Organophosphorus pesticides(OPs) used in agriculture intensively has led to the contamination in aquatic environments and affected the non-target aquatic organisms, particularly fish. Therefore, rapid, sensitive, and accurate determination of OPs is necessary in order to take immediate necessary action. In recent 20 years, many researchers have studied the biomarkers related to detection of OPs in water. In this paper, the distribution, structure of these biomarkers and their response features to OPs are reviewed, the remained problems in monitoring OPs by biomarkers and the future research direction are also proposed.

In order to study the effect of deletion of crp (cAMP receptor protein) gene and insertion of gfp (green fluorescence protein) gene to the virulence and immunogenicity of Salmonella choleraesuis attenuated vaccine C500 strain, and to constuct crp-deleted C500 as a carrier of multip oral vaccine, crp-/gfp+mutant of deletion of crp and insertion of gfp was constructed. Firstly, the recombination suicide vector with 320 bp-deleted crp gene and 720 bp-insert gfp gene was constructed and conjugated with C500. The unmarked crp deleted ang gfp inserted strain was selected by two-step method. crp deletion and gfp insertion on the genome was determined by PCR and fluorescence microscope. In conclusion, the crp-/gfp+mutant was successfully constructed.

In this study, the gene encoding for bovine interferon-tau (bIFN-τ), with signal sequence, was obtained through PCR from bovine early embryos and subcloned into pGEM-T vector. After being verified, the bIFN-τ fragments with signal sequence or without signal sequence were inserted into the expression vector pET-30a(+), respectively. Two recombinant plasmids were induced to express the recombinant proteins by Isopropyl β-D-1-thiogalactopyranoside, respectively. The expressed products were then denatured, renatured and purified. The biological activities of purified rbIFN-τ were identified by assaying its antiviral activity and its effect on the morphology of bovine endometrial epithelial cells during in vitro culture. The results showed that bIFN-τ gene could be obtained from five bovine blastocysts by PCR without extraction of genomic DNA. Its homologies were 99% in nucleotide acids and 97% in amino acids to the sequence in GenBank (XM593584). The products of rbIFN-τ with minus signal sequence expressed in pET-30a(+) were analyzed by SDS-PAGE, and a new protein of 20kD was detected. Its molecular weight was as the same as the designed. The antiviral activity of rbIFN-τ was 1×104IU/mg using standard cytopathic reduction assay. The rbIFN-τ induced obvious morphological changes in bovine endometrial epithelial cells in vitro. The cell volume was larger than the control and a lot of vesicles appeared in the cytoplasms after 24 h culture in presence of 2.9µg/ml rbIFN-τ. In conclusion, the purified rbIFN-τ with biological activities was obtained in this experiment.

PCR-SSCP were used to analyzed the mitochondrial DNA(mtDNA) encode region variation of 465 individuals of 9 native goat breeds from 9 provinces and autonomous region in China and 1 foreign breed Angora goat. Accoding to the result of PCR-SSCP,we sequenced the mitochondrial hypervariable region 1 (HVR1) of 118 individuals. The analysis of these sequences reveals the goat breed were classified into four distinct lineages A, B, C and D, The fixation index (Fst =77.77%) suggested that most of the total genetic variation was due to variation within populations,and revealed a weak phylogeographical structure in the goat breeds. Mismatch distribution analysis showed that 2 population expansion happened among goat population.

The nonstructural gene 3D of foot-and-mouth disease virus was amplified by polymerase chain reaction (PCR) from the recombinant plasmids T-3D. A point mutation was conducted at the EcoRⅠrestriction site of 3D gene by means of spliced ovelapping extension PCR. The mutated 3D gene was inserted into the pSOC expression plasmid and the T4 bacteriophage recombinant integration plasmids pR to obtain the recombinant plasmids pSOC-3D and pR-3D. The recombinant plasmids were selected and confirmed by PCR screening and EcoRⅠrestriction endonuclease digestion. The expression plasmid pSOC-3D was used to transform E. coli BL21 and the expression product was analyzed by SDS-PAGE. The recombinant integration plasmid pR-3D was transinfected E.coli E2 strain to obtain the recombinant phages T4-3D, and the protein bands could detected by SDS-PAGE and reacted specifically to serum from FMDV-infected animals.

