To know the energy budget of adult Tegillarca granosa, the sort, the activity, and the tissue distribution of some isozymes related with energy metabolism were tested in Tegillarca granosa by using the poly acrylamide gel electrophoresis technology. The result was that: quite like other biosome, Tegillarca granosa, a kind of lower mollusk, possessed aerobic respiration, anaerobic respiration, and hexose monophosphate pathway, three kinds of energy metabolism methods, and each way takes a significant role in the metabolic activity of adult Tegillarca granosa. However, aerobic respiration is obviously the most important of the three, while the other two are less important and only play auxiliary roles in its energy metabolism. As to the way of the energy absorption and expenditure, the degradation of esters and carbohydrates may be the main method of the energy intake, while the main channel of energy exhaustion are activities such as food digestion and muscle movement.

Abstracts：A new modified two-temperature and multiple polymerase chain reaction (PCR) was developed for the simultaneous detection of WSSV( white spot syndrome virus) and TSV(taura syndrome virus ). Two sets of specific primers were designed based on the gene sequences of WSSV and TSV. It is shown that all samples which contained WSSV and TSV can be simultaneous amplified by the two-temperature and multiple polymerase chain reaction (PCR), but no specific band was amplified from control group of shrimp vibrio pathogen. The sensitivities for detection of WSSV and TSV are 10pg and 100pg respectively by this PCR. It was suggested that this two-temperature and multiple polymerase chain reaction (PCR) has high specificity and sensitivity.

The Pseudorabies virus (PRV) Fa strain gC gene was subcloned into adenovirus shutter vector pDC316 through double enzymatic digestion. After cotransfection of the shutter vector pDC316-gC and adenovirus DNA helper plasmid pBHGloxΔE1,3Cre into 293 cells, the recombinant adenovirus expressing gC protein was preliminarily obtained through homologous recombination with observation of remarkably cytopathic effect. Afterwards, high titer of recombinant adenovirus designated as Adv-gC were prepared after one round of PCR identification and virus plaque purification. Glycoprotein gC gene expression was confirmed in Marc145 cells by immunofluorescent staining assay. Intramuscular immunization of Adv-gC could induce PRV specific humoral and cellular response with assays of anti-gC protein IgG antibodies、neutralizing antibodies and delayed-type hyper-sensitivity(DTH) in mice. In the challenge experiment, the group immunized by Adv-gC presented 100% protection efficiency as contrast to 0% obtained in the negative control group. Totally, the present result will provide a good foundation for developing a novel type of PRV live vector vaccine.

According to feline IL-18 gene sequence reported in GenBank, feline IL-18 gene was amplified by RT-PCR from the PBMCf stimulated by ConA. The PCR product was purified and ligatured with pMD18-T. The recombinant plasmid was used for sequencing. The full length of feline IL-18 gene was 579 bp , which encoded 192 amino acids. There were no signal peptide and N-glycosylation sites, while had 4 conservative Cys amino acid in the deduced amino acid sequence. Compared with IL-18 genes of other different species, the nucleotide sequence of feline pIL-18 gene shares higher identities with IL-18 genes of canine, ovine, bovine and porcine, but displays significant differences with mouse and chicken IL-18 gene sequence which published in GenBank. Meanwhile, the feline IL-18 gene was subcloned into the prokaryotic expressing vector pET28a and recombinant vector further transformed into host E.coli strain BL21 (DE3) for expression under the induction of IPTG. The results of SDS-PAGE revealed that it had a molecular weight of 27.5 kDa, which amounts to 13.6% in the total protein of the induced bacteria by the assaying of gel scanning.

