In this paper, we investigated the secondary somatic embryo induction from primary somatic embryo which developed at globular stage to cotyledonary stage. The highest secondary embryo induction rate of 85.61％ was obtained from the treatment with 1µM 6-BA. High content of the sucrose induced repetitive somatic embryogenesis which made the somatic embryo conservation easier. The addition of activated charcoal could increase the secondary somatic embryo induction rate but lowered the quantity of the induction. And the dedifferentiation step was achieved from different stage of the embryos. The optimal secondary somatic embryo induce treatment was obtained, which lays the foundation for genetic transformation.

Proteome difference between high royal jelly producing bees (A. mellifera L.) and Chinese honeybees (A. cerana cerana F.) was investigated by two-dimensional gel electrophoresis, and some proteins were deduced by comparing with the already identified royal jelly proteome . Significant higher protein spots (93) in royal jelly of high royal jelly producing bee were found than those of in Chineses honeybee’s (70). Their molecular weight ranged from 50 to 80 kDa, and pI 5.0-8.0. Thirty protein spots were resolved to two images, while others were unique to each sample. Comparing with the already identified royal jelly proteome, it can be deduced that these well resovled proteins were likely MRJP1 (major royal jelly protein 1), MRJP2, MRJP3 and glucose oxidase. Protein abundance analysis of proteins present on two samples showed that 4 proteins of Chinese honeybee significantly higher than those of high royal jelly producing bees, but 7 spots showed a reverse relation. The results suggest that there are significant differences between royal jelly from high royal jelly producing bees and Chinese honeybees in term of proteins composition and abundance.

The antagonistic strain (Bacillus subtilis) DS45-2 were cultured in NB medium for 48h at 30℃. The supernatant was collected by centrifugation at 8000r/min for 15 min at 4℃. The crude extracts of antifungal protein was obtained from the cell free supernatant of fermentation liqour of DS45-2 strain by ammonium sulphate precipitation. Assays for the antifungal activity of crude extracts were done on agar plates. The antifungal protein was found to be thermostable, resistant to pepsinum and trypsin, and partially sensitive to proteinase K. Its active pH range was wide, from 4.0 to 10.0. A antifungal protein purified by DEAE Sepharose Fast Flow chromatography and reversion phase chromatography, It’s molecular weight was estimated to be 23 k daltons by SDS-PAGE. It is significant to clone the genes because the antifungal proteins were greatly inhibitory to Verticillium dahliae Kleb.

Transgenic poplar (Populus tremula x Populus alba) with antisense CCoAOMT was planted in field for four years. The Klason lignin content evaluation showed that the transgenic tree had decreased lignin content by about 13% comparing to the wild control, which means that the decrement of lignin content was maintained in 4-year-old transgenic poplar. The lignin structure analyses by Thioacidolysis and GC displayed that G/S ratio in transgenic poplar was slightly decreased comparing to control. Histology examination of stem section showed that most of vessels in transgenic tree were regular similar to that in wild control, but some vessels transgenic tree were deformed with inward sunk walls. There were no difference in plant height and stem diameter between transgenic trees and wild control during four years of growing.

Here, we report a loop-mediated isothermal amplification (LAMP) assay with high sensitivity and specificity for the detection of Actinobacillus pleuropneumoniae. A set of six primers were designed, based on the ApxIV gene of A. pleuropneumoniae serotype 1. The detection limit of the LAMP assay completed within 45min at 63℃was 102 copies of the target sequence, which is 10 times more sensitive than the PCR assay. High species-specificity of the LAMP method were confirmed by the assay of 18 pathogens such as A. pleuropneumoniae serotypes 1~10, Pasteurella multocida, Streptococcus suis, Mycoplasma hyopneumoniae . In addition, All the nasal swab samples from 8 experimental infected pigs and 127 clinical cases were detected by LAMP assay, with a higher sensitivity than the PCR assay.