IGF2 gene plays an important role in the development of skeletal muscle. In order to investigate the effect of IGF2 on the regulation of development of prenatal skeletal muscle in pigs, the SYBR-Green real-time PCR method was established and used for expression analysis of IGF2 in the prenatal skeletal muscles from Tongcheng and Landrace pigs at 33, 65 and 90 days post coitus (dpc). The results indicated that the real-time PCR method established in this study was effective for the expression analysis of IGF2 gene. IGF2 had the highest expression at mRNA level in skeletal muscle at 65 dpc among selected stages and exhibited the wave expression pattern in two breeds. Interestingly, the IGF2 expression was higher in Tongcheng than that in Landrace pigs at all three prenatal stages studied, but the reason is still unknown.

In order to amplify the complete ORF of the porcine TNNC2 ( Fast skeletal muscle troponin C2 ) gene, one pair of primers was designed according to porcine TNNC2 mRNA ( GenBank accession No. DQ629177 ). A 617 bp TNNC2 cDNA fragment including the complete open reading frame ( ORF ) was obtained by RT-PCR. The nucleotide sequence identity of TNNC2 cDNA of 617 bp ( GenBank accession No. EF673726 ) with the porcine TNNC2 mRNA ( GenBank accession No. AY575058 ) was 99 %. Five nucleotide mutations were found in TNNC2 ORF. 319 T→C, 320 G→A and 321 C→T, resulted in amino acid substitutions 107 Ala→Met. 322 A→G was a silent mutation. 433 A→T resulted in amino acid substitution 144 Glu→Asp. The pig TNNC2 complete ORF, containing BamH I and EcoR I restriction sites, was amplified with one pair of primers designed according to TNNC2 complete ORF in this study. Then the PCR product was cloned into pMD18-T vector. In order to produce the recombinant expression vector pRSET A-TNNC2, the T-TNNC2 recombinant plasmid was digested with BamH I and EcoR I after colony PCR and restriction analysis, and the fragment was inserted into pRSET A vector digested with the same enzymes. The pRSET A-TNNC2 recombinant plasmid was transformed into E. coli BL21 ( DE3 ) and induced to express fusion protein with IPTG on different induction conditions. SDS-PAGE and Western blot of pRSET A -TNNC2 indicated that about 24 kD fusion protein was obtained. Further, the effects of concentration of IPTG and induction time on the yield of pRSET A -TNNC2 fusion protein were studied, the results of SDS-PAGE indicated that the best expression time of pRSET A -TNNC2 fusion protein was 4 hours and the best concentration of IPTG was 0.6 mmol/L. Analysis of the protein solubility showed that pRSET A -TNNC2 fusion protein was expressed in the form of soluble protein.

Through PCR amplification, 5’-flanking region and partial open reading frame of the β-actin gene of Nile tilapia（Oreochromis niloticus）was obtained. The sequence includes 1643 bp regulatory sequence and 90 bp of partial ORF which encodes a 30 amino acids peptide. The 1643 bp regulatory sequence which contains 108bp 5’ proximal promoter，the first untranslated exon and the first intron of β-actin gene. The proximal promoter region contains elements that were critical for transcription activity, including the CCAAT Box，TATA Box，CArG Box which respectively located at -92，-29，-62bp upstream of transcription initiation site. The regulatory sequence was inserted into the promoterless pDsRed2-1 vector. The linearized recombinant plasmid was microinjected into the fertilized eggs of white cloud mountain minnow. DsRed2 expressed in transgenic fish and red fluorescence could be observed by micro fluoroscope and anatomical lens. The results showed that the β-actin gene promoter possess effective transcription activity. The present study lays a foundation for the further autotransgene nile tilapia research.

