As a popular leafy vegetable, lettuce(Lactuca sativa L.) has been considered by scientists to be an ideal platform for plant bioreactor. In the present study, 2 lettuce cultivars(Grand Rapid and Hong Kong Glass)commonly cultivated in the production were evaluated which one was feasible to be served as explants for chloroplast transformation. To do this, the tissue culture regeneration systems of both kinds of explants were developed separately in which adventitious shoot buds were induced using young leaf explants of 2 cultivars placed on induction media supplemented with different concentration of hormone and plantlets were obtained on rooting medium. The Grand Rapid cultivar was chosen to provide explants for subsequent chloroplast transformation approach because of its higher frequencies of induction, regeneration and rooting. According to the published sequence of 16S-trnI-TrnA-23S region located in lettuce chloroplast genome, the homologous recombination sequence covering most of 16S-trnI-TrnA-23S region was obtained by PCR amplification using lettuce-specific designed primers, and then inserted T vector to form an intermediate cloning vector. Chemically synthesized expression cassette harboring gfp reporter gene and aadA resistant gene were inserted to the recognizing locus of Ecl136Ⅱ restriction enzyme located in above-mentioned homologous recombination sequence so as to finally finish the construction of specific expression vector PTLE. In the expression cassette, gfp gene represents the green fluorescent protein was used as a visible marker, and aadA gene confered the resistance to spectinomycin and streptomycin by adenylylation. The PTLE vector was transferred into chloroplast genome via particle bombardment, after 2 rounds of selection on medium supplemented with spectinomycin at concentration of 30 mg/L, the surviving plants were obtained on the resistant medium. PCR amplification and green fluorescence analysis confirmed that the regenerated plants were still heteroplastomic, meaning more works of selection were needed to reach the homoplasmy. Taking together, these results suggested that the basic approach of lettuce chloroplast transformation was established and pave the road for further achieving homoplastomic plants and extending to comprehensive understanding of plant bioreactor.

Homologous recombination (gene targeting) is an important technique that is used to modify mammalian genome. We constructed an efficient common gene targeting vector pGT-C1 based on the plasmid pEGFP-C1. The vector consisted two different multiple cloning sites(MCS), two locus of X-over P1 (LoxP) and positive and negative selection markers. Two positive selection markers, neomycin resistance gene (Neo) and aequorea coerulescens green fluorescent protein gene (AcGFP) flanked by two LoxP sites. The two synthesized multiple cloning sequences MCS1 and MCS2 were placed in outside of LoxP sites. Additionally, a negative selection marker HSV-tk (herpes simplex virus thymidine kinase) gene was located adjacent to MCS2. Each element was detected by enzyme digestion, and positive and negative selection markers were verified by adding geneticin(G418) or ganciclovir (GANC) to cell culture media, respectively. The cutting efficiency of Cre-LoxP system was measured by semi-quantitative PCR after transfected Cre expression vector. Finally, we built the cow (Bos taurus) beta casein targeting vector pGT-L-R, and tested the targeting efficiency after transfection and drug screening. Targeting vector pGT-C1 had the following unique features: it contains two multiple cloning sites (MCS), which facilitated the directional insertion of homologous arms and then enhanced the versatility of this vector. The green fluorescent tag AcGFP between two LoxP was used to monitor instantly the transfection efficiency and integration efficiency, and the selection markers was able to be removed from the recipient cells by Cre-LoxP system to avoid the potential damage of selection markers to the recipient cells and increase the efficiency of gene knockout. Collectively, we constructed a gene targeting vector pGT-C1 with high efficiency and flexibility, and subsequently produced the transgenic bovine fibroblasts via constructing the vector pGT-L-R target the β-casein gene. This work provides basic tool for the development of transgenic animal models.

In the insect growth and development stages, insect chitinase and chitinase-like proteins play an important regulatory role. In recent years, new technologies were applied for the structure of insect chitinase, time and space expression, tissue specific expression and biological function of insect chitinase, and got many new research achievements. In this paper, the structure of insect chitinases classification, physiological function, recombinant expression, methods of detection of enzyme activity and influencing factors of enzyme activity and other aspects are discussed and reviewed.

