Porcine circovirus type 2 (PCV2), the essential causative agent of PCV2-associated disease (PCVAD), has made a grave threat to the worldwide swine industry, but no efficacious therapy has been found to treat this viral infection. To explore a new approach to treat PCVAD, plasmids (pGensil-R259, pGensil-C297 and pGensil-C490) encoding siRNA to PCV2 replication-involved (rep) and capsid (cap) protein genes, which encode virus Rep protein and Cap protein respectively, were constructed with a plasmid (pGensil-SCR) encoding PCV2-nonspecific siRNA as a negative control. PK-15 cells were respectively transfected with different PCV2-specific siRNA expression plasimids and negative plasmids by liposome and these cells were inoculated with PCV2 at 20 h of post-transfection. Plasmid-based PCV2-specific siRNA significantly inhibited DNA synthesis, Rep and Cap protein expression of PCV2 and reduced virus titers markedly. The inhibition changed along with different positions in siRNA target gene sequences. Within 36 h post-infection, PCV2-specific siRNA conducted the strongest inhibition and it could significantly inhibited PCV2 replication until 60 h post-infection in spite of a slight decline. These results showed that plasmid-based specific siRNA can strongly inhibit PCV2 replication while the inhibition is in negative correlation with action time.

This work aimed to develop a SYBR Green Ι Real-time fluorescent quantitative PCR assay for detection of bovine toll-like receptors mRNA. According to the gene sequences of bovine's Tol1-like receptors (BoTLR-2, 3, 4, 5, 7, 8 and 10) available in GenBank, eight pairs of primers based on their conserved regions were designed with bovine glyceraldehyde-3-phosphate dehydrogenase (BoGAPDH) as an interna control to construct Real-time fluorescent quantitative PCR assay. The results showed a good linear relationships(r2>0.991) between the Ct value and the concentration of positive plasmid for each gene on the condition that the concentration of positive plasmids were limited from 1×102 to 1×109 copies /μL. The melting curve analysis showed the product was specific to a single peak, with high sensitivity and specificity. Reproducibility showed that the minimum detectable concentration of positive plasmids of TLRs and GAPDH gene were 100 copies /μL and 10 copies /μL. And the intra-assay and inter-assay coefficient of variation values were maintained at less than 3.5%. The clinical sample test showed that TLR3 and TLR8 mRNA levels in the induction of early high peak at 4h, while the level of TLR4 and TLR7 contrast, higher late in the induction, peaked at 24 h.These data showed that the detection method in this study successfully can be use for the detection of clinical samples and provids a technical platform in the quantitative analysis of BoTLRs on the mRNA levels.

To better understand the transformation events of transgenic cottons in China and to develope the rapid and accurate identification methods, the integrated structure and genomic flanking sequence of transgenic cotton(Gossypium hirsutum L.) Ezamian 1, the new event of transgenic insect-resistant cotton in china were identified using the long distance PCR(LD-PCR) and the genome walking methods. The event-specific qualitative PCR method for Ezamian1 was established based on the flanking sequence through the optimization of primers, testing of the specificity and the sensitivity. The sensitivity of the method was 40 copies, corresponding to 0.1% in 100 ng template. Twenty one samples from the market was tested with this method, three samples were identified having the same event structure of the Ezamian1. All of these indicated that the event-specific detection method established this study may be useful for the identification of transgenic cotton in China.

For establishing an assay of Real-time PCR to detect distribution and expression level of sheep Food and mouth disease virus (FMDV) integrin receptor αv submit in different tissue, in this study, a pair of primers was designed according to the published nudeotide sequence of integrin receptor αv submit. And tissue expression profile of sheep (Ovisaries) integrin αv in different tissue was detected by Real-time PCR. The results showed that the integrin receptor αv submit was generally expressed in 19 kinds of tissues with different level. The highest expression level was showed in breast, the next was in hoof, and the lowest was in muscle tissue. We have established the Real-time PCR method which can be used to confirm tissue profile of sheep FMDV integrin receptor αv submit. This study provides basic data for further study tissue tropism of FMDV and molecular detection method.