In order to find mutations in porcine IFN-γgene, genomic sequencing was employed in different breeds. Three SNPs were identified for IFN-γgene. For these mutations, two were found only in Erhualian. And the other one was polymorphic in every breed, with a molecular markers of PCR-SSCP developed accordingly. Association was not detected between markers and health levels recorded in herd, but detected between makers and porcine reproduction traits.

Nine prepubertal gilts (JunMu No.1) were randomly allocated to three groups (n=3) and fed with a high level digestive energy (Group H), a low level digestive energy (Group L), and a control group digestive energy (Group M) diet, respectively, for 14d. Free access of water was provided throughout the whole research period. Ovaries and uteruses were collected, after 2 weeks energy treatment, and processed for determination of the absolute quantities of IGF-IR and EGFR mRNA by using real-time PCR. The ovaries and uteruses in the Group H expressed significantly greater amount of IGF-IR and EGFR mRNA than in the Group M and Group L (p＜0.05). But only the ovaries in the Group L expressed significantly less amount of IGF-IR and EGFR mRNA(p＜0.05) than in the Group M. The present study demonstrated that high energy intake markedly enhances the ovarian and uterine expression of IGF-IR and EGFR in prepubertal gilts, whereas, insufficient energy intake markedly inhibits such expressions. The result suggests that IGF-IR and EGFR might be involved in mediating the effects of energy intake on the development of reproductive system in prepubertal gilts.

The gonadotropin releasing hormone receptor (GnRHR) gene was studied as a candidate gene for the prolificacy of Small Tail Han sheep. Eight pairs of primers were designed to detect single nucleotide polymorphisms of exon 1, exon 2 and exon 3 of GnRHR gene in both high fecundity breeds (Small Tail Han and Hu sheep) and low fecundity breeds (South African Mutton Merino, Corriedale and Chinese Merino sheep) by PCR-SSCP. Only the products amplified by primers P4 and P7 displayed polymorphisms. For primer P4, two genotypes (AA and BB) were detected in Hu sheep, only one genotype AA was detected in other four sheep breeds. Sequencing revealed five mutations (+692G→A, +706T→A, +747T→C, +748A→T and +802T→A) of exon 1 in genotype BB compared with genotype AA, which gave rise to amino acid changes (Gly→Ser, Asp→Glu and Leu→Pro). For primer P7, two genotypes (CC and DD) were detected in prolific Small Tail Han and Hu sheep, only one genotype CC was detected in three low fecundity sheep breeds. Sequencing revealed two mutations (+50A→G and +101A→C) of exon 2 in genotype DD compared with genotype CC, which resulted in amino acid changes (Glu→Gly and Gln→Pro). In Small Tail Han sheep, frequency of CC and DD genotypes was 0.87 and 0.13, respectively. The Small Tail Han ewes with mutant homozygous genotype DD had 0.81 (P<0.01) lambs more than those with wild type CC.

The bone morphogenetic protein 4 (BMP4) gene was studied as a candidate gene for the prolificacy of Small Tail Han sheep. Single nucleotide polymorphisms of exon 2, exon 3 and exon 4 of BMP4 gene were detected in high fecundity breed (Small Tail Han sheep) and low fecundity breeds（Chinese Merino, Corriedale and South African Mutton Merino sheep）by PCR-SSCP. No polymorphism was detected for exon 2 and exon 4 of BMP4 gene in four sheep breeds. Polymorphism was detected for exon 3 of BMP4 gene in four sheep breeds. For exon 3, three genotypes (AA, AB and BB) were detected in four sheep breeds. In Small Tail Han, Chinese Merino, Corriedale and South African Mutton Merino sheep, frequency of genotype AA was 0.017, 0.216, 0.115, 0.429, frequency of genotype AB was 0.102, 0.317, 0.269, 0.500, frequency of genotype BB was 0.881, 0.467, 0.616, 0.071, respectively. Sequencing revealed one singlenucleotide mutation C→A at 305 bp of exon 3 of BMP4 gene in genotype BB in comparison to genotype AA, and this mutation resulted in an amino acid change of alanine→aspartic acid. The Small Tail Han ewes with genotype BB had 0.61 (P<0.05) or 1.01 (P<0.05) lambs more than those with genotype AB or AA.