A metallothionein gene from Tamarix. sp was directionally cloned into the pBI121 binary vector, in place of the XbaI – SacI GUS cassette. The Cauliflower mosaic virus (CaMV) 35S promoter/-nopalin synthase (nos) terminator system and kanamycin resistant gene (NPT II; neomycin phosphotransfers II) were used for these constitutive expression systems. The plasmid was then introduced into Agrobacterium tumefaciens (strain EHA105) by electroporation. Tobacco primary transformants were produced by leaf disc transformation. Kanamycin tolerance analysis showed that most transgenic plants have one copy of the exogenous gene in tobacco transformants. Southern and Northern hybridization indicates that exogenous gene has been integrated into the transgenic tobacco plants, and has been correctly expressed under the control of 35S promoter. Transgenic tobacco plants showed a better tolerant ability to cadmium than that of non- transformant.

Caenorhabditis elegance is one of the model organisms that could be raised with Escherichia coli in lab. Crystal proteins from B. thuringiensis are pore-forming toxins used as insecticides around the world . A collection of Bt strains were screened for toxic activity against the nematodes .Six strains were identified with significant toxicity to the adult nematodes in vitro. By assaying their toxicity on free-living nematodes ， within 36 hours ， the LC50 of the nematicidal toxins achieved 0.498 µg/ml . It’s concluded that Bt crystal proteins have potential in controlling nematodes . Toxic proteins were purified crudely by sulfate and the sizes were estimated by comparison with protein standards. DNA ladder survey showed that the toxic protein did not damage the DNA of nematodes as chemical pesticides.

After seven generations of self-crossing assisted by GUS detection, 26 homozygous transgenic rice lines were obtained. The effects of the transgene on the iron content, agronomic traits, rice quality and stress tolerance were studied taking Xiushui 11 as the control. The results indicated that the average iron contents of the transgenic rice in the milled grain was 10.37 ug/g, among all the homozygous lines, there are only four lines (Fer34, Fer36, Fer39 and Fer65) whose iron contents were significantly higher than that of the control rice (6.46ug/g); And significant differences in main agronomic traits such as days from sowing to heading, plant height, main panicle Length, total grains of main panicle, seed setting rate, 1000 -grain –weight, yield per plant were found among a few of homozygous lines, except flag leaf length and number of panicles per plant. It showed that inserting foreign gene（Fer）had a slight impact on the main agronomic traits, but the transgene had no negative effects. The homozygous rice lines quality such as the rate of brown rice, milled rice, full milled rice, grain length, grain width, and width ratio were all similar to the control, whereas, variations occurred in chalkiness, translucency, alkali spreading value, gel consistency, amylose content among partial homozygous lines, and did not show obvious regularity. The damage of the homozygous lines treated with low and high temperatures were not significantly correlated with the Fe contents, and the resistance to rice blast, bacterial blight and brown planthopper were enhanced with the advanced of Fe content. Furthermore, the possible causes of the biological characteristics variation among transgenic rice and the effects of foreign gene were discussed.

SLR1 gene is a stigma-specific S-locus related gene. Primers derived from the SLR1 promoter sequence of Westar were used to perform PCR analysis of several B. napus varieties, so as to isolate corresponding promoters of self-compatible B. napus. The fragments achieved were cloned and sequenced, and then fused to the GFP gene, replaced the 35S promoter in the plant expressed vector pFGC-eGFP. The vector was transformed to Arabidopsis by agrobacterium-mediated transformation. By sequences analysis and fluorescence detection, we concluded that the SLR1 promoter in self-compatible B. napus had similar structure and function with the self-incompatible B. oleracea. The results would lay a foundation for further study on the S-locus genes in Brassica napus.

Torenia fournieri genomic DNA was digested with DraI、EcoRV、PvuII、SmaI respectively. A special adaptor was ligated to the ends of the digested DNA fragments as a template for adaptor PCR. With the adaptor and TfPLC1 gene-specific primers, bands of 798bp and 813bp upstream of TfPLC1 were obtained successively. After sequenced, blastn and combined, a TfPLC1 promoter of 1432bp was gained. TATA box and CAAT box-like elements were found in the sequence, and TA domain was rich in the far upstream of the promoter. And also some endosperm-specific elements were found in the sequence. PBI121 and the promoter were double digested to construct plant expression vectors PPP1326 and PPP700 by 5’ deletion. Then the vectors were transformed to Torenia fournieri to validate the function of the promoter.