Cellulose is the earth's most abundant resources of renewable resources, Cellulase enzymes and the production cost restricts the use of cellulose. In this study, a fungi G-1 was isolated from rotten wood(Populus bonatii) placed outdoor for years and indentified as Trichoderma viride through 18S rDNA sequence analysis. A endoglucanase gene(EG) (GenBank Access No.HM116999.1) was cloned from G-1 and a mutant sequence (EG-mut) was obtained by overlap primer extension method for site-directed mutagenesis of EG. Both EG and EG-mut were inserted into the secretion expression vector pPIC9K and imported in Pichia pastoris, and two strains were screened by Congo red staining and enzyme activity assay DNS method. Results showed that the activity of enzyme was respectively 20.915 and 24.110 U/mL, significantly higher than the other strains obtained in this experiment. The results indicated that some site-directed mutagenesis can reveal important functional domains of enzyme activity.

LuxR homologues are the main component of LuxI/LuxR circus in quorum sensing (QS) of Gram- negative bacteria. In this study, an internal fragment of luxR homologue from Acidovorax avenae subsp.citrulli was amplified by PCR, the luxR insertion mutant was constructed by homologue recombination. In comparison with the wild-type strain, the luxR deletion mutant(△luxR) had a significantly reduced ability to form acyl homoserine lactones (AHLs), which are quorum-sensing molecules. Tolerance to antibiotics were enhanced in the △luxR than that of the wild-type strain. △luxR had reduced virulence on melon(Cucuumis melo var. cantalupensis) leaves. RT-PCR indicated that LuxR protein regulated expression of the luxI gene. The study of luxR gene is to provide basic data for the prevention and control of Acidovorax avenae subsp. citrulli using the technology of signal interference.

Tetracycline waste residue is considered to be a good protein source for animal feed, since it contains a high proportion of protein and a relatively nutritional balance in amino acids composition. On the other hand, tetracycline waste residue also has a high level of tetracycline, which will limit its usage in animal feed industry. It may be feasible to eliminate tetracycline in the waste via microorganism fermentation method. Thus, the present study was conducted to obtain tetracycline-degrading strains. Two tetracycline-degrading strains, named TD2 and TD3, were isolated from soil contaminated by tetracycline. Based on the results of phenotype features, physio-biochemical identification and the phylogenetical analyses of 16S rDNA sequence, TD2 and TD3 were identified to Brevundimonas diminuta and Ochrobactrum anthropi, respectively. Both TD2 and TD3 could grow in the medium containing tetracycline as only carbon source. The highest efficacy of TD2 was achieved when it was incubated in the medium consisted of 0.5% peptone and 0.015% CuSO4, whereas that of TD3 was obtained in the medium composed of 0.5% glucose, 1.5% beef extract, 0.015% CuSO4. Identically, both TD2 and TD3 had an optimal inoculation ratio of 1%, and tetracycline degradation rate proportional to ventilation, as well as an optimal culture condition of 30℃ for five days. After the optimal incubation, the tetracycline degradation ratio for the two strains could be up to 90%. Our results indicated that TD2 and TD3 were the microorganism candidates for the tetracycline waste residue fermentation process by which tetracycline can be eliminated.

To speed up the pace of industrial application of cellulose, and solve the low yield, high production cost issues in fermentation industry, orthogonal design methodology was applied to optimize the fermentation of Bacillus megaterium genetic engineering bacteria for neutral cellulase. Results indicated that optimal medium compositions were 2.5% wheat bran, 1.5% corn thick liquid, 0.75% KH2PO4, 0.04% MgSO4, and 0.25% NaCl; The optimal fermentation conditions were fermentation temperature of 37℃, pH 7.0, tank pressure 0.03~0.05 Mpa, rotation speed of shaker 600 r/min, 3 L of packing volume, 10% inoculation amount, fed 10 h xylose by 0.25 g/L/h flow rate after 4 h cultured, constant dissolved oxygen fed-batch 8 h after 16 h of fermentation, dissolved oxygen≥30% during the fermentation process. The activity of neutral cellulase obtained at this optimal fermentation condition was 3 846.48 U/mL, which was 4.3 times of the shaking flask culture. This research can provide the basis for the cellulase manufacture.