By using microsatellite markers, the genetic structure of 9 domestic duck populations in East china, including Weishan sheldrake, Chaohu sheldrake, Shaoxing, Gaoyou, Dayu, Jinding, Liancheng white, Putian black and San sheldrake, was analyzed in this experiment. The results showed that the average heterozygosity was higher in 9 populations，the lowest is Jinding, the highest is San sheldrake, which ranged from 0.5137~0.6055. The average heterozygosity of 9 duck populations was 0.5523, which reflected the rich diversity. Considerable breed differentiation was observed and 25.65% of the total genetic variation came from breed differences, this result affirmed each breed was of own genetic diversity. The DA genetic distances suggested the longer differentiation existed between those breeds. The domestic duck breeds in East China were clustered into four groups based on the NJ clustering, the clustering results had some relationship with the distributions and economic utilizations of these duck breeds. All results not only fully analyzed the genetic structure of domestic duck breeds in East China, but also was of important value to transform resource advantage to economic advantage.

This paper reports on our observation that progenies derived from rice plants pollinated by pollens of Oenothera biennis exhibited distinct variations in numerous morphological and developmental traits. Through pedigree selection on these plants, genetically stable lines were obtained, which are useful as new germplasms for rice breeding. Several representative rice lines were selected for AFLP analysis. Results showed that these lines indeed contained extensive genetic variations resolvable by the AFLP marker, which included lose of rice parental bands and/or appearance of novel bands. . Tthe characteristics and usefulness of this new distant hybridization, i.e., repeated pollination, was discussed.

Two recombinant plasmids pPM1 and pPM2, containing different -10 consensus sequences of tac promoter, were constructed by PCR amplification. The results of endonuclease digestion showed that tac promoter of plasmid pPM1 can be deleted in E. coli.. The total cell lysate of the host bacteria was extracted after cotransforming into Escherichia coli. HB101 respectively with pPM1+ pAX1, pPM2+ pAX1. The quantity of activator and 3α- HSD was tested by ELISA. The results indicated that -10 consence sepuence TATATT of tac promoter had higher transcriptional activity than TATGAT. The quantity of activator and 3α- HSD was pPM1＞pPM2＞pb. Meanwhile, recombinant plasmids were integrated into C. testosteroni chromosome through homologous recombination by electroporation respectively. Their degradation ability was determined by HPLC. It showed that gene engineered bacteria had higher degradation ability than cb7（C.T.+pb）and C.T..

Bacillus thuringiensis WB9, isolated from Wuyi Mountain of China, showed high insecticidal activity against several agricultural pests and was identified for cry2Ac gene by PCR-RFLP. According to the published sequences of cry2 genes, a pair of primers was designed for full length DNA cloning of cry2Ac gene by PCR using the plasmid DNA of WB9 isolate as the template. Subsequently, the amplified fragment of cry2Ac gene was inserted into Escherichia coli cloning vector pMD18-T and sequenced. The gene had been registered in GenBank with accession number DQ361267 and designated as the novel gene cry2Ac4 by International Nomenclature Committee of Bt. An expression plasmid pHT2Ac was constructed by subcloning the cry2Ac4 gene into shuttle expression vector pHT315. pHT2Ac was transformed into Escherichia coli SCS110 and acrystalliferous Bt HD73 Cry-, respectively. SDS-PAGE analysis showed that the cry2Ac4 gene could be expressed as 70 kD peptide. In addition, square crystal was observed. The bioassay results indicated that the Cry2Ac toxic protein was distinctly insecticidal activity against Bactrocera dorsalis Hendel larvae. However, it exhibited little toxicity towards the larvae of Plutella xylostella and Culex fatlgans.

