY-5 was inoculated in Newhall navel orange (C. sinensis) and the HinfⅠrestriction fragment length polymorphism(RFLP) and single-strand conformation polymorphism(SSCP) of its coat protein(cp) gene was comparatively analyzed. The results of RFLP analysis revealed that the single group of the CTV isolate in Jianyang Tangelo changed into mixed groups when it inoculated in Newhall navel orange. DNA bands of cp/SSCP of CTV isolates increased when the CTV isolate was graft inoculated from Jianyang Tangelo to Newhall navel orange. The results of HinfⅠRFLP and SSCP showed that the replication of some composition of CTV isolate was inhibited in Jianyang Tangelo but enhanced in Newhall navel orange. By analyzing and comparing the nucleotide and amino acid sequences of cp, p23 and k17 of isolate JY-5 in Jianyang Tangelo and isolate JY-5R from inoculated Newhall navel orange, the identity of genomic regions were 92.5%, 90.3% and 82.3%, respectively, and of their amion acid sequences were 95.9%, 90.9% and 77.9%, respectively. Phylogenetic analysis revealed that the isolate JY-5 in Jianyang Tangelo and JY-5R in inoculated Newhall classified into different clusters in phylogenetic trees of cp, p23 and k17. Sequence comparisons and phylogenetic analysis indicated that no high homology and closer relationships between the isolates JY-5 and JY-5R, the isolate JY-5R was inhibited in Jianyang Tangelo but enhanced in Newhall navel orange. The results suggest that if the CTV isolate from Jianyang Tangelo infects Gannan's Newhall navel orange and spreads in Ganzhou, maybe cause some damage to Gannan's navel oranges. Therefore, it is necessary to take a survey on the CTV occurence and Jianyang Tangelo plantation in Ganzhou.

To identify grape (Vitis vinifera L.) genes in response to exogenous gibberellins (GA) treatment, a large number of grape EST sequences collected at NCBI were analyzed. Forty-five non-redundant EST sequences that only expressed after GA treatment were obtained by using local Blast, Blast2go and other programs. Blastx annotation results indicated that twenty-five sequences(55.6%) were of hypothetical or unknown protein, fourteen sequences(31.1%) did not have available information, and six sequences(13.1%) had predict functions. The six sequences with annotated functions carried genes encoding integrase (EE077049), SPX domain (EE085000), serine carboxypeptidase-like 44(EE091188), CHY1 (EE092187), pseudouridine synthase/ transporter (EE106096) and K+ channel protein (EE108944). Blast2go analysis showed that only twenty-nine sequences (64.4%) had available Gene Ontology (GO) annotations, belonging to categories of molecular function, cellular component or biological process. Taken together, the GA-responsive gene products mainly had binding activities (46.34%) or catalytic activities (39.02%), distributed in the whole cell (44.68%) or in specific organelles (40.43%), participated1 in cellular processes (28.57%) or metabolic processes (25.71%) in responding to the exogenous GA treatment. This study provides basic information for further analysis of gene expression in response to exogenous GA treatment.

Ochratoxin A(OTA) is a widespread mycotoxin. It can contaminate cereals, animal feed, animal foodstuffs et al. OTA has been shown to be nephrotoxic, hepatotoxic, teratogenic, immunotoxic, mutagenesis and carcinogenesis to several species of animals. The plant toxicity of OTA has been discovered in recent years. In this research we focused on the involvement of salicylic acid(SA) in OTA toxicity to Arabidopsis thaliana, which is an important endogenous signal molecule of plants. SA content of A.thaliana leaves exposed to OTA was measured by high performance liquid chromatography. The results showed that OTA treatment increased SA content in A.thaliana leaves. Quantitative Real-time PCR was performed to determine the relative expression level of pathogenesis-related gene 1(PR1) which is an important gene in SA pathway. We found that PR1 expression was induced by OTA, moreover the effect of OTA was related to treatment time. When exogenous SA was treated with OTA, aggravated lesions were observed. According to results of relative leakage rate and reactive oxygen species content measurement, it was showed that SA could enhance the damage of leaves and promote reactive oxygen species production caused by OTA. The research indicates the involvement of SA in plant toxicity of OTA which can be enhanced by SA.

High abundance and tissue-specific gene expression are important for gene therapy and preparation of transgenic animals. Cre-LoxP system is effective to improve transcriptional activity of tissue-specific promoter. However, it is not known if the Cre-LoxP system affects the tissue-specific expression regulated by the specific promoter.. In this study, intestinal mucin 2 promoter was used to regulate the Cre recombinase expression base on Cre-LoxP system. Then luciferase activity was detected after transfection. The results showed that Cre-LoxP system increased the expression of mucin 2 promoter -mediated target genes (6-fold) in intestinal cells. However, cell-specific tests showed that the system decreased the specificity of mucin 2 promoter. The results suggest that the use of Cre-LoxP system requires higher specificity of the promoter although the system can significantly improve the weak promoter activity.