To investigate the mechanism of B function genes for double flower development in Camellia japonica, CjDEF-1 was cloned and its expression characteristic was analyzed. CjDEF-1 fragment was cloned from C. japonica Hongshibaxueshi flower buds based on homologous sequence from online blast results, and the full length of CjDEF-1 cDNA was amplified by RACE method(GenBank Accession No.HM773024.1). The spatialtemporal expression pattern of CjDEF-1 gene was studied by Real-time PCR. The results exhibited that CjDEF-1 cDNA sequence length was 1 013 bp and included an completed ORF(open reading frame) and encoded 226 amino acids. The sequence analysis showed that there were four encoding amino acid differences between C. japonica subsp. rusticana and C. japonica Hongshibaxueshi. The highest expression was in small buds, the lowest was in middle buds, while the expression went up at flower stage. The expression results from highest to lowest at different flower tissue were central petals, outside petals, middle petals, sepals and bracts. These differences with single flower showed that CjDEF-1 gene may be involved in formal double thigmomorphogenesis in Camellia.

The V-H+-ATPase in plant cell transports can proton into the vacuole and forms the electrochemical gradients across the membranes, which energizes the secondary transport systems presented on the membranes. We applied plants Mesembryanthemum crystallinum, Zea mays, Aplum graveolens, and Daucus carota, and initially observed that the V-H+-ATPase from different plant species performed special active peaks of the proton transportation and vary isoforms of subunit A during circadian rhythm and/or stress. Repeat experimental results showed that there were two strong peaks of proton transportation in the leaves of M.crystallinum occurred at the time of 3 and 18 o'clock, respectively. In the leaves of A.graveolens, D.carota and Z.mays only one strong peak was observed: the active peak in the leaves of A.graveolens and D.carota was observed at 15 and 12 o'clock, respectively; the active peaks in the leaves of Z.mays was revealed during the time periods of 15~18 o'clock. The expressions of the isoforms of the subunit A of the V-H+-ATPase from above plant were observed: two isoforms were revealed in the leaves of M.crystallinum, which was not affected by salt stress and day/night change; four isoforms of the subunit A were observed in the leaves of Z.mays, the number of the isoforms was not affected by salt stress; three isoform of the subunit A from the leaves of A.graveolens did not show much differences during day/night change, but appeared a new isoform after salt stress; In the leaves of D.carota, the isoform of the subunit A was changed during day/night time period, at 8 o'clock two isoforms were observed, while at 18 o'clock only one was revealed, the isoforms of the subunit A from D.carota were not affected by salt stress. The proton transportation and the isoforms of the subunit A of the V-H+-ATPase from above plant species suggest the specific phenomenon related plant species, which may be an interesting base for the studies of the role of gene silencing and proteases.

Different Agrobacterium-mediated protocols were used to evaluate the transformation efficiency of gloxinia(Sinningia speciosa), and CFL(Cucumber-FLO-LFY) gene was introduced into gloxinia to investigate its potential to promote early flowering. The plant expression vector pCA-CFL was constructed by inserting CFL gene into the plant high-efficient expression vector pCAMBIA13011. To restrain the growth of untransformed cells, we confirm the hygromycin(Hyg) filtration pressure(20mg/L) through explants culture treated with different Hyg concentrations. CFL gene was transformed into leaves of gloxinia mediated by Agrobacterium tumefaciens and GUS(β-glucuronidase) test showed that the protocol of ultrasonic 10 s treatment was the most efficient. After two selection cultures, dozens of Hyg-resistant regenerated green shoots were obtained. PCR and Southern dot blot analysis revealed that CFL gene had been integrated into the gloxinia genome. Transgenic seedlings were grown under long-day condition until maturity.The analysis showed that most of transgenic gloxinia plants(71%) flowered 26~32 days earlier than wild-type plants, and had terminal flowers emerging directly from shoot apex with no inflorescence branches. Our results imply that CFL act as a functional homolog of LEAFY(LFY) in gloxinia.