MxA was a new kind of antivirus protein that was induced by IFNα/β.It’s very useful and necessary to study for the mankind. We induced CEF with IFNβand Poly:IC in order to get MxA gene. Then we abstracted total RNA from the CEF. A pair of primers was designed to carry through RT-PCR. The PCR product was sequenced and then they were inserted into a expression plasmid vector. The full-length recombination MxA protein was got by induced E.coli .Then we puried and disposed the product in order to get the active recombination protein.We can know the density of the MxA was 10mg/ml by analyse. The active experiment done in and out of the body proved that the recombination MxA protein had biological activity of interferon virus .We have enough reasons to believe that the recombination MxA protein will has a good and useful prosepect.

To study the mechanism of Changes of the ability of testosterone synthesis in different periods , kunbai mouse of different peroids wereused as experimental animals . the ability of testosterone synthesis was analyzed by RT-PCR and Western Blot.The results were as followed: (1) In all peroids, the expression of P450scc ,3β-HSD，StAR mRNA and protein had no significant change.(2)The expression of P450c17 mRNA had no significant change in 10d,60d,120d.however, The expression of P450c17 mRNA decreased significantly in 180d.(3) From 30d to 270d, the activity of ERK hadno significantchange. These results inferred that activity of ERK had no indirect. relationwith the decrease of P450c17 mRNA and the level of testosterone

To optimize the enucleation protocols of mouse oocytes, the study examined the effects of the enucleation methods of abradeding the zona pellucida(AZP) or sticking the zona pellucida (SZP)straightly、sucrose treatment or not、concentration of CB and inner diameter of pipettes on enucleation efficiency，and the effects of the concentration of CB on parthenogenetic embryos removed partial cytoplasm from 1/8 to 1/12. The results showed (1) there were no significant differences between supplemented 3% sucrose group and control group on manipulating time and enucleation rate achieved by AZP or SZP（P＞0.05）; (2) 5μg/mL、10μg/mL and 20μg/mL CB treatments had no significant effects on manipulating time（P＞0.05）. AZP had no significantly different enucleation rates among 5μg/mL、10μg/mL and 20μg/mL CB groups（P＞0.05）；however, in SZP, the enucleation rates of 5μg/mL、10μg/mL CB treatments were significant higher than that of 20μg/mL（P<0.05）; (3) the cleavage rate of parthenogenetic embryos had no significant difference between various groups（P＞0.05）. Blastocyst rate of 20μg/mL CB group was significant lower than that of other groups（P<0.05）; (4) the enucleation rates of 10μm、15μm and 20μm inner diameter of pipettes were no significantly different in AZP（P＞0.05）; the enucleation rates of 10μm and 15μm inner diameter of pipettes were higher than that of 20μm in SZP（P<0.05）. The enucleation rates achieved by AZP was significant higher than that achieved by SZP （P<0.05） . The results indicate that the optimized protocol combining AZP, 10μg/mL CB with 15μm inner diameter of pipettes significantly improves the efficiency of enucleation, and can be used for nuclear transfer on mouse.

Five loose-curd-type cauliflower hybrids were applied to induce embryogenesis and plant regeneration via isolated microspore culture. The results indicated that the embryogenesis was mainly genotype-dependency. The highest embryo production was found in Chinglong 65, which produced an average yield of 15.5 embryos per flower bud. The germinated rate for microspore-derived embryos in loose curd cauliflowers was normally at 30%, meanwhile a great number of doubled haploid plants were obtained. Cold pretreatment to flower buds had a significant influence upon microspore embryogenesis, and a contradictory result was given regarding to tested genotypes. A big variation in growth stage and fertility for regenerated plants at heading stage and anthesis was observed, showing above 50% plants giving normal seed set and fertile capacity, which implies no longer doubling treatment in cauliflower microspore culture.