Mammalian thymus is the central immune organ. In order to investigate the immune response of the central immune organ in fetal rhesus macaques when aborted, spontaneous aborted fetuses were used as experimental material and the distribution and expression of their interleukin-2 and -10(IL-2+ and IL-10+) cells were determined by HE staining and immunohistochemical staining in different gestational age of fetal rhesus macaques. Results: Interlobular dividing lines of the fetal thymus in macaques in each gestational age group were clear. The number of lobules and lymphocytes in cortical medulla were increased by month-to-month. In two-month embryo age, medulla was formatted and diffuse thymus vorkommen was appeared. , In three-month embryo age, typical thymus vorkommen was observed in medulla and the thymus structure was organized. Thymus IL-2 and IL-10 expression in the 1-month-old was negative. After that, the optical density and the area ratio of positive cells were increased with the development of age. Positive cells were distributed in the cortical edge, the junction of cortex and medulla, the central area of the medulla, and the thymus body without degradation and blood vessels around the thymus. Furthermore, the expression of IL-2 was high, whereas the expression of IL-10 was low. The results suggest that two cytokines, especially IL-2, were mainly expressed in thymocytes' growth area. In addition, secretion levels of IL-2 and IL-10 can be as a predictor of premature of pregnant women.

In order to find the molecular marker used in marker-assisted selection (MAS) to accelerate goat (Capra hircus) breeding progress, the goat transforming growth factor-β1 gene(TGF-β1) was selected to study the association between its polymorphism and litter size. According to the sequence of bovine TGF-β1 gene, three pairs of primers were designed to detect SNPs of intron 3 and exon 2 of TGF-β1 gene in two goat breeds by PCR-SSCP and DNA sequencing. The least square mean and genetic variance of different genotypes at polymorphic loci were analyzed. The results showed that the PCR products of primer 1 (P1) had polymorphism. Three genotypes (AB, BB, and AA), and one single nucleotide mutation (A→G) was detected in two goat breeds, and this mutation resulted in an amino acid change: Thr→Ala. Allele A was the dominant allele. The frequencies of allele A were 0.530 and 0.648 in two goat breeds, respectively. In Boer goat, AB genotype had significantly higher (P<0.05) litter size than that of BB and AA genotypes from the first to third parity. AB genotype was very significantly higher (P<0.01) than that of AA genotype, and significantly higher (P<0.05) than that of BB genotype, and BB genotype was significantly higher (P<0.05) than that of AA genotype in the fourth parity and average litter size. In Shaannan goat, AB genotype had significantly higher (P<0.05) litter size than that of AA and BB genotypes in the second, third, fourth and average parity, and AB and BB genotypes had significantly higher (P<0.05) litter size than that of AA genotype in the first parity. The TGF-β1 gene had significant effects on litter size in two goat breeds. Therefore, these results suggest that the TGF-β1 gene is a strong candidate gene that affects litter size in goats.

Insulin-like growth factors (IGFs) Ⅰ and Ⅱ (IGF-Ⅰ and IGF-Ⅱ) play important roles in fish growth and development. In this study, cDNAs of both IGF subtypes were cloned and sequenced from zanzibar tilapia (Oreochromis hornornum), the expression patterns of the two IGF were detected using semi-quantitive RT-PCR. Total RNAs were isolated from liver of zanzibar tilapia. The cDNAs encoding IGF-Ⅰ and IGF-Ⅱ were amplified by RT-PCR, 3'RACE and 5'RACE. The amplified cDNA fragments were inserted into pMD-T vector. Sequence analysis revealed that the IGF-Ⅰ and IGF-Ⅱconsisted of 1 305 and 1 091 bp, respectively. They all had the characteristic landmarks of IGF, for example, they all possessed signal peptide and B, C, A, D and E domains and six cysteine residues, but their features were distinctly different from each other, the overall deduced amino acid sequence identity between IGF-Ⅰand IGF-Ⅱ was rather low(26%). Under normal physiological conditions, both IGF-Ⅰ and IGF-Ⅱ were present in all tissues examined. IGF-Ⅰwas most highly expressed in liver, muscle and gonad but lowly expressed in kidney. IGF-Ⅱwas most highly expressed in kidney, stomach, intestine, spleen and pituitary, but low in muscle. The mRNA levels of IGF-Ⅱwere higher than that of IGF-Ⅰ in all tissues tested, but significantly difference (P<0.05) only observed in intestine, spleen, stomach, kidney and pituitary. IGF-Ⅰ expressions in females were lower in most tissues examined than those in males except for in spleen, stomach, kidney and pituitary. But significant difference existed in muscle(P<0.05). No significant difference was found in IGF-Ⅱexpression in all the tissues of male and female zanzibar tilapia. This observation can contribute to further investigation of growth regulation of zanzibar tilapia, and the results will give us a better understanding for the physiologyical role of IGFs in fish.