At present, the most widely used gene in commercialized transgenic glyphosate-resistant crops is 5-enolpyruvy-shikimate-3-phosphate synthase (EPSPS) genes from Agrobacterium tumefaciens CP4 and Escherichia coli, which results in narrow gene resource. In this study, aiming at developing new genes, Arabidopsis EPSPS gene was cloned and two amino acids (T178I, P182S) were point mutated via overlap extension PCR. The wild-type gene (AtEPSPS) and the mutant gene (AtTIPS) were introduced into EPSPS-defective strain E. coli ER2799, respectively. The glyphosate resistance of AtEPSPS and AtTIPS transformed ER2799 strains were tested at 0, 5, 10, 20, 50, 100 and 150 mmol/L glyphosate. AtTIPS transformed ER2799 grew normally in the medium containing 20 mmol/L glyphosate, while the growth of AtEPSPS transformed ER2799 was inhibited by 5 mmol/L glyphosate(P＜0.01). When the concentration of glyphosate was higher than 50 mmol/L, the growth of AtTIPS transformed ER2799 was also inhibited, without difference with AtEPSPS transformed ER2799. Meanwhile, we constructed two plant expression vectors containing AtEPSPS or AtTIPS, and introduced them into wild Arabidopsis thaliana via Agrobacterium mediated flower dipping approach, respectively. Seven AtEPSPS transformed lines and ten AtTIPS transformed lines were gained. PCR and RT-PCR analysis suggested that the AtEPSPS and AtTIPS genes were successfully integrated into the Arabidopsis thaliana genome and expressed correctly. We transplanted the transgenic plants to MS media containing 0.3, 0.5 and 1.0 mmol/L glyphosate, respectively, to test their resistance to glyphosate. All three glyphosate concentration led the leaves of AtEPSPS transformed plants and wild type plants to turn yellow, whereas AtTIPS transformed plants grew normally. RT-PCR results showed that there was no significant difference of transcriptional level of AtEPSPS and AtTIPS in transgenic plants. The survival rate and fresh weight of AtTIPS transformed plants were significantly higher than that of AtEPSPS transformed plants (P＜0.05). Whereas, the survival rate and fresh weight of AtEPSPS transformed plants were higher than that of wild type plants. These results suggest that the overexpression of AtEPSPS could only confer transgenic plants with low level of glyphosate tolerance; however, transgenic plants overexpressing AtTIPS could tolerate high concentration of glyphosate. So we can conclude that AtTIPS transformed plants performed higher glyphosate resistance than that of AtEPSPS transformed plants and wild type plants, and AtTIPS can be a candidate for the development of transgenic glyphosate-resistant crops.

To enhance the safety of piggyBac (PB) transposon system and weaken the genotoxicity of the helper plasmid in gene transfer, we try to use PBase N-terminal fused Human immunodeficiency virus (HIV)transactivator (TAT) peptide with prokaryotic expression to replace the helper plasmid and verify its activity in human embryonic kidney 293 cells (HEK 293 cells). Furthermore, we hope to establish an approach that TAT peptide mediates foreign proteins into eukaryotic cells. In present study, we constructed two prokaryotic expression vectors containing the plasmid pET28A-Pbase 1 to generate PBase N-terminal fused TAT peptide and the plasmid pET28A-Pbase 2 to generate PBase C-terminal fused TAT peptide. The PBase with N-terminal fused TAT peptide was successfully expressed by the plasmid pET28A-Pbase 1 in Escherichia coli BL21 (DE3) strain. Polyacrylamide gel electrophoresis (PAGE) and Western blot (WB) were used to confirm the PBase expression. After protein quantification, the PBase with 100 mg/mL in cell culture medium was transduced with the donor plasmid into HEK 293 cells. Furthermore, HEK 293 cells transfected the dual plasmid system of PB transposon were the positive control. PBase were determined by immunofluorescent staining (IF) in HEK 293 cells. Thermal asymmetric interlaced PCR (Tail-PCR) was used to detect the flanking sequences of PB integrate sites from purified genome of HEK 293 cell clones. After methylene blue staining for HEK 293 cell clones, Student’s t-test was employed to analyze the colony count. Results showed that the PBase was successfully expressed by the form of soluble protein. Simultaneously, we found that the PBase was localized in the nucleus from the result of immunofluorescent staining. Detection of flanking sequences indicated that the integration mechanism of PB was regarded as random recombination. Compared the dual plasmid system of PB, HEK 293 cell clones generated by the PBase with prokaryotic expression were significantly reduced and similar to negative control. These observations suggested that the PBase with prokaryotic expression lost its natural activity and caused the failure of transposition. Hence, further studies are necessary for this PB transposon system lead to transposition in other eukaryotic cells. However, the comprehensive information from this study supplied a reliable method for foreign proteins mediated with TAT peptide across eukaryocyte membranes.