采用PCR技术从质粒pET-ChIFN-γ中扩增得到IFN-γ基因，将其亚克隆到真核表达载体pCAGGS中。将鉴定正确的克隆命名为pCAGGS-ChIFN-γ，体外转染CEF细胞，24小时后经间接免疫荧光（IFA）和免疫印迹（Western-blot）检测，表达蛋白具有良好的免疫原性。然后利用表达绿色荧光蛋白（GFP）的重组水疱口炎病毒（VSV*GFP）在CEF细胞上检测表达的ChIFN-γ抗病毒活性（AVA），经检测其抗病毒活性为2×103AU/mL，并且其活性可被抗鸡IFN-γ的多克隆抗体阻断。结果表明：ChIFN-γ在鸡胚成纤维(CEF)细胞中成功表达，并且表达的ChIFN-γ具有良好的抗病毒活性。

Five pairs of primers (P1-P5) were designed to detect single nucleotide polymorphism of neuropeptide Y-Y1 receptor (NPY-Y1R) gene in sexual precocity and high fecundity breed (Jining Grey goat) and sexual precocity and moderate fecundity breed (Boer goat) and sexual late-maturing and low fecundity breeds (Angora and Inner Mongolia Cashmere goats) by PCR-RFLP and PCR-SSCP. The results showed that only the products amplified by primers P2, P4 and P5 displayed polymorphisms. As for P2, three genotypes (AA, AB and BB) were detected in Jining Grey, Inner Mongolia Cashmere and Angora goats, only two genotypes (AA and AB) were in Boer goats. Sequencing revealed one mutation (C625G) in genotype BB compared with genotype AA, which caused no amino acid change. No significant difference (P>0.05) was found in litter size between AA, AB and BB genotypes in Jining Grey goats. As for P4, two genotypes (CC and CD) were detected in four goat breeds. Sequencing revealed one mutation (G1141A) in genotype CD compared with genotype CC, which caused no amino acid change. No significant difference (P>0.05) was found in litter size between CC and CD genotypes in Jining Grey goats. As for P5, two genotypes (EE and EF) were detected in Jining Grey goats, only one genotype EE was in other three goat breeds. Sequencing revealed one mutation (C1330T) in genotype EF compared with genotype EE, which caused no amino acid change. The differences of genotype distribution were highly significant (P<0.01) between Jining Grey goats and three late-maturing breeds, and no significant difference (P>0.05) was found between three late-maturing breeds. The Jining Grey goat does with genotype EF had 0.58 (P<0.01) kids more than those with genotype EE. These results preliminarily indicated that F allele of NPY-YIR gene was associated with sexual precocity and prolificacy in Jining Grey goats.

Antimicrobial peptides (AMPs) were regarded as important components of the host innate immune system and play crucial roles in host defence against microbial invasion. Hepcidin was an antimicrobial peptide and iron regulatory molecule primarily in liver. A number of hepcidin AMPs had been isolated from teleosts. In this study, one hepcidin gene was amplified from the liver of yellow catfish(Pelteobagrus fulvidraco)by means of reverse transcription polymerase chain reaction(RT-PCR).The length of hepcidin cDNA fragment （GenBank accession number EU257703）was 282bp with an ORF ranging from 1st to 279thbp, which encoded a peptide of 93 amino acids. According to the blast result in GenBank database, the cDNA nucleotides and the deduced amino acid sequence of ORF share 80-82% and 69-71% identity with those of other species in Siluriformes.The prepropeptide contained a signal peptide (23 amino acids),a prodomain(45 amino acids) and a mature peptide(25收 amino acids).The predicted 25-amino acid hepcidin mature peptide included 8 conserved cysteine residues which were proposed to form four disulphide bonds. RT-PCR demonstrated that the hepcidin gene was expressed in a wide range of tissues of yellow catfish. And the hepcidin transcripts were highly abundant in liver,abundant in head kidney, intestine, muscle, brain, stomach, gill, heart, ovary, less abundant in skin, spleen and trunk kidney. Challenge of yellow catfish with Aeromonas hydrophila and Bacillus cereus significantly elevated hepcidin mRNA levels in liver. The new member of hepcidin discovered in this study could contribute to the research of immune function and mechanism in yellow catfish.

采用磁珠富集法结合放射性同位素杂交法分离出鲤鱼(Cyprinus carpio)三、四核苷酸重复的微卫星阳性克隆1248个。对其中的384个克隆进行测序分析，共得到微卫星序列325个，其中完美型244个（75.08%），非完美型59个（18.15%），混合型22个（6.77%）。依据得到的微卫星序列，用Primer 3软件在线设计并合成引物145对，检测结果显示71.43%（80对/112对）的引物在野生个体间表现出多态性，等位基因数在3~12之间，多态信息含量在0.2109~0.8607之间，80%的位点处于高度多态水平。对群体的遗传结构评估结果显示，四核苷酸重复的微卫星标记在黑龙江鲤野生群体表现出的多态性水平（PIC=0.7740）高于三核苷酸重复（PIC=0.5161）和双核苷酸重复（PIC=0.49），适用于群体间遗传差异分析和遗传连锁图谱的构建。