A new cDNA, named Dd-ace-1, encoding an acetylcholinesterase (AChE, EC3.1.1.7) was isolated from the sweet potato stem nematode, Ditylenchus destructor. The full-length cDNA, carrying the trans-spliced SL1 lea- der sequence, is 2517bp long with an open reading frame of 836bp encoding 611 amino acid residues (GenBank a- cession no. EF583057). The complete amino acid sequence of AChE deduced from the cDNA consists of 21 resi- dues for the putative signal peptide and possesses a potential cleavage-addition site(ω) for a glycophosphatidylin- ositol anchor(GPI) at position 583 and 562 residues for the mature protein with a predicted molecular weight of 70144.12D. The conserved motifs involved in the catalytic triad, the choline binding sit and 11 aromatic residues lining the catalytic gorge were present in the Dd-ACE-1 deduced protein. The prediected protein shared with str- ong homology with Caenorhabditis elegans ACE-3 and C. elegans ACE-4. Phylogenetic analysis based on other nematodes and species AChEs showed that the deduced AChE formed a cluster with ACE-3s.

EMSA (electrophoresis mobility shift assay) is recently the most popular method to study the binding properties of nucleic transcription factors in vitro, meanwhile isotopes can cause damage to human body, which increases the complexity of operation. In this study, radioactive EMSA system was optimized to improve the stability of EMSA. Results indicated that, the purification of probes effectively reduced the background of autoradiography; the centrifugation of purified proteins facilitated to produce neat and clear bands; the use of a 350V voltage and 4°C temperature insured the stabilization of DNA-protein complexes during electrophoresis; the removal of radioactive liquid from gel and the adherence of gel to a piece of paper helped obtain clear autoradiography. Finally the optimized system effectively improved the stability of EMSA.

Abstract: The parent strain Aspergillus niger LW-1 producing acidic β-mannanase was isolated naturally and then treated with vacuum microwave irradiation and EMS. A high- and stable-yield acidic β-mannanase producing strain Aspergillus niger E-30 was obtained through screened by solid-state fermentation on basis fermentation medium and many times of subcultures. Its enzyme activity was increased by 2.15 times from 17 048 U/g to 36 675 U/g. The E-30 properties of highly producing acidic β-mannanase remained stable after storage for two months.

Abstract: To compare four methods that were used to isolation and primary culture goat corneal endothelial cells（GCEC）, and research characters of goat corneal endothelial cells in vitro culture. The endothelial cell ,and layers including the Descemet’s membrane were sterilely stripped from 1～2 months guanzhong diary goats cornea, primary culture established by four methods as explant culture，trypsin digestion, dispase digestion and dispase combined trypsin digestion, the ability of dispersion and adhesion of the cells were compared. Results showed that dispase combined trypsin digestion was the optimum method to isolate and purify GCEC.The Morphology, immunostaining, Giemsa staining, NSE staining, scaning and Transmission electron microscope characters proved that the isolated cells were GCEC. It indicated that dispase combined trypsin digestion was optimum method to isolate and purify GCEC, and this method could provide seed cells for theoretical researches of mammalian corneal endothelial cells and tissue engineering corneal endothelium.

The purpose of this study is to establish an optimized protocol to cryopreserve boar semen. The results showed that the optimal procedure should be conducted as follows. Firstly, boar semen were pre-diluted with ZO solution and pre-equilibrated at room temperature for 1 h. After adding extender I, spermatozoa were equilibrated at 5℃ for 1.5 h and then the equal volume of extenderⅡ was added, holding 2 h for equilibration. The resulting spermatozoa were loaded into 0.25 mL straws and equilibrated for 10 min at 3 cm above the surface of liquid nitrogen (LN) and then put into LN promptly. When thawing, straws were put into water bath at 37℃ for 30 s, resulting in optimal post-thaw motility of 0.58.