OsPHD1 is a member of the plant homedomain(PHD)-finger transcription factor family in rice(Oryza sative). Expression of OsPHD1 is induced under various stresses, including drought, high salinity and cold treatments.By using Agrobacterium-mediated method, OsPHD1 was successfully introduced into rice cultivar Zhonghua 11(Oryza sativa ssp. japonica) and T1 generation of transgenic rice was achieved. The results indicated that overexpression of OsPHD1 in rice could significantly improve more than 43.3%, 60 % and 25% respectively in drought(70%~95% relative water content), high saalinity(200 mmol/L) and cold tolerance(4~8℃) of transgenic plants, and this phenotype was well confirmed in transgenic plants of T2 generation, meanwhile, transgenic plant was almost not different to control in some field characters. Subcellular localization assay of OsPHD1 by using transient expression of OsPHD1::GFP fusion protein in onion(Allium cepa) cells revealed nuclear localization of OsPHD1 protein. Results of qRT-PCR suggested that OsPHD1 protein regulated the anti-reversibility of plants by regulating the stress response of gene expression. Results imaged that OsPHD1 gene has an important application prospect for the anti-reversibility of palnt breeding.

Nicotinamide adenine dinucleotide phosphate(NADPH) thioredoxin reductase from chloroplast contains reductase domain and thioredoxin domain. Each domain has the two catalytic cystein residues. Using RT-PCR method, the gene encoding the mature NADPH dependent thioredoxin reductase from chloroplast(NTRC) was cloned from maize(Zea mays) young leaves. The catalytic cysteine residues in reductase domain, and thioredoxin domain were mutated into serine residues, respectively. The wild and mutated genes were inserted individually into the expression vector pET-28b and the constructed vectors were transformed into Escherichia coli strain BL21 (DE3). The recombinant proteins were fused with histidine-tag at N terminus. SDS-PAGE analysis showed that the soluble expression level of the recombinant NTRC wild type was affected by the inducing temperature, and co-expression of molecular chaperones including GroEL, GroES and GrpE. The mutated proteins were expressed mainly as inclusion bodies. The recombinant NTRC wild type was purified by Ni-NTA affinity chromatography. The molecular weight of the NTRC subunit was estimated about 52 kD, as shown by SDS-PAGE. The activities for both domains of NTRC using DNTB and insulin as the substrates were determined respectively. The results indicate that maize NTRC is overexpressed in the soluble form in E. coli. The purified recombinant protein display the dual function of thioredoxin and thioredoxin reductase.

Wheat 1B/1R translocation lines have been widely used in China because of their high yield and wide adaptability. However they have a common defect that is bad grain processing quality. ω-secalin is believed to be an important factor for the bad quality. It is a good strategy to silence these ω-secalin genes through RNAi. Using ω-secalin gene's promoter to construct the RNAi expression vector can make the introduced gene to express in needed tissue and needed time and thus raise the effect of molecular breeding. In order to get a active promoter from ω-seclin genes, one pair of specific primers was designed. Promoter cloning was carried out with Lankao 906 as the material, a wheat (Triticum aestivum) 1B/1R translocation line. Five promoters A9-1, H2-1, H7-1, C11 and F11-1 corresponding to three active ω-seclin genes were obtained. Two promoters A9-1 and F11-1 corresponding to two active ω-seclin genes were chosen to make expression vectors with GUS as the marker gene. Transient expression analysis of the GUS gene was made by bombarding young wheat seeds and one promoter F11-1's activity was confirmed. The results provide the basic data for the construction of RNA interfering expression vector driven by the ω secalin gene itself promotor.