本试验以来自安徽省郎溪县雁鹅原种鹅场的雁鹅为研究材料，应用ISSR技术对雁鹅种群的遗传多样性进行研究。筛选出43个ISSR引物，对94个供试样本进行了扩增，共扩增出216个位点，每条引物扩增出的谱带数在2~7之间，平均为5.0条；多态条带比率在16.67~100%之间，平均为65.60%，材料间遗传距离为0.2～0.45。用不加权算术平均组对法（UPGMA）对94个个体间的关系进行聚类分析，构建了雁鹅种群的遗传系谱图。

This study was conducted to determine the effects of supplementing different level of malic acid (MA) and unsaturated fat acid, including oleic acid (OA), linoleic acid (LA) and linolenic acid (LNA) on rumen fermentation and functional microbe in vitro. The result showed the concentration of propionate acid was significantly increased (P<0.01), total gas and CH4 gas production were obviously decreased, and other VFA were significantly decreased accompanied with increased unsaturation of fatty acid; Methanobactrium formicicum in LA and LNA was obviously increased （P<0.01）, Ruminococcus flavefaciens and Ruminococcus albus were significant increased (P<0.01) compared with CK. However, Anaerovibrio lipolytica and Butyrivibrio fibrisolve were decreased (P<0.05) than CK. Methanobactrium formicicum in 10 mmol/L MA-OA which was decreased obviously (P<0.05). The conclusion was unsaturated fatty acid and malic acid could effectively change rumen fermentation profile but there were no directly relationship between them to inhibit CH4 production, and no obviously effect of supplementing malic acid and unsaturated fat acid on main rumen microbe.

In order to develop a high effective gene transformation protocol of cotton hypocotyl pieces via Agrobacterium tumefaciens, an alflafa antifungul peptide gene (alfAFP) was used as a foreign gene, and effect factors of transformation, including Agrobacterium strains, bacterium density, infection time, acetosyringone (AS) concentration in co-culture medium, co-culture time and co-culture temperature, were devalued by the expression detection of GUS and hygromycin resistant gene in transgenic callus. Optimum conditions for transformation was that cotton hypocotyl pieces from 5 to 7 days-old sterile seedlings were dipped into Agrobacterium LBA4404 suspension (OD600=0.5-0.7) for 10-15 min, then were transferred on co-culture medium with 200µmol/L As and were co-cultured at 21℃ for 60h in the dark. The most amount of transgenic calli was gained by culturing hypocotyl pieces on callus introduction medium with 50mg/L hygromycin. 15 regenerative plants were developed from somatic embryos differentiated from transgenic calli on embryogenesis medium. PCR and PCR-southern detection proved that the antifungal gene was introduced in 12 regenerative plants. They might be used as germplasm resources for breeding resistant-fungal disease cotton in the near future.

To investigate the role of lipid transfer proteins in chilling resistance of blossom of Chimonanthus Praecox , we cloned three members of the LTPs family, namely CpLTPI.1, CpLTPI.2 and CpLTPI.3, by randomly cloning and sequencing, based on the cDNA library constructed from Chimonanthus Praecox flower and its EST analysis. The three genes have full-lengths of 611 bp, 1016 bp and 656bp, respectively, with similar opening reading frame(ORF) of 360 bp, 360 bp and 351bp, and have different length of 5′and 3′ untranslated region with different regulation motifs. The three genes, which belong to the nsLTPI family, have the typical structure of the plant nonspecific lipid transfer protein including 4 pairs of disulfide, 4 α-helices and 1 tunnel-like hydrophobic cavity which can contain lipid molecules. Real-time quantitative PCR analysis revealed that the three genes were differently regulated by drought, salinity, cold and ABA (abscisic acid), which suggested that the three genes would play different roles in water balance, chilling tolerance, metabolism and other physiological processes in Chimonanthus Praecox.

