5-enolpyruvylshikimate-3-phosphate synthase(EPSPS), which has an active site with glyphosate, can catalyze 5-enolpyruvylshikimate-3-phosphate(EPSP) synthesis with shikimate-3-phosphate(S3P) and phosphoenolpyruvate(PEP), and the enzyme activity can be inhibited when combine with glyphosate which are important in herbicide resistance genetic engineering of plants. To cultivate the maize(Zea mays) having resistance to glyphosate, we cloned Sorghum bicolor chloroplast transit peptide (CTP) of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. Expressional vectors were constructed after fused the CTP and Agrobacterium sp. strain CP4 EPSPS gene together, while using Ubiquitin as promoter and 35S polyA as terminator. The vectors were transformed into maize, with vectors that did not contain CTP fragment as control. Analysis of the transgenic plants by PCR, Southern blot and ELISA showed that: Although the gene expression level of plants from CP4 EPSPS and CP4 EPSPS with CTP was almost the same, plants having the CP4 EPSPS without CTP did not have resistance to glyphosate. On the contrary, plants having the CP4 EPSPS with CTP had strong resistance to glyphosate. These results indicated that CTP did not affect the expression of CP4 EPSPS, but played an important role in the resistance of glyphosate. In addition, we confirmed that the CTP we cloned had the right biology function. In this study, we provide a framework and inspiration for cultivating the crops with resistance to glyphosate by EPSPS.

Transcription factors WRKY are plant specific genes and participate in response to various biotic and abiotic stress. To investigate the role of the WRKY gene in wheat and explore their role in the respons to abiotic stress, in this study, a new wheat(Triticum aestivum L.) WRKY transcription factor, designated as TaWRKY51 was cloned by using RT-PCR, combined with RACE method. The full-length cDNA of TaWRKY51 was of 1 295 bp with a 942 bp of open read frame(ORF) encoding a protein of 313 amino acid residues. Length of 3'UTR and 5'UTR were 73 and 280 bp, respectively. Sequence and structure analysis indicated that TaWRKY51 possessed one WRKY domain and one C2H2 type Zinc-finger domain. BLAST analysis revealed that TaWRKY51 was most homologous to OsWRKY51 from rice(Oryza sativa)(66%), and AtWRKY11, AtWRKY17 from Arabidopsis thaliana(57%). Phylogenetic analysis revealed that all WRKY domain containing proteins could be grouped into three classes and TaWRKY51 belonged to the ClassⅡ because TaWRKY51 contained one WRKY domain and one C2H2 type Zinc-finger domain. Semi-quantitative RT-PCR analysis exhibited that the mRNA abundance of TaWRKY51 was relatively higher in leaves, root and inter-node, as compared to in seeds of 12 d after pollination. The expression level of TaWRKY51 gene was also higher in mature leaves and aged leaves than in young leaves. The results indicated that TaWRKY51 could be a positive regulator in leaves maturing and aging. And expression level of TaWRKY51 gene was upregulated after drought treatment which suggested that TaWRKY51 might be involved in abiotic stress response. Moreover, ecotopic overexpression of TaWRKY51 in A. thaliana significantly increased the number of lateral root, suggesting TaWRKY51 might participate in lateral root regulation in A. thaliana. Remarkably, transgenic lines grown on MS medium containing ABA, mennitol and NaCl were much more sensitive to ABA, drought and salt stress as compared to WT, and transgenic lines grown on soil could not survive after salt and drought stress. These results indicated that TaWRKY51 could be a negative regulator in stress signal transduction pathway. All together, in the present study, we identified a new wheat WRKY gene and analyzed its mRNA expression pattern in different tissues, in leaves at different growing period, and in response to abiotic stress. The results revealed that the gene may play a role in abiotic stress response to environment. These data enable us to better understand the underlying molecular mechanism of TaWRKY51 in plant lateral root development and abiotic stress response.