For investigated the expression level of adipocyte fatty acid binding protein gene (FABP4) at different stages of sheep longissimus muscle, in this paper, the cDNA of sheep(Ovis aries) adipocyte fatty acid binding protein gene (FABP4 ) was cloned by the method of RT-PCR using designed primer pairs of Ff and Fr according to the FABP4 gene cDNA conservative region. The putative protein spatial structure of FABP4 was analyzed by biology software. Additionally, the expression of FABP4 in sheep longissimus muscle and the correlation between FABP4 expressions of longissimus muscle and intramuscular fat contents at different days were tested by real time RT-PCR. The results showed that the sequence of cDNA of sheep FABP4 was 464 bp containing a 399 bp of open reading frame which encoded 132 amino acids and the amino acid of FABP4 was conserved in evolution, and the FABP4 protein was assembled as a barrel conformation by two α helixes and ten β folds. The real time RT-PCR revealed that expressions of FABP4 in longissimus muscle from 160 and 200-day's sheep were higher than those from 90-day's (P<0.05), expectively. The expression of FABP4 longissimus muscle at different days was positive correlated with longissimus muscle intramuscular fat contents (R2=0.7196, P<0.01). The result indicated that regulated the expression of FABP4 in sheep longissimus muscle is a potential approach for improving sheep meat quality traits.

The study is conducted to investigate the association of B2M polymorphism with FcRn expression, in hope of improving the transportation of IgG in milk. The mammary gland samples were collected from 40 healthy Chinese Holstein cows immediately after slaughter and preserved in liquid nitrogen. We extracted total DNA from the frozen samples and designed specific primers to amplify B2M gene, followed by SNPs identification according to the sequencing results. The total RNA was purified from animal tissues according to the haplotypes of B2M, and then was transcripted reversely into cDNA. And the level of FcRn mRNA expression was detected by Real-time PCR. The results showed that there were three SNPs in B2M gene, which assembled four haplotype combinations. The expression of FcRn mRNA in H4 was significantly higher than that of the others(P<0.05), but there were no significant differences(P>0.05) between H1, H2 and H3. We conclude that the haplotype combinations of B2M have the effect on the expression of FcRn mRNA.

For deeply researching parthenogenetic development of cashmy goat oocytes, and offerring an efficient in vitro activated method for the somatic cell nuclear transfer of cashmy goat, the ovaries from slaughterhouse were used as material and cutted to collect oocyte, then the oocytes were matured in vitro. In this experement the matured oocytes were treated with IA23187＋ 6-dimethylaminopurine(6-DMAP), Ionomycin＋ 6-DMAP, 7% ethanol＋ 6-DMAP, IA23187＋ CHX, Ionomycin＋ CHX and 7% ethanol ＋ cycloheximide (CHX) respectively. Activated oocytes were cultured in vitro in modified synthetic oviductal fluid (mSOF) medium and assessed for cleavage, 8-cell stage (48 h) and the blastocyst stage (7~9 d). The cleavage rates were lower significantly in IA23187＋ 6-DMAP(89.6±4.9%) compared to Ionomycin＋ 6-DMAP(99.1± 0.8%), IA23187＋CHX(97.6±1.2%), Ionomycin＋ CHX (99.3±0.6%) and 7% ethanol＋ CHX(97.5±1.4%)(P＜0.05), but no significantly different with 7% ethanol＋ 6-DMAP(96.3±0.5%, P > 0.05); The 8-cell stage rates were higher significantly in IA23187＋ CHX(81.3±5.8%) and Ionomycin＋CHX (82.5±6.6%) compared to other treatments(48.4± 4.3, 42.5± 1.8, 38.2± 2.1 and 62.5± 2.2%, respectively, P＜0.05); The blastocyst rates were higher significantly in IA23187＋ 6-DMAP(22.8± 2.4%) and Ionomycin＋ 6-DMAP(20.6±4.8%) compared to other treatments (10.5±1.8, 8.4±1.2, 11.8±2.0 and 8.8±2.5%, respectively, P＜0.05); The cell number of blastocysts were not significantly different among treatments 200.0 ±14.0, 169.6 ± 11.4, 187.0 ± 8.8, 201.3±16.5, 179.6±5.2 and 177.6±20.8, respectively, (P > 0.05). All groups of IA23187, Ionomycin and the combination of ethanol and CHX used to activate goat oocyte could obtain higher rate of oocyte cleavage and higher 8-cell rate, the combination of IA23187 and 6-DMAP group and the combination of Ionomycin and 6-DMAP group could obtain higher blastocyst rate, and the blastocyst from different actived group all had higher cell number, the group of either IA23187 or the combination of Ionomycin and 6-DMAP was the best method to activate goat oocyte, and they were also the better in vitro activated method for the technology of somatic cell nuclear transfer, the results of this resarch can offer reference for parthenogenetic development and in vitro activation of goat oocyte.