SdiA, belongs to luxR family involved in quorum sensing, is present in Escherichia coli and Salmonella typhimurium genome. A homolog of SdiA was cloned from Erwinia amylovora and named as EASdiA, which shares 45.42% amino acid identity with Es. coli and 43.33% with S. typhimurium. Based on regions of luxR-homolog in Er. amylovora where divergent from other luxR-homolog reported, specific primers F-EAluxR and R-EAluxR were designed to identify Er. amylovora. Ten tested Er. amylovora strains were successfully identified using these primers from a range of closely related bacteria. Less than 10 cells can be detected, when tested with extraction of pear tissue, as few as 102 bacteria can be detected. This is the first report of cloning EAsdiA from Er. amylovora and applied for molecular detection of this bacterium .

Stripe rust, caused by Puccinia striiformis f. sp. tritici (PST), is one of the most devastating diseases in common wheat (Triticum aestivum L.) in China. Chuanmai 47, a new wheat variety derived from synthetics (Triticum durum×Aegilops tauschii), was highly resistant to Chinese newly predominant races of Chinese Yellow Race (CYR32) at adult stage. To map the resistant gene in Chuanmai 47, a total of 355 F2 progenies derived from the cross between resistant cultivar chuanmai47 and susceptible variety Taichang29 were used for the resistance gene identifying and genetic analysis. Results indicate that the stripe rust resistance of chuanmai47 conferred by a single dominant gene. And the resistance gene close to the centromere of chromosome 1B and closely linked to the marker Xgwm11, Xgwm498 and Xgwm273 with a genetic distance of 2.2cM, 3.9cM and 4.5cM. Chuanmai47 could be used as strip resistant germplasm in wheat breeding by molecular assistant selection(MAS).

Based on the consensus of the S-adenosylmethionine synthetase(SAM) and the sequence of the Bacillus thuringiensis genome, we designed a pair of primer for the S-adenosylmethionine synthetase gene of the Bacillus sphaericus.The fragment of S-adenosylmethionine synthetase gene of the Bacillus sphaericus was amplified by PCR. Sequence analysis was done after the fragment was inserted into the the expression vector pGEX-6P-1 to construct the plasmid pGEX-SAM. The results show that the homologous of the fragment with the SAM synthetase gene from E. coli, Bacillus subtilis, Saccharomyces cerevisiae, rat and Homo sapiens is 63.9%，75.4%，58.8%，59.1%，55.7% respectively. For the difference between the fragment and the reported SAM synthetase gene, we expressed the gene in E.coli DH5α and the purified 43KD proterin was acquired. The activity of catalyzing the formation of SAM was identified by determining the produced SAM through High Performance Liquid Chromatography.

BoMF9 (BoMF9c and BoMF9l) gene, homologous to the BcMF9 gene encoding pollen-specific polygalacturonase of Chinese cabbage，were cloned from Brassica oleracea var. capitata and B. oleracea var. alboglabra by PCR amplification. Sequence analysis and phylogenetic analysis revealed that the deduced amino acids sequences of BoMF9c and BoMF9l contained characteristic sequence of polygalacturonase expressed in pollen. RT-PCR analysis again showed that BoMF9c and BoMF9l were expressed in flower buds but not other tissues. These two genes may act as polygalacturonases involving in pollen development.

According to the two distal and conserved regions of known α-gliadin genes, gene-specific primers for α-gliadin were designed to amplify the full gene coding regions. The amplifled fragments were cloned and sequenced. Three clones with the GenBank accession No. DQ417343, DQ417344 and DQ417345 were screened out, among which, the coding regions of DQ417343 and DQ417345 are 942 and 921bp, enloding the proleins with 313 and 306 amino acid residues, respectively; DQ417344 is a pseudogene by the definition of two in-frame stop codons. Multi-alignment analysis indicated that these sequences had higher similarity with other α-gliadin genes, and the differences mainly existed in the N-terminal repetitive domain and two polyglutamine regions.