RNA interference (RNAi) is a powerful technique for functional genomics research in insects. Feeding dsRNA via an artificial diet has been successfully applied in many insects, including Hemipteran insects. However, transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been only reported for Lepidopteran and Coleopteran insects. In this study, we synthesized the dsRNAs of two genes (tubulin alpha-1 chain-ds1 and tubulin-specific chaperone c-ds10) of Nilaparvata lugens. When nymphs were fed on an artificial diet including the dsRNA, expression levels of the targeted genes were reduced; lethal phenotypic effects after dsRNA feeding were observed simultaneously. The reduction of target genes in N.lugens was observed only in a few strains after the nymphs were fed on rice(Oryza sativa L. ssp. japonica cv. Zhonghua 11) plant materials expressing N.lugens homologous dsRNAs. In this study, the generated dsRNA transgenic rice haven't shown lethal phenotypic effect, but when insects were fed on artificial diet with dsRNAs, the target genes transcript levels were suppressed and it shown lethal phenotypic effect. This result indicated potential of practicability on cultivating transgenic rice resisting to brown planthopper depends on techinical improvement, so it provides the basis for the cultivation of Brown planthopper-resistant transgenic rice.

It has been documented that pyruvate dehydrogenase kinase(PDK) is involved in the regulation of pyruvate dehydrogenase complex activity in mitochondria. However, very little is known about the regulatory mechanism of PDK. Our previous study showed that there was difference in the expression of TaPDK between genetic sterile line and physiological sterile line. To further study the acting factor regulating TaPDK during anther abortion induced by SQ-1, the interaction protein of TaPDK was screened via yeast two-hybrid technique. The yeast (Saccharomyces cerevisiae) strain Y2HGold containing pGBKT7-PDK plasmid and, library yeast strain Y187 were cultured by yeast mating. Then, the mated culture was incubated on SD/-Ade/-His/-Leu/-Trp plate before selection of clones with diameter larger than 2 mm, and further incubated on SD/-Ade/-His/-Leu/-Trp/AbA/X-α-Gal plate for screening blue clones. A new binding protein proliferating cell nuclear antigen was obtained, and was designated as proliferating cell nuclear antigen(TaPCNA). The results from pollen development and quantitative expression indicated that the expression of TaPCNA was scarcely expressed in leaf, and mainly expressed in different stage of pollen development. Especially, its transcripts accumulated predominantly in physiological sterile line at bicellular and tricellular stage, which suggested that TaPCNA was closely involved with the cell cycle of microspore development, and provided the basic date for further study of the abnormal cell cycle of microspore development in physiological sterile line.

α-mannosidase(α-man), a key enzyme in the process of N-glycan, was reported to play an vital role in controlling fruit softening and extending shelf life. This is of important significance to make clear the physiological function and regulation mechanism of α-man during peach fruits softening for further clarifying the softening mechanism. A full length of α-man gene was cloned from mature peach (Prunus persica cv. Shahong) fruit by RT-PCR and RACE(rapid amplification of cDNA ends) methods, and its transcript levels under different treatments (ethrel and 1-methylcyclopropene(1-MCP) at room temperature) on peach fruit were further detected by Real-time quantitative PCR(qRT-PCR). The results showed that the obtained α-man gene, named Pp-α-man (GenBank accession No.JX310861), was 3 491 bp in length, with a single 3 075 bp opening reading frame that predicted to encode 1 024 amino acids, which contained a NXT/S glycosylated site in the N-terminal region, and belonged to glycosyl hydrolase 38 family. qRT-PCR showed that the expression of α-man in control fruits increased to the first peak at 2 d after harvest. While in ethrel treatment, it reached to the maximum at the first day, and the peak was significantly higher than that in the control. While in 1-MCP treatment, the peak time of α-man's expression was delayed 1 day and the α-man's expression was suppressed through the whole softening progress, especially at later stage. Therefore, the expression of α-man in peach fruits is concluded to be regulated by ethylene and ethylene signal transduction, and then participates in the softening process.