The selection of a suitable reference gene is an important prerequisite for precise gene expression analysis by quantitative Real-time PCR (qRT-PCR). Ten houskeeping genes were choosed for reference genes selecetion based on the research on reference genes. After calculations of PCR efficiencies, eight houskeeping genes were retained and their expression stabilities were evaluated by the computer algorithms geNorm. These genes included ubiquitin-conjugating enzyme(UBC), E2 ubiquitin-conjugating enzyme(UBCE2), ribosomal protein S5(RPS5), α-tubulin (TUBA), β-actin(ACTB), β-tubulin(TUBB), elongation factor1(EF1) and elongation factor3(EF3) . Using qRT-PCR, we investigated the expression stability of 8 housekeeping genes of Puccinia striiformis f.sp. tritici (Pst) in different samples including fresh spores of CYR32 and Pst78, germinated tubes of CYR32 and Pst78, inoculated wheat (Triticum aestivam) leaves sampled at 0.5, 3 and 14 days post inoculation(CYR32/XZ9104 and Pst78/AvS). The analysis with geNorm algorithms revealed that the expression profiles of ACTB, TUBB and TUBA were consistent with the infection process of Pst. The combination of ACTB, TUBB and TUBA can be used as reference genes for gene expression analysis of Pst.

Protein elicitor from Verticillium dahliae(PevD1, UniProtKB accession No. P86840) was isolated from V. dahliae culture filtrate. To functionally investigate the mechanisms of disease resistance induced by the elicitor, we have expressed and purified the recombinant protein, 6His-PevD1 in Escherichia coli, The recombinant protein could cause cell death in tobacco (Nicotiana tabacum cv. Samsun-NN) cell suspension and enhanced the systemic resistance of tobacco to Tobacco mosaic virus (TMV). The rate of necrosis reduction by the elicitor treatment could be up to 46.64%. The activities of phenylalanine ammonia lyase(PAL), polyphenol oxidase(PPO) and peroxidase(POD) were increased significantly upon PevD1 treatment in tobacco leaves. The maximal activity of PAL appeared at 144 h post the treatment, which was 3.3 times as high as that of the untreated control. And the activities of PPO and POD increased by 236.8% and 204.6% after 120 h post the treatment, respectively. Transcription of acid pathogenesis-related gene 1(PR1-a), basic pathogenesis-related gene 1(PR1-b), non-expresser of pathogenesis-related gene 1(NPR1) and phenylalanine ammonia lyase gene (PAL) was upregulated after PevD1 treatment as well. Taken together, these results demonstrate that the activity enhancement of defense-related enzymes and the induction of resistance-related genes' expression are the key mechanisms underlying the systemic acquired resistance (SAR) induced by PevD1 in tobacco.

Antimicrobial peptideis is a kind of bioactive polypeptides induced by pathogens in organisms, which is an important component of innate immunity. In order to improve the expression of antimicrobial peptide and easily detect the expression of antimicrobial peptide. The red fluorescent protein dTomato and muti-copy MagaininⅡgenes were fused into a single open reading frame (ORF) with a copy of the Foot-and mouth disease virus (FMDV) 2A region placed between the two genes. The fused genes were placed under the control of the alcohol oxidase (AOX1) gene promoter in pPIC9K vector to construct the recombinant expression vector T-dTomato-2A-3M. The linearized plasmid T-dTomato-2A-3M was transformed into Pichia pastoris GS115 strain by electroporation to construct the recombinant expression yeast strain. Through G418 screening and PCR identification, the positive clones were transferred into shake flasks at 30℃with 0.5% methanol to induct for 3 d. SDS-PAGE analysis showed that the recombinant had expressed a 31 and a 9.5 kD proteins in supernatant of GS115/pPIC9K-dTomato-2A-3M. Observed under a fluorescence microscope, supernatant of GS115/pPIC9K-dTomato-2A-3M appeared red fluorescent. The activity assay demonstrated that the recombinant antimicrobial peptide had antibacterial activities against Escherichia coli and Staphylococcus aureus. The results show that the fused genes (red fluorescent protein dTomato and muti-copy MagaininⅡ) linked by 2A region of FMDV are expressed in P. pastoris successfully and the polyprotein is "cleaved" to each functional protein, which provides an effective method to detect the expression of antimicrobial peptided.