摘要：以龙眼正常成花花芽和成花逆转花芽为试材，应用cDNA-AFLP技术，研究龙眼正常成花与成花逆转花芽cDNA的差异表达，并利用RT-PCR技术验证，探讨了龙眼成花逆转的分子机制。通过64对引物组合的cDNA-AFLP分析获得了2 560条扩增片段。选择其中有明显差异的片段进行回收、测序，获得了14条核苷酸序列。其中有13个片段在GenBank数据库中发现同源序列，1个功能未知。通过核酸和氨基酸比对分析，与龙眼成花逆转相关的112基因片段同源于银灰杨（Populus tremula x Populus alba）中编码Nek激酶家族(NIMA related protein kinases)基因（83 ％）， 331的核苷酸序列（80％）同源于与拟南芥（Arabidopsis thaliana）中编码内切-1,4-β-葡聚糖酶(EGases)的基因，313的核苷酸序列（78%）同源于与柑橘（Citrus sinensis）中的几丁质酶（chitinase CHI1）基因。

The conservative region of Lipoxygenase（LOX）gene from peach(Ruipan 5) was amplified from fruit cDNA, which was combined with the ESTs of LOX genes from GeneBank, and design the GSP primers after Blast aligning and sequence install. Three full-length different cDNA sequences were obtained, which were named PpLox1-3. The result of analysis indicated the three members of peach LOX gene family have high homogeneous with other plant LOX genes in protein level, but high heterogeneous in nucleotide level. Phylogenetic tree based on LOXs in Peach and other species indicated that PpLox1 was belonged to sort of 13-LOX and PpLox2-3 were belonged to sort of 9-LOX.

Abstract: In research paper, by BLAST(Basic Local Alignment Search Tool) and HMW(Hidden Markov Wodel)analysis,a full set of disease resistance candidate genes encoding nucleotide-binding sites (NBS) in a complete genome(Mt2.0) of Medicago truncatula was identified and characterized by structural diversities, gene duplication，physical positions, phylogenetic relationships. We found 469 NBS-coding sequences which contain 446 regular NBS genes and 23 non-regular NBS genes.Meanwhile, We found significant excesses of duplications in the genome of Medicago truncatula after analysis of NBS standard physical locations and phylogenetic tree .These results suggested that gene duplication played a major role in disease resistance genes expansion .

Based on integrated DNAs construct of seven genetically modified maize, Bt11, Bt176, Mon810 , Mon863, TC1507, GA21 and NK603,event specific primers were designed for polymerase chain reaction .In addition, event specific oligo probes were constructed by analyzing integrated DNAs structure of genetically modified maize. All the event specific probes could be completed in one microarray . The results indicate that the sensitivity of oligonucletide microarray (0.01%) was higher than that of the electrophoresis method (0.1%),meanwhile the detection efficiency was highly improved by using multiplex PCR.

Construction of a mutant library is a fundamental approach to new genes and functional genomics of a plant pathogen. To rapidly and accurately identify a Tn5 insertion position in a corresponding gene in Xanthomonas oryzae pv. oryzicola, the critical pathogen of bacterial leaf streak in rice, thermal asymmetric interlaced PCR (TAIL-PCR) and Tn5 transposon rescue were adopted to verify 8 virulence-reduced mutants screened from the Tn5 inserted library of the pathogen based on virulence assay in rice. TAIL-PCR revealed that 5 virulence-reduced mutants were due to that the Tn5 transposon was inserted in the general secretion protein F (gspF), cAMP-regulatory protein, integral membrane protease subunit, S-adenosylmethionine decarboxylase proenzyme, and gpsJ genes, respectively. Tn5 transposon rescue indicated that the genes in other three mutants were inserted by Tn5 were pathogencity protein, conserved hypothetical protein, and flagellum-specific ATP synthase genes, respectively. Homology analysis demonstrated that eight genes inserted by Tn5 were 100% identities to the corresponding genes in the genome sequence of the strain BLS256. The results above suggest that TAIL-PCR and Tn5 transposon rescue are effective tools for identifying Tn5 insertion position of a target gene of a plant pathogen and for isolating the target and can be used coordinatively and cooperatively.