Abstract: To search for an effective culture method for zona-free embryos and provide technical support for handmade cloning, this study displayed three methods for the culture of zona-free embryos and improved the more effective well of the well (WOW) culture method that embedded the well with calcium alginate gel and tried to combine with the co-culture technology. The results indicated that the blastocyst development rate(54.7 %) and the average cell numbers of blastocysts (54.3) culturing with calcium alginate gel embedding well(AEW) culture method was significantly higher than culturing with the micro-drops single culture method and agarose-pillar embedding culture method ( P＜0.05) and there was no significant differences (P＞0.05) with the WOW culture method(P＞0.05); This study also found the cleavage rate and the blastocyst development rate culturing with this new culture method were no significant differences(P＞0.05) when the concentration of the sodium alginate was used between 0.5 %～1.0 % and the concentration of the calcium ion was used between 1.0 %～2.0 %, and the optimal concentration of the sodium alginate and calcium ion were 0.7 % and 1.5 %; The study also found the cleavage rate(89.7 %) and blastocyst development rate (67.9 %) culturing with the AEW culture method combining with co-culture technology was significantly higher than culturing with the AEW culture method alone (P＜0.05). The whole results indicated that the effect of AEW culture method on culturing zona-free mouse PN-stage embryos was better than the other culture methods; this method combining with granule cells co-culture technology could greatly raised the cleavage rate and blastocyst development rate for the culturing of zona-free embryos..In conclusion,this method is suitable for the batch zona-free cloned embryo production.

A totle of sixty one-day-old Yellow brioler chickens were alloted into treatment and control groups. The treatment group was fed with the diet supplemented with 3% conjugated linoleic acid(CLA) for 48d, while control group was fed with the diet supplemented with 3% rapeseed oil. Chickens were slaughtered in each group at the age of 49 d, and abdominal adipose tissue was sampled. The total RNA was extracted from adipose tissue to measure the abundance of the chicken growth hormone receptor（cGHR）, insulin-like growth factor 1（cIGF-1）, insulin-like growth factor Ⅰreceptor（cIGF-ⅠR）, peroxisome proliferator-activated receptor gamma（cPPARγ）, cAdiponectin and cAdipoⅠR mRNA by RT-PCR using β-actin as an internal standard. Results showed that the CLA decreased the abdominal fat index by 20.93%（P<0.05）. CLA down-regulated the relative abundance of cGH-R mRNA and PPARγ mRNA in abdominal adipose tissue by 24.74%（P<0.05）and 66.52 %（P<0.01）respectively. However, no differences in the relative abundance of cIGF-1, cIGF-ⅠR, cAdiponectin, and cAdipoⅠR mRNA in abdominal adipose tissue between CLA treatment group and control group were found（P>0.05）. The data demonstrated that CLA inhibitted abdominal fat deposition preadipocytes via down-regulation of PPARγ, in broiler chicken may determine by decreasing the GH-R available for GH, and by inhibiting the differentiation of but independent of IGF and (or) GH-IGF pathway or adiponectin action.

Four breeds of chickens (White Leghorn, Yangshan, Taihe Silkies, White Recessive Rocks) with different egg production were applied to screen potential haplotype related to reproduction trait in part region of prolactin gene based on DNA pooling and sequencing. Two common haplotypes, CCCG and TGGA, of the most three ones CCCG, TGGA and TGCA which consisted with four SNPs sites (C-2402T, C-2161G, C-2062G and G-2040A) were found successfully in the fragment of 1028bp at the distal part of 5′flanking region. CCCG could be considered favourable for egg production since its frequency was estimated as 1 in White Leghorn which was verified by the results of PCR-RFLP and PCR-SSCP. Its effect on egg production was tested in the chicken reference of Nongdahe × Taihe Silkies, higher egg production was found in the haplotype combinations with CCCG, but no significant differences between them and the other haplotype combinations. The haplotype H3 (CCTCTG) which resulted from CCCG combining the other two sites (T-2101 and T-2054) was associated with egg production significantly in the chicken reference of Nongdahe × Taihe Silkies, and could be considered as a candidate marker for egg production. So rapidly screening potential causative haplotype could be succeeded applied DNA pooling and sequencing based on our researches.