Leaf area is critical to photosynthesis efficiency and a major trait affecting crop yield. Wild cucumbers (Cucumis sativus var. hardwickii) bearing little leaves, after domestication, the leaf area of cultivated cucumber (Cucumis. sativus var. sativus) is enlarged around 2 to 3 times. Previously, a major gene ll (little leaf) was mapped on chromosome 6. In this study, F2 population of 205 individuals segregating at leaf area by crossing from 2 cucumber lines XF-24 (little leaf) and DF-32 (large leaf). The result of normal distribution test by SAS showed that the area data of mature leaves from the same nodes of 205 individuals were in normal distribution, which characterized the trait of quantitative inheritance. In order to effectively accelerate the process of study, we utilized insertion and deletion(InDel) markers for gene mapping after the whole genome sequencing of parents of F2 population. InDels of the whole genome sequencing data from parents of F2 population were found in bioinformatics analysis. Eighty and eight InDel markers were evenly designed on all chromosomes by Primer Premier 5.0. These InDels were acted as primers to amplify the two gene pools of both large and little leaves, which came from bulked segregant analysis, the polymorphic InDels screened from the previous step were used to amplify DNA of F2 population, and 7 InDel markers identified on chromosome 7 were InDel-1, InDel-2, InDel-3, InDel-4, InDel-5, InDel-6 and InDel-7, respectively. Genetic linkage map was constructed by JionMap4 .0 and QTL locus was found by MapQTL4.0, using the result of PCR and traits of F2 population. Seven InDel markers listed above were contained in the genetic linkage map, spanning 22.1 cM. This result led to the discovery of a second major QTL (gene) controlling leaf area in cucumber, here designated ll2 (little leaf 2). Further analysis delimited ll2 into a 1.24 Mb genomic interval between the 2 markers of InDel-2 and InDel-4. ll2 was the first QTL found on chromosome 7 with a narrower interval, compared with the previous studies. Two major QTLs, ll and ll2, controlling leaf area in cucumber (Cucumis sativus L.)were found on chromosome 6 and 7, respectively. It is the 2 major ones that show the complexity of the genetic and molecular mechanisms of cucumber leaf area. Genetic mapping of ll2 will pave the way for deciphering the genetic and molecular mechanisms of leaf area in cucumber as well as for developing tools for molecular-assisted breeding.