Resistin play important roles in organism lipometabolism and energy metabolism. The present work was used to study the effects of resistin on proliferation and differentiation of porcine preadipocyte. In this paper, the preadipocytes were isolated from subcutaneus adipose tissue of 1-day-old crossbred piglet, and the preadipocytes were respectively cultivated in the DMEM/F12 culture media containing 0, 50, 100, 150 and 200 μg/L recombinant resistin. The changes of the cell morphology were observed, and the triglyceride content and the activity of lipoprotein lipase of the cells were determined to evaluate proliferation and differentiation of porcine preadipocyte. Results showed that the proliferation of porcine preadipocytes were significantly promoted by 0 to 100 μg/L resistin supplementation in the media, the activity of lipoprotein lipase of the cells was also significantly increased. As the resistin concentration in the culture media came to 100~200 μg/L, the facilitory effect of resistin on proliferation of porcine preadipocyte was declined. Moreover, as resistin concentration increased, the differentiation of porcine preadipocyte was significantly strengthened, the intracellular triglyceride synthesize and fatty droplet aggregation were promoted simultaneously. The results indicated that the resistin has dual effects on growth of porcine preadipocyte.

In order to execute the labeling regulations and solve the inadequate availability of positive plant genome DNA, the existence of insect resistant rice (Oryza sativa L.) Bt Shanyou 63 has to be monitored, In this study, the complete sequence of foreign DNA insertion of transgenic cry1Ab/cry1Ac fusing gene rice line Bt Shanyou 63 transformed by particle bombardment method was determined using thermal asymmetric interlaced PCR (TAIL-PCR), Genome Walking and long distance PCR (LD-PCR). The Real-time PCR assay was established using the standard plasmid pMDBt63 containing 3' flanking sequence of Bt Shanyou 63 and rice endogenous gos9 as the reference material. The length of the complete sequence of foreign DNA was 9 818 bp, located at position 5348630 of chromosome 10 (AP008214) in rice genome and composed of eight recombinant fragments including two expression boxes of cry1Ab/cry1Ac fused gene at tail to head direction. The square regression coefficients (R2) of the two standard curves for gos9 and 3' flanking sequence were 0.9997 and 0.9992 and the efficiencies (E) were 98.05% and 99.46%, respectively. The detection limit (LOD) was 10 copies and the quantitative limit (LOQ) was 100 copies in 100 ng of DNA template for one reaction. In addition, two mixed samples with known Bt Shanyou 63 contents (5% and 1%) were quantified using the established Real-time PCR and the mean values were 4.98% and 1.07%, respectively. The above results showed that the established method employing pMDBt63 as the reference material can be used reliably for Bt Shanyou 63 quatification.

Adiponection (AdipoQ), a protein hormone secreted from adipose tissue, modulates a number of metabolic processes, including fatty acid catabolism and regulation of insulin sensitivity. The expression changes of AdipoQ and its receptor genes (AdipoR1 and AdipoR2) in longissimus dorsi and psoas major muscles of Rongchang (a fatty, indigenous, Chinese breed) and Landrace pigs (a leaner, Western breed) were measured using a Real-time fluorescence quantitative PCR approach. Results showed that the AdipoQ, AdipoR1 and AdipoR2 were ubiquitously expressed in all tissues analyzed, nonetheless, their mRNA abundances existed notable differences among pig breeds, genders and tissue types. The mRNA abundances of AdipoQ and AdipoR1 genes exhibited a negative correlation with the POI (porcine obesity index) and IMF (intramuscular fat) content traits, and a positive correlation with longissimus dorsi muscle area. Additionally, the mRNA abundance of AdipoR2 gene exhibited a strongly negative correlation(P<0.05) with IMF content. These results are in accordance with the well-annotated function of AdipoQ and its receptor genes, which increase fatty acid oxidation and decrease fat deposition, and highlight AdipoQ, AdipoR1 and AdipoR2 as candidate genes for porcine meat quality and carcass traits.