从实验室现有菌种资源中筛选出木聚糖酶高产菌株，发酵液酶活达到408U/ml；仅采用超滤和凝胶层析2个步骤即从发酵液中提取到电泳纯木聚糖酶，回收率高达69.17%，比活达到28453.6U/mg；用ESI-MS/MS法测得该酶氨基酸序列，与该研究室登陆GenBank的木聚糖酶基因表达的氨基酸序列一致；其酶学性质为：采用SDS-PAGE和凝胶层析测得分子量为22.54 kD、21.89 kD；IEF-PAGE测得等电点为9.63；以桦木木聚糖为底物时，Km值和Vmax分别为1.0mg/ml和33.3μmol/（min.ml），燕麦木聚糖的Km值和Vmax分别为0.5 mg/ml和33.3μmol/（min.ml）；最适作用温度60℃，稳定温度0℃~60℃；最适pH5.8，稳定pH3.4~6.4；Fe2+、Co2+有激活作用，Cu2+有强烈抑制作用。

The Mn-superoxide dismutase encoding gene(Mn-sodA) from Bacillus sp. 110-2 was amplified by PCR. The Mn-SOD gene was inserted in pET-30a(+) to yield the recombinant expression vector pET-sodA. Over-expression of Mn-SOD in E.coli BL21(DE3) was achieved with pET-sodA, which was induced by Isopropyl-β-D-galactosidase(IPTG). SDS-PAGE analysis showed an over-expressed recombinant product at about 26.5 kDa, consistent with the molecular weight predicted from gene sequence. The expression of Mn-superoxide dismutase were also discussed. The recombinant protein was soluble in BL21(DE3). The specific activity of Mn-SOD was 252 U/mg, which was 2.1 times than wild type, so the Mn-sodA had a high expression in E.coli; Furthermore, the recombinant enzyme reserved the significant alkali-resistance of wild enzyme. From the engineered strain-BL21（pET-sodA）, we could obtain a protein resources and a methods to extreme-enzyme production.

To clone and prokaryotic express the gene encoding Inosine 5-monophosphate dehydrogenase (impdh) of the Streptococcus suis serotype 2, and to investigate the immune antigenicity and enzymatic activity of gene product．The encoding impdh gene of 05ZYH33 strain isolated from patients in Ziyang county of Sichuan Province was amplified by PCR with the designed primers, and then cloned into prokaryotic expression plasmid pET28a, and the recombinant plasmid pET28a-impdh was transformed into E.coli BL21(DE3). After induced expression by IPTG, the fusion protein was purified by chromatography. The isolated IMPDH protein was analyzed with SDS-PAGE and Western blotting, and its enzymatic activity was measured at different pH and temperatures. High expression of IMPDH was obtained and the strongest enzymatic activity displayed at the optimal temperature and pH condition. The experimental results showed that the impdh gene 05ZYH33 can be highly expressed in prokaryotic system, and the recombinant protein showed specific antigenicity and had best enzymatic activity in optimal pH and temperature．

为研究鸡卵细胞卵黄生成受体（Occyte vitellogenesis Receptor , OVR）对鸡产蛋性能及蛋品质的影响，本试验以六个鸡种（洪山鸡、江汉鸡、双莲鸡、郧阳大鸡、郧阳白羽乌鸡、海兰褐）的一代混交群体390个个体为研究材料，采用PCR-SSCP、PCR-RF-SSCP及测序的方法进行鸡ovr基因内含子单核苷酸多态性（SNPs）位点筛选，并与鸡群蛋用性状进行关联分析。结果表明，在鸡ovr基因中发现内含子2 T3967G，内含子7 C8099T、A8296G，内含子9 A8839T、G9084A共5个突变位点；其中内含子2 HinfⅠ突变位点与开产日龄、蛋壳厚度相关极显著（p<0.01），与产蛋量、蛋重、蛋黄比例相关显著（p<0.05）；三种基因型中TT个体在开产日龄、平均产蛋量、蛋重、蛋黄比例等方面都优于其它基因型个体。本研究结果证实了其他学者研究的结论，即ovr基因对鸡的产蛋性能和蛋品质性状有重要的影响。

We analysised the genetic diversity and phylogenesis of 225 individuals of 9 sheep populations using mitochondrial D-loop sequences and microsatellite DNA. The results of the two methods in genetic diversity analysis were consistent. It was that Qinghai Tibetan sheep had rich genetic diversity, but Hu sheep and Minxian Black Fur sheep's genetic diversity were lower than others. While in phylogenesis analysis, phylogenetic trees based on mtDNA D-loop sequence and microsatellite DNA were different. Because microsatellite DNA accords with Mendelian inheritance and can reflect population relationship more reliability. mtDNA is extranuclear inheritance and belongs to maternal inheritance and is not superiority in population relationship analysis. Furthermore, the breeding background of Qinghai Merino and Gansu Alpine Merino showed that they had close relationship. This was accorded with phylogenetic tree constructed by microsatellite DNA. Thus we conclude that microsatellite DNA is more reliable in population relationship analysis. Further network analysis of the phylogenetic relationship of the 172 haplotypes identified from 9 sheep populations supported there are three distinct maternal lineages.