The idea and the elements for semliki frost virus(SFV) RNA replicon, a kind of new generation vector, come from the Alphavirus genus. It was designed to overcome the poor efficacy of some current DNA-based and RNA-based vector. Genes coding for viral replicases are reserved while genes coding for structure proteins are replaced by foreign gene in RNA replicon. High level replication of RNA and expression of foreign gene in cytoplasm are regulated by the replicases.To evaluate the effects of the SFV RNA replicon on the efficiency of gene expression, LacZ gene was inserted into pIRES-neo which digested by BamHⅠand dephosphorylated by shrimp alkaline phosphatase, creating pIRES-neo-LacZ in the present study. RNA replicon vector pCMV-rep-LacZ and two conventional CMV promoter-based vector pLNCX-LacZ, pIRES-neo-LacZ were transfected by Lipofectin to prepared 293 cells respectively. RNA replicon vector pCMV-Rep-GHRH and two conventional CMV promoter-based vector pCDNA3.1(+)-GHRH, pIRES-neo-GHRH were transfected by Lipofectin to prepared 293.

Activation tagging is very important in plant genomics study. In this paper, an activation tagging library containing independent 30000 Basta-resistance lines was constructed by Agrobacterium tumefaciens-mediated transformation which used Arabidopsis bzr1-D as materials. Among these transformants, 47 lines showed obvious phenotype which is different from bzr1-D, containing late flowering time、dwarf、different shape of leaf、longer leaf petiole、trichomeless leaf 、sterile and no kink between stem/leaf. some T-DNA flanking sequences were obtained by Tail-PCR and Nested PCR which is the base for cloning mutant gene and co-segregation analysis.

It has been demonstrated that plant receptor-like kinases play important roles in growth and development and environmental stress. Juxtamembrane region, the fragment between transmembrane and kinase domain in receptor-like kinases, spans about 20 amino acids in length, but the sequences are not conserved. It was demonstrated in animal receptor kinases that juxtamembrane region regulates autophosohorylation activity. Rice kinases were cloned and expressed and analyzed autophosphorylation activities in our previous study. To investigate the effect of juxtamembrane region on the autophosohorylation activity of rice receptor-like kinases, truncated kinases without juxtamembrane region were expressed, and their autophosphorylation activities were analyzed. The results showed that, the absence of juxtamembrane region did not affect the expression of kinase proteins, while 14 out of 17 truncted kinases abolished the activity as detected by autophosphorylation, indicate that the juxtamembrane region may play vital role for the regulation of plant receptor-like kinases.

HbUEP（Hevea brasiliensis ubiquitin extension protein）gene , a latex expressed gene, has been cloned and sequenced from latex in H.brasiliensis, based on the analysis of differentially expressed cDNA subtractive library induced by ethphon, indicating that the UEP gene cDNA has 771bp nucleotides, composed of 226bp 3`UTR,77bp 5`UTR and a 468bp length open reading frame encoding 156 amino acid peptide. Southern blot analysis showed this gene is a kind of low-copy genes in H. brasiliensis genomic. UEP gene lowly expressed in latex from control and rubber trees 6h after application of ethphon, and strongly expressed in latex from rubber trees 12h after application of ethphon within 24h after application of ethphon, showing that this gene expression can be regulated by ethphon. Ethphon can increase the latex yield and induce the senescence of latecifer in H.brasiliensis. It is suggested that UEP gene may be involved in the regulation of ethphon-induced high latex yield or senescence of latecifer in H.brasiliensis.

Abstract: Amylose content (AC) and protein content (PC) are important traits influencing rice quality, so it is important to find effective methods for their prediction. In this work, effect-increasing loci for AC and PC screened by SSR markers were used in their prediction using two-group method and three-group method respectively, and corresponding prediction model based on three-group method was also established. The results indicated that: 1) the prediction efficiency of AC and PC was high enough for application, and the prediction effect with effect-increasing loci (IL) was better than that with positive loci (PL). 2) The prediction efficiency varied with different traits and parents, better with fixed restorer lines than with fixed sterile lines, as well as better for AC prediction than for PC prediction. 3) With fixed restorer lines, prediction efficiency by two-group method was better than by three-group method; prediction values for AC and PC by two-group method were 0.8076 and 0.6722, while 0.6823 and 0.4174 by three-group method, respectively. With fixed sterile lines, prediction efficiency showed no significant difference between two-group method and three-group method.