Genetically modified maize with high lysine LY038 has been widely applied in animal feed, but so far there has been no report about the method of amplification for flanking sequence of LY038 and its event-specific qualitative PCR detection. In this study, the 5'- flanking sequence of integrated gene-construct of genetically modified maize LY038 was characterized by modified adapter-linked PCR (M-AL-PCR). According to the 5'- flanking sequence, the event-specific primers were designed，and the event-specific qualitative detection of genetically modified plant LY038 was established to produce a 175 bp product. With genetically modified maize (Zea mays L.) LY038, MIR604, Bt176, Bt11, MON810, MON863, GA21, NK603, non-genetically modified maize, genetically modified rice (Oryza sativa L.) Cry1C* and Cry2A*, genetically modified soybean (Glycine max) Roundup Ready and genetically modified oilseed rape (Brassica campestris L.) GT73 as materials, this method developed in this work proved to be highly specific and sensitive, and the limit of detection was 0.1%. It is suggested that the developed qualitative detection system can accurately, fast and efficiently identify the genetically modified maize LY038 and its derivates.

Due to the characterization of infect a broad range of cell strains and high efficiency of integrate into genome of the cells, lentivirus vector has been widely used in preparation of transgenic animals. This research aims to produce a high level titer lentivirus with relatively lower cost, we constructed the lentivurus vector which contained the green fluorescent protein and red fluorescent protein, after transfect the three lentivirus vectors into the 293T cells with calcium phosphate transfection mathod, the transfection efficiency up to 72%, this method reduced the cost to produce lentivirus vector. We used high speed centrifugation and ultracentrifuge method to purify the lentivirus vector, after the purification and concentration we got the 80 μL purified lentivirus vector with the titer up to 2.38×109 TU/mL from 150 mL packaged culture medium. This lentivirus vector can be used for producting double foreign genes transgenic animals and comparing the effect of the two different report genes in screening of the transgenic offspring.

As one of the backbone parents of hybrid rice breeding at present, Shuhui 527(Oryza sativa) has many merits, such as high general combining ability, strong heterosis of hybrid combination, and many derivative R-lines. In order to clarify the genetic composition and the key genome regions related yield characters of Shuhui 527, the heredity and the entire genome of Shuhui 527 were researched in this study. The results showed that seed-setting rate, filled grain number per panicle, grain number per panicle and panicles were possibly derived from the pedigree of IR24-Shuhui527; the weight of a thousand seeds, plant weight and panicles were possibly derived from the pedigree of Gui630-R1318-Shuhui 527. The genomic map of Shuhui 527 and its related ancestral parents were constructed by scanning the entire genome with 1050 SSR primers, the results revealed that (1) the shared fragments of all varieties (i.e. none polymorphic fragments) accounted for 62.94%; (2) putative multi-originated fragments (polymorphic marker was unable to identify its origin) accounted for approximately 17.53%; (3) in the breeding process of Shuhui 527, R1318 had contributed 13.68% fragments, Fu36-2 had contributed 0.53% fragments, and IR24 had contributed 1.32% fragments; (4) Shuhui 527 had contributed 4% of itself fragments. The results indicated that the key genome regions of Shuhui 527 is initially determined, and some influencing output candidate QTL position spots of key fragments are found according to the analysis of the results of reported yield QTLs. The present study will benifit to the core parent development and supper hybid rice breeding.