The complete mitochondrial genome(mtDNA) of mallard was determined by DNA sequencing based on the PCR fragments of 15 pairs of primers.The entire genome was 16 606bp in length.It contained 37 genes(13 protein coding genes,2 rRNA ,22tRNA)and a non-coding control region(D-loop).The characteristics of mtDNA resembled to birds on Anseriforms. Based on the control region in GenBank, Phylogenetic trees constructed using the N-J method indicated that the 13 species of Anatini clustered in three major clades. Among them, the mallards(Anas platyrhychos) ,domestic duck and spot-billed duck(Anas poecilorhyncha) clustered in ones. The results support that domestic ducks origined from mallard and spot-billed duck. Both of mallard and spot-billed duck contributed to domestic duck evolution.

The relationship between [Ca2+] in activation medium and electrical pulse strength on parthenogenetic activation of porcine oocytes was examined , and the application of sorbitol was also discussed. It was showed that:(1) The cleavage rates (73.75% and 74.70%) and blastocyst rates（37.50% and 36.83%) obtained under 0.05 mmol/L [Ca2+] and 1.6 kV/cm pulse strength, and 0.1 mmol/L Ca2+ and 1.2 kV/cm, respectively, were significantly higher than that under other conditions (P < 0.05). However, under excessive level of [Ca2+], increase electrical pulse strength led to high degradation and low development rate. (2) Using sorbitol as electro-activation medium, the highest cleavage (77.23%) and blastocyst rates (34.15%) could be obtained under 1.6 kV/cm DC. (3) Be differ from mannitol electro-activation medium, development of parthenogenetic embryos of sorbitol group was not improved with the addition of alternating current (AC) pulse. (4) Using equally mixed sorbitol and mannitol as electro-activation medium, the cleavage rates were 72.33% and 70.03% under 1.2 and 1.6 kV/cm electrical pulse, and blastocysts rates were 31.03% and 29.60%, respectively. The development of mixture group was a little lower than mannitol group (77.07% and 36.03%), and higher than that of sorbitol group (69.63% and 26.93%), but no significantly difference (P > 0.05). These results demonstrated that parthenogenetic activation of porcine oocytes needs a threshold level of [Ca2+]. Under this level, embryo developmental ability can be improved by increase the electrical pulse strength. Sorbitol can replace mannitol as fundamental component in electro-activation of porcine oocytes. Mixture of sorbitol and mannitol also get a satisfied activation results.

Endoglucanase secretions from the oesophageal glands of plant-parasitic nematodes have played important roles in invasive plant.. A new β-1, 4-endoglucanase gene, named Dd-eng-1b,was isolated from Ditylenchus destructor,by RT-PCR and RACE. the full length cDNA of Dd-eng-1b consists of 1640 bp, with a 1443 bp ORF encoding a 481 amino acid protein (GenBa nk acession No. FJ430142) . The putative protein with theoretical molecular weight is 50.89kD and pI 6.94. The sequenceing analysis indicated that this gene is classified as a member of Cellulase (glycosyl hydrolase family 5, GHF5) and has a 19 amino acids long singal sequence at N terminal and a high similarity to a bacterial type cellulose-binding domain Ⅱ at C terminal Comparative analysis between cDNA and Genomic showed that Dd-eng-1b contain four introns，demarcated by 5'-GT...AG-3' in the slpice sites，and phylogenic analysis suggested that Dd-eng-1 had a strong homology to Bacillus.subtilis and Erwina carotovora, which may be indicative of an ancient horizontal gene transfer from bacteria.

In this study, the high molecular weight glutenin subunit (HMW-GS) composition of 282 common wheat (Triticum aestivum L.) landraces originated from Xinjiang Uygur Autonomous District were analyzed by using sodium dodecyl sulphate polyacrylamide-gel electrophoresis (SDS-PAGE) method. The number of alleles on Glu-A1, Glu-B1, Glu-D1 loci was 3, 6 and 5. The predominant subunits at the Glu-A1, Glu-B1, Glu-D1 loci were "null", "7 + 8", "2 + 12" and their frequencies are 75.5%, 90.8% and 72.0%, respectively. A total of 20 kinds of HMW-GS combinations were observed in the 282 common wheat landraces tested. The most frequent combination was (null, 7+8, 2+12) (52.8%), followed by combinations (null, 7 + 8, 2.6 + 12) (14.1%) and (2*, 7 + 8, 2 + 12) (11.0%). The frequencies of other combinations were all less than 10%. In addition, a novel subunit 2.6+12 at the Glu-D1 locus was detected in this study. In the 282 common wheat landraces tested, two landraces harboring high processing quality HMW-GS combination, (2*, 7 + 9, 5 + 10) and (1, 7 + 9, 5 + 10) were identified. These materials might be used as potential genetic resources for improving processing quality in a wheat breeding program.