Ethylene is an important plant endogenous hormone which controls fruit ripening and senescence, and 1-aminocyclopropane-1-carboxylic acid oxidase(ACO) is one of the key rate-determining enzymes in the biosynthesis of ethylene. VvACOs is coded by a small gene family, and the highest expression of VvACO1 and VvACO2, which are key genes regulating ethylene production changes during berry fruit development, is observed in the stage of veraison. In order to verify the influences of over-expression of VvACOs on ethylene release rates in tomato(Solanum locopersicum), Grapevine 1-aminocyclopropane-1-carboxylic acid oxidase (VvACO) genes were introduced into tomato genome by Agrobacturium mediated transformation method. The transgenic tomato plants were further verified with PCR and RT-PCR analysis, and ACO enzyme activities and ethylene release rates in the leaves of transgenic tomato plants were measured by gas chromatography. RT-PCR analysis indicated that three, four and six transgenic tomato lines with over-expressing VvACO1, VvACO2 and VvACO3 were obtained, respectively. The ACO enzyme activities and ethylene release rates in the transgenic plants were higher than those in non-transgenic tomato leaves and fruits. The highest ACO enzyme activities and ethylene release rates in the leaves and fruits of tomato transgenic lines with over-expressing VvACO1 and VvACO3 were observed, respectively. The transgenic tomato plants which over-expressing both VvACO1 and VvACO3 were normal morphological growth. The lowest ACO enzyme activities and ethylene release rates were observed in the tomato transgenic lines with over-expressing VvACO2, and the VvACO2 over-expressing plants were shown dwarf phenotype. Transgenic tomatoes that over-expressed of the each of the VvACO genes were analyzed for their growth physiological indexs, ethylene release rates, and ACO enzyme activies. Based on these results, functions of VvACO genes were preliminarily predicted.

A method for producing triptolide and alkaloid by Tripterygium. wilfordii Hook.f. suspension culture had been founded, but the secondary metabolites content is low that produced by this method, so the terpenoids secondary metabolites content in T. wilfordii Hook.f. suspension culture system were detected and analyzed by using ultraviolet-b(UV-B) radiation and ultrasonic treatment in this study. UV-B radiation and ultrasonic treatment were conducted to the cell suspension culture system of T. wilfordii at different culture period and treated time. To get the optimal treatment condition, HPLC(high performance liquid chromatography) was used to determine the content of triptolide, wilforgine and wilforine in suspension cell and culture medium of T. wilfordii. The results showed that UV-B radiation and ultrasonic treatment could improve the biomass of suspension cell and the content of the secondary metabolites in T. wilfordii cell suspension culture. After UV-B radiation and ultrasonic treatment, the biomass of suspension cell of T. wilfordii was about 1~2 times higher than that of control. After UV-B radiation for 90 min or ultrasonic treatment for 40 s at 13 d in the cell suspension cultivate period of T. wilfordii, the content of secondary metabolites production in the suspension cell of T. wilfordii reached to the highest of 5.185, 39.747, 42.189 μg/L and 5.185, 40.080, 45.472 μg/L, respectively. It was about 1~3 times higher than that of control. Ultrasonic treatment could promote the secretion of the secondary metabolites of T. wilfordii into the cell culture medium. After the detection found that the conversion rate of triptolide(93.7%), wilforgine(86.6%) and wilforine(88.1%) reached to the highest when treated by the ultrasonic treatment for 40 s respectively. Compared with the conversion rate of control, those conversion rate increased 1~3 times. The results provide foundation for solving the shortage of natural resource and production of triptolide, wilforgine and wilforine in T. wilfordii by using T. wilfordii cell suspension cultivation.