The trichome has diverse biological functions in tomato, in order to explore the gene functions involved in trichome formation, a novel gene (named as ShMYB1) related trichome initiation was isolated from LA1777, using RACE (rapid amplification of cDNA ends) technology. The full-length sequence of ShMYB1 cDNA was 1 350 bp and encoded a protein of 338 amino acids. The ShMYB1 had a conserved R2R3 MYB domain and a specific subgroup 9 motif. The expression profiles of ShMYB1 were analyzed with Real-time PCR in different tissues and developmental stages, the results showed that the gene was expressed at a high level in leaves and flowers, but no expression was detected in roots, stems or fruits, and no significant differences at different stages of leaf development, but the highest expression occurred in young buds, gradually declined during bud development. Also significant differences of ShMYB1 expression were detected between several trichome mutants and their wild types. These results suggested that the ShMYB1 gene may be involved in the initiation of trichome and the development of flowers.

Quality properties of starch are strongly affected by the compositions of Wx genes in wheat. It is very important to establish stable and efficient methods and systems to detect Wx genes for improvement of quality and rapid evaluation of germplasm resources in wheat breeding programs. In this study, based on the compositions of Wx proteins in 173 wheat (Triticum aestivum L.) cultivars and advanced lines using SDS-PAGE method, the validity of sequence tagged sites (STS) markers for Wx genes were evaluated, and two types of multiplex PCR systems were built up. The results using the SDS-PAGE method showed that twenty of these cultivars and advanced lines were the null Wx-B1 type (11.6%), and one of them was waxy wheat lacked all three Wx proteins (Wx-A1,Wx-B1 and Wx-D1) (0.6%). The results identified by four co-dominant and two dominant STS markers for Wx genes were all identical to those detected by the SDS-PAGE method. Using the six effective molecular markers above, two multiplex PCR systems were established. The multiplex PCRⅠ could simultaneity identify four alleles at Wx-B1 and Wx-D1 loci, and multiplex PCRⅡ were used to detect four alleles in Wx-B1 and Wx-D1 loci. The genotypes of all wheat cultivars and advanced lines identified by the two multiplex PCRs were coincided with those detected by the SDS-PAGE and corresponding single PCR method, respectively. The six STS markers and two multiplex PCRs can be used to increase the efficiency of evaluation and improvement of wheat starch properties in breeding programs.

With the development of genetic modified plants and products, the urgent need is the standard reference materials for genetic modified detection. This work is about genetic modified maize NK603 standard plasmid construction and application. Genetic modified (GM) maize (Zea mays) line NK603 event specific product and endogenous gene zSSIIb product were amplified and ligated to T vector to constructed plasmid pNK603. And the pNK603 plasmid was successfully proofed by PCR amplification, digestion and sequencing. Results showed that the pNK603 plasmid was very specific for GM maize NK603 since there were no any other PCR product amplified using different primers except the specific main bands. Three different level of GM matrix samples were detected by quantitative PCR when pNK603 plasmid DNA and GM NK603 genomic DNA were used as quantification standards, respectively. The result showed that there was no statistic significance between the two different standards. T test showed the PCR efficiency, slop and intercept of the two standards for NK603 and endogenous gene respectively were not significantly different. The results indicated that pNK603 plasmid can be used as a standard for GM maize NK603 detection.

The aim of this study is to prepare the antiserums against chicken liver bile acid binding protein (L-BABP) and analyze expression characteristics of L-BABP. Specific primers were designed to amplify the coding region of chicken L-BABP by RT-PCR. And the L-BABP gene was then inserted into pGEX-4T-1 vector and expressed in Escherichia coli BL21 (DE3) with IPTG induction. Then the fusion protein was purified by Glutathione Sepharose 4B affinity chromatography and the antiserums against L-BABP was produced by immunizing rabbits. The results showed that a 38 kD (12 kD L-BABP+26 kD GST) fusion protein was induced. And the titer of the antiserum against GST/L-BABP was 1∶100 000 detected by indirect ELISA. Analysis of tissue expression showed that L-BABP specificly expressed in liver, and there were no detectable signal in kidney, lung, crureus, glandular stomach, pectorales, heart, fat, spleen, ileum, muscle stomach, jejunum and duodenum, respectively. In the current study, preparation of high titer and high specificity of the chicken L-BABP antiserum provides a good foundation for studying the biological function of L-BABP in the protein level.