The project was to distinguish accurately homozygous regenerants using molecular markers in early stages of rice anther culture and to analysize distroted segregation in genotype of the population , to constuct DH population quickly and exactly. Hybrid of japonica/indica combination Zhonghua 11/Zhongzu 14 was constructed and anther culture of the hybrid was carried out. 46 polymorphic markers from 228 ones between the two parents had been selected. All of 112 regenerants were distinguished homozygous using 22 of the 46 polymorphic markers. It suggested that the regenerants population was accuratedly obtained from microspore. The 22 markers were used to analysize distorted segregation of the obtained population in genotype. Results showed that it was in continuous segregation and showed normal distribution. The project indicated the DH population was constructed accurately with microspore-derived regenerants and normal sgregation using SSR polymorphic markers, and which could be efficiently carried out earlier using SSR markers.

The optimal SRAP-PCR system in Chinese date was established with orthogonal design. In a total volume of 20µL SRAP-PCR system, it contains1×buffer, 200µmol/L dNTP, 30ng Primer, 2.5mmol/L MgCl2, 1.0U TaqDNA polymerase and 20ng template DNA. The suitable PCR procedure was initially denaturing at 94℃ for 5min; then 94℃,1min, 33℃,lmin, and 72℃,1min for the first five cycles; then the annealing temperature is raised to 53℃ for another 30 cycles. The new established SRAP-PCR system of Chinese date was fully reproducible and good stability.

Fire blight, cause by the Gram-negative bacterium Erwinia amylovora, is a destructive bacterial disease of pear and apple and other fruit trees and ornamental plants. Molecular detection protocols based on the information of the plasmid pEA29 sequence are impractical when the E. amylovora strains without the plasmid pEA29 exist in nature. This report developed the duplex-PCR method, which combined the primers from chromosomal regions and from the plasmid pEA29 in a PCR reaction, is fast, simple and accurate to identify this plant pathogen.

In the present investigation an optimized semi-quantitative RT-PCR was applied to detect the PR-39 gene expression difference in femur marrow of piglets at different ages and weaning by using 18S rRNA as inner marker. Samples were taken at birth ,3 ,14 , 23 ,28 days of age respectively. The results showed that age affected PR-39 mRNA levels in femur marrow significantly (P < 0.05), the expression of PR-39 mRNA was the lowest at birth, then increased remarkably at 3 days of age, but the expression at 14 d-old was lower than that of 3 days (P < 0.05),and it was increased thereafter. Weaning affected PR-39 mRNA levels significantly also (P < 0.05). Weaning decreased the PR-39 mRNA levels at the same day.

The full-length genome of porcine reproductive and respiratory syndrome virus (PRRSV) NB/04 strain isolated from Zhejiang was sequenced and analyzed. The results showed that the complete genome of NB/04 strain is 15408 nucleotides(nt) long , including 28nt Ploy(A) tail. The entire genome of NB/04 strain contains about nine open reading frames(ORFs), with 189nt in the 5’UTR and 150nt in the 3’UTR. Comparative analysis with the genomic sequences of domestic and abroad North American isolates showed that it seemed to be more extensive in Nsp2 of nonstructural regions and ORF5 of structural coding regions. Especially, Nsp2 and ORF5 share 79.3%-96.4 and 86.1%-95.5%of deduced amino acid identity with domestic and abroad North American isolates, respectively. Phylogenetic analysis based on Nsp2 deduced amino acid sequence suggested that domestic PRRSV isolates could be divided into two big genetic subgroups, the NB/04 strain belongs to the same subgroup as BJ-4 , the others including Ch-1a strain belongs to the other subgroup, which indicated that PRRSV has evolved to different extents . These results would enrich the information data of PRRSV genome and provide a valuable basis for further studay of biological characteristics , heredity and mutation of PRRSV.

S-nitrosylation, the prototypic redox-based signaling mechanism, is now established as a key post-translational modification in animals. The emerging evidence now suggests that S-nitrosylation may also have a central function in plant biology，especially in plant resistance to diseases.