Peroxisome proliferator activated receptor γ(PPARγ) plays an important role in the metabolism process of fat and glucose. In order to explore the function of PPARγ gene during lacation in dairy goat(Capra hircus), the experiments were designed to clone the whole cDNA of PPARγ gene by RT-PCR and RACE from Xinong Saanen dairy goat mammary gland, to analyze its expression in ten tissues and mammary tissue in different lactation periods and to detect the expressions of fatty acid metabolism related genes in mammary epithelial cell treated with rosiglitazone(ROSI) by qRT-PCR. The whole cDNA sequence of PPARγ gene was isolated from the dairy goat (GenBank accession NO.HQ589347). Homology alignments revealed that PPARγ gene was conservative among the mammalian. Structure prediction showed that there were two zinc finger structures and a ligand binding domain in the dairy goat PPARγ protein. Tissue spectral analysis showed that PPARγ gene had the most abundant expression in adipose tissue, followed by the rumen and the minimal expression was detected in muscle. Expression analysis in the two different lactation periods of mammary tissue revealed that the PPARγ mRNA level during lactation peak was about twice than that of dry period. After treatment with rosiglitazone， a specific agonist， in goat mammary epithelial cells, some fatty acid related genes such as LPL(lipoprotein lipase ), FABP3(fatty acid binding protein), ADRP(adipose differentiation related protein) and TIP47(tail-interacting protein 47) were up-regulated significantly. These results indicate that the PPARγ gene may play an important role in fatty acid metabolism during the dairy goat lactation process, and provide the basic data for further research in regulating milk fat, improving the quality of milk and developing mammary gland bioreactor in the individual level.

Serum albumin(alb) is the majority transport protein in serum, which mainly is synthesized by the liver. The objective of this study was to identify porcine serum albumin gene(alb) promoter region and its main regulation elements, to quest the alb gene’s expression and regulation mechanisms. Firstly, endogenous expression level of alb from different tissues of pig(Sus scorfa) and different cell lines of hepatic(hepa1-6) and porcine embryo fibroblkast (PEF) were analyzed by quantitative RT-PCR(qRT-PCR); Luciferase reporter vectors containing five different lengths of pig alb promoter region were constructed by cloning, DNA sequencing and other methods. Hepa1-6 and non hepatic PEF cell lines were transfected with these 5 vectors above-mentioned, then functions of different alb promoter region were validated through the observation of relative activities of luciferase reporter genes. Our results demonstrated that the pig alb gene showed a unique liver-specific expression pattern; the dual luciferase reporter assays revealed core region of alb promoter was located between -184 to -31 bp, deletion of this area would lead to loss of function. Between -184 ~ -2034 bp there existed some potential positive and/or negative regulatory elements involved in the regulation of pig alb gene expression. In this study five dual luciferase reporter vectors containing different fragments of Wuzhishan pig alb gene promoter were successfully constructed, core region of the promoter of the Wuzhishan pig alb gene were identified and the main regulatory elements within 2 kb upstream of the start codon were predicted. These studies may contribute to our understanding of the effective mechanisms of pig alb gene promoter, as well as construction of liver-specific expression vectors for further exogenous gene studies.

To establish DNA database for individual identification and parentage analysis system in Chinese Hostein bulls, eleven microsatellites(BM1824、BM2113、ETH3、ETH10、ETH225、INRA023、SPS115、TGLA53、TGLA122、TGLA126 and TGLA227)recommend by International Society of Animal Genetics(ISAG) and four microsatellites(MAF45、MCM158、UNM0108 and UMN0929) selected in Y chromosome were used to examine 2 150 Holstein bulls(Bos taurus) with fluorescence primer PCR and genetic analysis equipment of ABI3130, discussing feasibility on individual identification and parentage testing in cattle. Results showed that mean of heterozygosity was 0.766 and polymorphism information content(PIC) with 0.738 in 15 microsatellites markers. Largest mean of PIC in MCM158 marker was 0.866, and lowest mean of PIC in SPS115 marker was 0.588, belonging to high polymorphism markers. Combined power of discrimination, combined power of exclusion and probability of identity of 15 microsatellites markers were 99.99%, 99.99% and 1.62×10-17, respectively. Therefore, effectiveness and reliability of 15 microsatellites markers were high for individual identification and parentage analysis. In addition, DNA database for individual and parentage identification was established in this study, and progress was made in the standardization, normalization and simplification for individual identification and parentage analysis.