MdMYB1 is an important transcription factor to promote the accumulation of anthocyanin on apples. The yellowgreen apple turns red after bag removal. In order to study the regulatory mechanisms of anthocyanin biosythetic genes expression, we analyzed the effects on the expression level of the MdMYB1 gene in peels of yellowgreen apple Golden Delicious and Mutsu, red apple Fuji and deep red apple Starking by light; The studys showed that the colouring rate and area on different varieties were related with the transcript expression rate and cumulation of the transcription factor MdMYB1. We studied the changes on the relative expression levels of the structural gene phenylalanine ammonia-lyase (MdPAL), anthocyanin synthase (MdANS), Chalcone isomerase (MdCHI) and flavonoid-3-O-glycosyltransferase (MdUFGT) during the period of the pigmentation by paper-covering treatment on Golden Delicious and Starking fruits. The analysis results showed that the transcription factor MdMYB1 and structural gene MdPAL,MdANS, MdCHI and MdUFGT expressed continuously in the process of colouring, but the Golden Delicious not. The expression patterns of the gene MdMYB1 was the same as the structural gene MdCHI, and was similar to the MdPAL. By analyzing the transcription factor MdMYB1 and structural genes MdPAL and MdCHI promotor sequence of the apple Golden Delicious, its transcription factor was MdMYB1-2 that promoter region had many G-box binding sites of lights. We inferred the phytochrome regulated the transcription factor MdMYB1-2 by its G-box binding sites. The gene MdCHI promoter region contained the Cis-acting elements MBS, and the MdPAL promoter region contained the Cis-acting elements MBS and MBSⅡ. These results showed the transcription factor MdMYB1 regulated the expression of the structural gene MdCHI , and it likely regulated MdPAL or was closely related. These results showed the Starking color completely in that it was likely related with continuous and coordinate expression of the transcription factor MdMYB1 and structural gene during the period of coloring, while the structural genes that was regulated by light-induced transcription factor MdMYB1 played a decisive role during the Golden Delicious apples coloring.

Oxalic acid (OA) is an important virulence factor of many plant fungal pathogens, such as Sclerotinia sclerotiorum and Botrytis cinerea. These oxalic acid-producing pathogens can secrete OA to acidify the host organs, leading plant pathogenic cell-wall-degrading enzymes to degrade plant cells more quickly and synergistically. Moreover, oxalic acid can combine calcium to form calcium oxalate crystals, thus destroying the host cell walls. Plant microRNAs are involved in the regulation of plant growth and development and play important roles in the biotic and abiotic stresses. In this paper, the differentially expressed microRNAs of 3-week-old Arabidopsis thaliana under the stress of 30 mmol/L OA were uncovered based on plant microRNA microarray. Three differentially expressed microRNAs were obtained, that was miRNA-2988 (Ptc-miR3911), miRNA-3090 (Ath-miR858) and miRNA-3131 (Ppt-miR1211). Among them, one down-regulated microRNA was the homolog of Physcomitrella patens Ppt-miR1211, whose target mRNA was an unkown gene At5g35753 based on psRNATarget, the other down-regulated microRNA was the homolog of Populus trichocarpa Ptc-miR3911, which was identical to Ath-miR399b, and its target mRNA was a ubiquitin-conjugating E2 enzyme At2g33770, The only up-regulated differentially expressed microRNA was Ath-miR858, whose target mRNAs were transcription factors At2g47460 (AtMYB12), At5g49330 (AtMYB111), At1g06180 (AtMYB13) and At1g66230 (AtMYB20). qRT-PCR analyzed the expression profiles of the target genes of the two down-regulated miRNAs, the expression of At5g35753 and At2g33770 was induced at 2 h, peaked at 12 h, and decreased at 24 h with the challenge of oxalic acid. qRT-PCR analysis also showed that Ath-miR858 was induced at 1 h, peaked at 12 h, and decreased at 24 h under the stress of oxalic acid, however, the expression of most of its target mRNAs were decreased at the early stage (within 2 h) of OA stress, indicating the negatively-regulated function of microRNAs. Moreover, the bioinformatic analyses were conducted with the promoters of Ath-miR858 and Ath-miR399b. Our results provide the foundation of microRNAs in the role of oxalic acid stress.