Primer pairs were designed to amplify the flanking region of known gene by 2-3 round of PCR，3 nested primers ( p14, p15, p16) and a group of 9 restriction site primers in total were designed. Each restriction site primer consist of a random primer p17 plus a restriction site sequence at 3’ end, 3 nested primers were designed based on the sequence of known gene. First round of PCR was performed using outer nested primer p14 and 9 restriction site primers，its 5 initial cycles of amplification at low annealing temperature allows the incorporation of 5’ sequence of restriction site primer into the PCR products，then the annealing temperature of subsequent cycles of PCR can be increased. The later cycles were performed at a higher annealing temperature so that the entire restriction site primer could stably anneal only to PCR products from the initial cycles. In order to increase specificity and yield of the amplification, the primers p15 and p17 were used to conduct second round of PCR, primers p16 and p17 were used to conduct third round of PCR, the PCR products were purified and sequenced. In order to obtain correct sequence, new primer f3x was designed using sequenced DNA, then isolated DNA as model, primers f3x and p14 (or p15) were used to perform PCR and products sequencing. Using this method, the 528bp promoter and 142bp 5’UTR sequence of FPPS gene was obtained. The sequence has been registered in the GenBank (accession no. FJ2263960). It was found by Blastn tool that this is the first report of the FPPS gene promoter sequence. These results indicted that this is a suitable method for amplifying the flanking region of known gene.

Bacillus TS01 is a functional strain separated by our laboratory. TS01 strain and other 15 bacillus type strains (B. Licheniformis, B. Subtilis, B. Pumilus, B. megaterium , B. Coagulans, B. cereus , B. lentus , B. thuringiensis , B. thermoaerophilus , B. amyloliquefaciens , B. circulans ,B. sphaericus , B. laterosporus , P. polymyxa , V. Pantothenticus.) were used to be differentiated with amplified ribosomal DNA restriction analysis (ARDRA). 16S rDNA was amplified by the general primer pair 16S-27and 16S-1525. The 16S rDNA PCR amplicon (about 1.5 kb) was digested by six restriction enzymes (Alu I,Taq I,Mse I,Bst UI,Hha I and Tsp509 I ), The characteristic ARDRA fingerprint of TS01 was obtained. The ARDRA patterns were analyzed by GelcomparⅡ software using UPGMA method. The homology tree based on ARDRA analysis shows that TS01 strain belongs to Bacillus licheniformis, this result is identical to the identification results before in our lab by using morphology, biochemistry and 16S rDNA sequence analysis to identify TS01, which shows that the established technique of ARDRA analysis has the dependability for the identification of TS01 strain on the specific level.

In this study, the genome DNA of a strain of Pseudomonas aeruginosa was used as the template, via PCR, a 1.67kb product was obtained. We discovered the nucleotide sequence of the cloned DNA shared 99% homology with lasB from P. aeruginosa PA01 (GenBank accession no. M19472). Then the correct plasmid was digested with BamHI and EcoRI and ligated into pPIC3.5K with the same enzymic sites, generating pPIC3.5K/PAE. The insertion was identified by restriction analysis and sequencing.pPIC3.5K/PAE was linearized with Sac I, and electroporated into P. pastoris KM71. Nearly 400 transformants were selected on the MD agar plates，and high-copy transformant weas screened by YPD plates containing G418.Transformant stains were further confirmed by genome PCR and casein-MM plate. After expression of recombinant elastase in shaking flask by methanol induction, we found the size of recombinant elastase was similar to that of the native elastase (approximately 34 kDa) by SDS-PAGE analysis. The highest expression level of the recombinant elastase obtained is approximately 400 mg/L and the specific activity of the recombinant elastase was 1060 U/ml, which was approximately 26-fold higher than that of elastase obtained from P. aeruginosa. The Pseudomonas aeruginosa elastase gene was successfully cloned in this study, which has laid a foundation for high level expression of elastase.