Bacterial fruit blotch(BFB) is a threatening disease caused by Acidovorax citrulli and it can seriously damage leaves and fruit of watermelons and melons, resulting in reduction of production. So far, the research on this disease has mainly been concentrated in the advances in the identification of pathogens, detection, prevention and the identification of cultivar resistance. However, pathogenesis of pathogens is still poorly understood. In this study, we generated a transposon (Tn5) mutant library on the background of strain xjl12 of Acidovorax citrulli and screened it for reduced virulence by seed-transmission and injection inoculation with melons (Cucumis melo var. saccharinus). Meanwhile, we obtained mutant and complementary type and determined pathogenicity, growth, motility, etc, in order to explain the pathogenic mechanism from the perspective of molecular genetics. The results showed that the identification of a Tn5 mutant with reduced virulence was impaired in pdxJ, which was involved in pyridoxal phosphate biosynthetic protein. Further characterization of this mutant revealed that Acidovorax citrulli required pdxJ for pathogenicity, flagellar, growth capacity and levels of biofilm formation. By exogenous VB6 the mutant could recover capacity for pathogenicity and growth. Therefore, it is demonstrated that biosynthesis of pyridoxal phosphate (commonly known as VB6) plays an important part in pathogenicity, growth, formation of flagellar and so on.

Albendazole which is a broad-spectrum antiparasitic drugs, plays an important role in aquaculture. But that is overused and inpoured into water directly can has bad effects on human health through the food chains. In order to isolate an albendazole-degrading bacterium, a strain named ZS-1 which could use albendazole as the sole carbon source was isolated from albendazole-contaminated samples. Strain ZS-1 was finally identified as Rhodococcus sp. based on its morphological, physiological,biochemical tests and 16S rRNA gene sequence analysis(GenBank accession: No. KC763472). Degrading characteristics of strain ZS-1 to albendazole showed that ZS-1 could effectively degrade 100 mg/L albendazole in mineral salts media(MM) with albendazole as the sole carbon. Strain ZS-1 could degrade more than 90.6% of 50 mg/L albendazole within 3 d. The best pH and temperature for the albendazole degradation by strain ZS-1 were 7.0 and 30℃, respectively. The ion Cu2+ had obvious inhibition on the degradation of albendazole by strain ZS-1. Our research provide theoretical basis to bioremediation technology of albendazole pollution.

Potato late blight caused by Phytophthora infestans is one of the most destructive diseases. Crop losses and costs of late-blight control constitute a significant financial burden on the potato industry. However, understanding the mechanisms, processes and rates of P. infestans evolution are important factors in predicting the durability and effectiveness of new management practices. Microsatellite marker is regarded as a perfect molecular marker for studying biogenetics, syngenetic succession of population and diversity. Hence, microsatellite marker developed based on the genome system is particularly important for understanding of the genetic diversity of P. infestans. Single-copy microsatellite (more than 25 bp in microsatellite sequence length and more than 20 bp in adjacent interval) sites and sequences at both ends were screened in the genome of P. infestans by using SciRoKo, Clustal X and Primer Pair Specificity Checking from Primer-BLAST. And then the microsatellite specificity and polymorphism were detected by using 9 P. infestans strains of different years and geographical origins. Finally, 115 appropriate sites were obtained for developing microsatellite markers. Among them, 64 microsatellite markers were validated by feasibility test, including 33 polymorphic ones (14 intergenic ones, 13 Flank5', 7 Flank3', 15 exons and 6 introns) and 31 non-polymorphic ones(whose electrophoretograms included 4 situations——cluttering bands, no bands, bands appearing in some strains as well as three or more bands appearing in some strains). The determination of the number of alleles and evaluation of polymorphism were made for polymorphic microsatellite markers developed by using 40 P. infestans strains of different years and geographical origins. It was found that polymorphism–tagged microsatellite polymorphic information content(PIC) values ranged from 0.164 to 0.614. There were 7 markers with PIC≥0.5 (highly polymorphism), most of which belonged to the intergenic region; I-00408, I-10840 and E-04958 had 4 alleles, and I-01408, I-07111, I-06861 and F5-22735 had 3 alleles. There were 25 markers with 0.25＜PIC＜0.5 (moderately polymorphism) and there was only one with PIC≤0.25 (low polymorphism). The 9 polymorphic microsatellite markers were selected for genetic diversity analyses on 35 (from Hebei, Heilongjiang, Jilin, Yunnan, Sichuan, Chongqing, Ningxia, Inner Mongolia and Fujian) and 5 (from Poland, Switzerland and the U.S.) P. infestans strains of different years. UPGMA cluster analysis found a high genetic diversity of P. infestans in China and a significant interpopulation differentiation in different regions. Moreover, it was also found that P. infestans genome microsatellite genotype was correlated to its mating type and geographical origin to some extent but not correlated to metalaxyl sensitivity. These genomic microsatellites could provide rich and polymorphic markers for gene location, clone and genetic diversity of P.infestans.