Basic helix-loop-helix proteins (bHLHs), as one of the largest families of plant transcription factors, play important roles in plant growth development, stress response and secondary metabolism. In this study, the codon usage patterns of 106 bHLH genes from Vitis vinifera were calculated and statically analyzed using CodonW and SPSS, as well as the major influence factors on codon composition and properties were discussed. The results indicated that there was a super significant level (P<0.01) between GC1 and GC2 codons of Vitis bHLH gene family. By two-tailed test, the correlation coefficient of GC3 and GC1 or GC2 was not significantly correlated (P>0.05). The correlation coefficients for ENC with GC1, GC2 and GC3 were 0.1439, 0.0463 and 0.1766, respectively, all of which did not reach the level of significance, and a significant one existed between ENC and GCtotal of 0.1989 (P<0.05) . Nineteen preferred codons were further determined to be bias toward the synonymous codons with G or C, suggesting that selection pressure and mutation may result in the codon bias in grapevine bHLH gene family. These results provide an important theoretical basis for further research on molecular evolution, gene expression regulation mechanism and higher accurate prediction of bHLH gene member, which will be applied in grape resistance improvement.

InDel(insertion deletion length polymorphism) is the third generation of molecular markers which is based on genome sequencing. It has already applied to the genetic studies. In this research, we use F1 of Chinese cabbage (Brassica rapa L. ssp. pekinensis) (Yuxin No.4), its parents and eight half-blooded hybrid combinations as to be experimental material.Using the third generation of molecular markers - InDel and primer combination method to identify Chinese cabbage hybrid's purity rapidly. The results showed that there were three pairs primers (BrID10667, BrID90107 and BrID90147) presented stable codominance among 104 pairs of screened InDel primers in Yuxin No.4. The purity of first batch of materials for three primers was 98.00%. The purity of second batch of materials for three primers was 100.00%, 98.30%, 99.10%, respectively. The results tested in field is 100%,which was highly consistent with the results tested in indoor. The match index of two results were up to 99.13%. The results showed that the InDel markers, as an accurate and cost- efficient means, could be suitable for rapid purity identification of Chinese cabbage hybrid seeds. Using three pairs of specificity InDel primers which were screened out from 104 pairs of InDel primers to analyse Yuxin No.4 and eight other half-blood cross combinations, respectively. None of this three pairs of primers could distinguish Yuxin No.4 and eight other half-blood cross combinations at the same time. However, in this three primers, the annealing temperature of primer BrID90107 and primer BrID10667 was the same, but their position of amplification production was different. So we used multiplex primers BrID90107 and BrID10667 to identify Yuxin No.4 from their parental lines and eight other half-blood cross combinations. The results showed that multiplex primers methods could be used for rapid indoor identification of Chinese cabbage hybrid purity. Using the method of multiplex primers to test different hybrid lines could identify the contamination which caused by F1- hybrid seeds effectively, which indicated that InDel markers an have a wide range of application in rapid laboratory test of seed purities of Chinese cabbage hybrid.

In order to study the mechanism of flue-cured tobacco aroma style formation that the same tobacco variety in different ecological area, we extracted chloroplast from tobacco (Nicotiana tabacum) leaves which were planted in the typical delicate aroma style ecological region Yuxi and the typical strong aroma style ecological region Guiyang, then used TCA-acetone method to extract chloroplast protein, by using two-dimensional electrophoresis combined with mass spectrometry to analysis chloroplast differential proteomics of typical strong aroma style and delicate aroma style tobacco leaves. Between the molecular weight 14.3~97.2 kD, in Guiyang the detectable protein spots was 1 270, of which the increased expression protein spots were 41, the specific expression protein spots were 10; in Yuxi the detectable protein spots were 892, of which the increased expression protein spots were 48, the specific expression protein spots were 4, the co-expression protein spots were 716. Using MALDI-TOF-TOF mass spectrometry and bioinformatics to compare the specific expression14 protein spots, Guiyang had 6 homologous matching protein, ATP synthase subunit beta, Rubisco and NADPH dehydrogenase; Yuxi had 1 homologous matching protein, plastidic aldolase NPALDP1. The closely related to the photosynthesis of protein specificity expressed in Hunan Guiyang; The closely related to glucose metabolism of protein specificity expressed in Yunnan Yuxi. To our knowledge，this is the first attempt to explore the mechanism of tobacco aroma style formation on a chloroplast proteome level, providing a theoretical basis for the breeding and production of Flue-cured tobacco.