The adenosine monophosphate activated protein kinase(AMPK)is a key regulator of catabolic and anabolic processes in energy metabolism. This study was conducted to construct and select the effective siRNA interference vector for porcine AMPKα gene. Two pairs of porcine AMPK α-specific double-strand siRNA(dsRNA) primers were designed and inserted into pSilencer 4.1-CMV neo Vector after annealing. The vectors were then transfected into porcine intramuscular preadipocytes by lipofection 2000, AMPKα and lipolitic enzyme genes HSL(hormone sensitive lipase), ATGL(adipose triglyceride lipase) and CPT1(carnitine palmitoyl transferase) mRNA expressions in these cells were detected by Real-time PCR. The results showed that the AMPKα-specific siRNA vectors RA1 and RA2 were successfully constructed and transfected into porcine intramuscular preadipocytes. The efficiency of RA2 was significantly higher than that of RA1. The mRNA expressions of AMPKα and the lipolitic enzyme genes HSL, ATGL and CPT1 decreased significantly after treatment of porcine intramuscular preadipocytes by vector RA2(P＜0.05). It indicated that decomposition of fat and fatty acid oxidation rate decreased in porcine intramuscular preadipocytes when AMPKα mRNA expressions was inhibited. These data will provide insight in the function of AMPK in fat deposition and manipulating gene expression of AMPK in regulating fat metabolism and meat quality in pigs.

Vibrio anguillarum, V. harveyi, Aeromonas hydrophila, Edwardsiella tarda and V. parahaemolyticus are five major bacterial pathogens isolated from marine aquaculture animals in China. In this report, five set of nested specific primers were designed based on the pathogenic factor genes and a specific amplicon rescue multiplex PCR (Arm-PCR) for the simultaneous detection of these five major bacterial pathogens were established. The concentration of primer Mix, Mg2+, dNTPs, Taq DNA polymerase and the annealing temperature in the first step of Arm-PCR were optimized in this study. With summarizing the results of the experiment, in 50 μL of reaction volume, the optimized parameters were 10×PCR Buffer(20 mmol/L Mg2+) 5 μL, dNTPs(2.5 mmol/L each) 5 μL, Taq DNA polymerase(2.5 U/μL) 0.6 μL, 10×primer Mix(2 μmol/L) 5 μL, each template 1 μL. The annealing temperature was 55℃. The result showed that the Arm-PCR reported here could produce specific amplicons in one tube simultaneously. The expected sizes were 144, 190, 266, 315 and 371 bp for V. anguillarum, V. harveyi, A. hydrophila, E. tarda and V. parahaemolyticus, respectively. The sensitivities of the Arm-PCR were 1.745, 1.847, 16.000, 28.126 and 369.900 pg to above bacterial genomic DNA. There were no cross reactions with genomic DNA from healthy fish and other common bacteria, such as Escherichia coli, V. alginolyticus, Pseudoalteromonas tetraodonis, Bacillus subtilis, accorrding to the Arm-PCR. In 2012, the established Arm-PCR method was applied to the detection of 24 bacterial isolates from diseased fish and five strains of E. tarda, three strains of A. hydrophila, two strains V. harveyi and two strains V. parahaemolyticus were identified. The results showed that the method was reliable and practicable. The Arm-PCR method reported here can be used not only for the simultaneous specific detection and epidemiological survey of above five pathogens in marine aquacultural animals, but aslo for the development of microarray detection methods in the future.