As a member of transforming growth factor(TGF-β), myostatin(MSTN) plays an important role for the specific regulation of muscle growth of animals. The purpose of this study is to verify the efficiency of zinc-finger nucleases(ZFN) gene editing on porcine(Sus scrofa) and to pick out the efficient gene whicn includes targeting sites. Using the method of microinjection, ZFN plasmid were injected that were specifically targeted on MSTN into porcine oocyte parthenogenetic embryo . Then the MSTN sequence of cultured embryos were amplified and analyzed by PCR. The results showed that totally 373 embryos were injected, and 2 mutate sequences were found after the specifically amplification by MSTN primers which disigned according JN630464(GenBank accession number), 14 single base mutations were found in No. 39 embryo, and 11 single base mutations were found in No. 321 embryo. Both mutations occurred in ZFN targeting sites, the targeting efficiency was about 0.54%. The results ensure the possibility and provide feasibility analysis for the preparation of MSTN knockout pig by ZFN gene editing technology.

The Leptin gene, which is previously known as obese gene is the major candidate gene for production traits research of cattle and sheep. To analyze the association of Leptin gene polymorphisms and sheep (Ovis aries)production in Gansu Alpine Merino, the genetic polymorphisms of part of exon 3 of Leptin gene in Gansu Alpine Merino were detected by PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) and effects of different PCR-SSCP genotypes and alleles of Leptin gene with related growth traits were analyzed. The results showed that 2 SNPs (G271A and G433A) corresponding to 4 alleles (A, B, C and D) and 4 genotypes (AA, AB, AC and AD) were detected in the exon 3 of Leptin gene in Gansu Alpine Merino. Two alleles (B and D) and 1 SNPs (G433A) were firstly reported. The alleles A with the frequency of 0.6586 and genotype AC with the frequency of 0.4095, respectively, were the highest. The association analysis results of polymorphism and growth rate indicated that genotype AC influenced significantly the individual weight of 1, 2 and 4 months samples (P<0.05) than that of other genotypes. The allele C associated significantly weight and average daily weight gain of 1, 2 and 4 months samples(P<0.05). The samples with the allele A or the genotype AC had better weight than samples with other alleles and genotypes. The results of this study suggest that the exon 3 of Leptin gene is polymorphic in Gansu Alpine Merino, and the allele C and genotype AC can be used as effective genetic markers to improve the group production performance of Gansu Alpine Merino. The findings also provide basic data for Leptin gene genetic characteristics research of sheep.

Seven-band grouper (Epinephelus septemfasciatus) is a seawater fish with high economic values that grow fast and tolerate strong with low temperature. Population quantity is less in the natural environment, and the germplasm resources for long-term preservation are imminent. Therefore, based on the cryopreservation of seven-band grouper embryos, the effects of cryopreservation (-196 ℃) on the enzyme activity (superoxide dismutase (SOD), creatine kinase (CK), Na＋/K＋-ATPase, lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione peroxidase (GSH-Px)) in Epinephelus septemfasciatus embryos were studied. In the present study, the changes of enzyme activity in embryos were analyzed by kits to clarify damage mechanism of embryos. The results indicated that the MDA enzyme activity of embryos after cryopreservation treated with the vitrifications solution of PM(24% 1,2 - propylene glycol(PG)＋16% methanol(MeOH))、PMG(15.75% pylene glycol(PG)＋8.75% methanol(MeOH)＋8.75% glycerol(Gly))和PMGT(15.75% pylene glycol(PG)＋8.75% methanol(MeOH)＋8.75%glycerol(Gly)＋5% Trehalose) significantly increased compared with no cryopreservation groups. However, the activity of some other enzymes descended significantly before and after cryopreservation compared with control group (P<0.05), and the latter was significantly lower than the former. However, there was no significant difference between Na＋/K＋-ATPase enzyme activity of no-frozen embryos treated with PM vitrification group and the control group. From various enzymes activity change from seven-band grouper before and after cryopreservation, cryopreservation and cryoprotectants have significant effect on the enzymes activity of seven-band grouper. Therefore, through the detection and analysis of the concentration of enzymes activity change from frozen embryos, freeze made MDA enzymes activity increase, other 5 enzymes activity decreased. PM vitrification solution had less effect on CK and Na＋/K＋-ATPase, which showed it had certain protective function. Therefore, the research results provide a theoretical basis for cryopreservation of seven-band grouper embryos.

How to use exogenous enzyme effectively to degradate the protein in tobacco leaf,improving tobacco quality is a new research task in tobacco industry. A protease high-yielding strain was isolated from the surface of Yuxi tobacco(Nicotiana tabacum) leaves.The strain was cultivated and the protease was concentrated from the strain fermentation metabolites, protease activities and characterization of the enzyme were introduced in this paper. Orthogonal test was designed to detect the effection of enzyme quantity(40, 80 and 120 U/g)，temperature (25, 35 and 45 ℃), relative humidity(50%、60% and 70%)) and reaction time(30, 60 and 90 h) to tabacco protein, aroma component was also analzsed under optimal conditions. Results illustrated that the activity of Bacillus pumilus strain B1 was 4 200 U/mL, the highest activity was under the conditions of pH 6.0 and 50 ℃. The optimal conditions for the enzyme was enzyme quantity120 U/g, temperature 35 ℃, relative humidity 70%, reaction time 60 h, the degradability rate of protein was 18.64% and the amino acid increased 12.72%, GC/MS showed that the amount of volatile aroma content increased significantly. The results suggested that the enzyme produced by the strain B1 isolated from the surface of Yu Xi tobacco leaves can degrade protein in flue-cured tobacco leaves, increasing the availability of lower - grade variety of tabacco, improving the qudlity of tabacco leave. And The enzymes can be applied in industrial production.

Canine distemper virus (CDV) is a pathogen of acute infectious disease, which caused fever, diarrhea, pneumonia and central nervous system disorders in Canis and Felis. The phosphorylation protein (P) is important to replication and pathogenicity of CDV. In this study, according to CDV-P gene (GenBank No. AF164967), primers were designed by primer primer 6.0 software. The P gene was cloned by reverse transcriptional polymerase chain reaction(RT-PCR), and subcloned into plasmid of pET-28(a). The pET-28(a)-CDV-P was constructed and identified by enzyme digestion and sequencing. The pET-28(a)-CDV-P was transfected into Escherichia coli BL21 (DE3). The his-CDV-P was expressed in E.coli BL21 (DE3) by isopropyl-β-D-thiogalactopyranoside (IPTG) inducing. High purified protein was obtained by his-band Ni+ affinity chromatography. The mouse anti-CDV-P antibodies were prepared from balb/c mouse immunized by his-CDV-P protein . In CDV infected cells, the expression of CDV-P protein was detected by Western blot, and subcellular localization was detected by indirect immunofluorescence assay using mouse anti-CDV-P antibodies. The results showed that his-CDV-P protein was expressed by inclusion body and its molecule weight was 65 kD by SDS-PAGE. The expression of his-CDV-P protein was more than 35% of total bacterial protein, and up to 60% of total protein after purification by grayscale scanning analysis. Mouse anti-CDV-P antibody specifically recognized the prokaryotic expression products and viral infections products by Western blot. In the CDV infected cells, the P protein expressed in the cytoplasm and nuclei by indirect immunofluorescence assay. The results showed that the P potein had 2 forms (phosphorylation and non-phosphorylation) and CDV-P protein expressed in the beginning phase and late phase of post-infection, and the phosphorylation and expression level of CDV-P were gradually increasing in infection stage by Western-blot. In this study, recombinant protein CDV-P and mouse anti-CDV-P antibodies were prepared, expression and subcellular localization were detected. Therefore, those results provide basic date for studying phosphoprotein P functions and CDV pathogenic mechanism.