In our study, the first discovered dwarf mutant（Ai 10）of the C.moschata Duch was used as the offerer parent, and the normal C .maxima Duch (MengRi) was used as recurrent parent. Nine pure lines of the NILs (Near isogenic lines) named S1—S9 was got through the 6 times backcrosses and twice selfcrosses. The 306 F2 individuals derived from the single cross between the NIL S2 and MengRi. All of the 640 random primers were screened for the marker linked to the dwarf gene D using RAPD and NILs approaches. The results show that the RAPD marker S1225-548 is linked to the dwarf gene. The genetic distance is 2.29cM, and it has been transformed to SCAR marker SCAR3-398.

Twenty-five simple sequence repeat (SSR) markers were isolated and characterized in Castanea mollissima from the cultivar Yanhong. For the identification of SSR loci, primer were designed on each side flanking the repeat region and they were initially tested on 6 chestnut samples using chemiluminesence detection. Eighteen loci where shown to be polymorphic and the number of alleles detected per locus varied from 2 to 6. Twelve loci were chosen for the analysis of 24 cultivars grown in North Chinese using the semi-automatic system ABI PRISM 377. These 12 markers showed a high level of genetic polymorphism with a total of 75 alleles; the number of alleles ranged from 4 to 10 per locus, with an average level of 6.3. The mean expected and observed heterozygosity were 0.743 (range: 0.680－0.845) and 0.829 (range: 0.730－0.930) respectively. The estimated frequence of null alleles showed a positive value for 3 loci, but except for 1 locus, the values were very low. The total value for the probability of identity was 7.01×10-11. Paternity exclusion probability was very high (0.999), sufficiently high to study pollen flow.

Zwittermicin A (ZwA) is a broad spectrum, novel antibiotic which shows diverse inhibitory activity against a broad target of plant pathogens; and Acyl-homoserine Lactonase (AHL lactonase) can attenuate expression of virulence genes in bacterial pathogens and then to suppress certain plant diseases by degrading N-acyl-homoserine lactones (AHLs) which are involved in the inducement of the expression of many virulence genes in bacterial pathogens. In order to increase disease suppression by combining the two different disease suppression mechanisms of ZwA and AHL lactonase, AHL lactonase gene was introduced into ZwA+ Bacillus cereus strains, and the recombinant stains were attained and showed higher expression level of AHL lactonase compared to their parental strains, without affecting the expression level of ZwA. Furthermore, the recombinant stains exhibited stronger ability of AHLs inactivation and higher restraint to the potato rot disease caused by Erwinia carotovora than their parental stains. Thus, it was indicated it was feasible to increase disease suppression by combining the biological functions of ZwA and AHL lactonase.

The research and development of the hybrid rice had paid great contribution to the development of the national economy, and had been popularized in the Southeast asia. How to build up a set of fast, accurate ture-false and purity test system of hybrid rice has been technique hard nut to crack which perplexed quarantine researchers for long time. The real-time fluorescent PCR technique is a newly arisen detect technique in recent years. Using PCR amplification, The reliability of DNA fragment R2-630 was confirmed which related to male sterity reported in 1995.Based on this fragment, one TaqMan probe and one pair of primers were designed, which can detect 17 hybrid rice samples. The result shows: this probe can with peculiar to amplify, hybrid rice and sterile lines peculiarly, and can be used in the purity identification basically. Thus the first step of one time- saving, accurate and quick real-time fluorescent PCR test system was built up.

Progressive atrophic rhinitis is an important respiratory disease in swine industry, caused by toxigenic Pasteurella multocida. The OmpH of Pasteurella mumtocida is a major outer membrane protein involved in the pathogenesis and immunity. In this study the ompH gene of P. multocida strain HN-13 of swine origin was cloned, sequenced and highly expressed in E. coli, and an indirect enzyme-linked immunosorbent assay (ELISA) was established using the recombinant protein. The structure of the ompH gene and its protein was analyzed using bioinformatics methods, suggesting that the OmpH is a porin.

Five primers were synthesized using yeast biased codons, and a 210bp DNA fragment encoding mature peptide of porcine Insulin-like Growth Factor I was amplified by Over Lap Extension Polymerase Chain Reaction. The fragment was then inserted into pPIC9K vector, with the insert being downstream of AOX1 promoter and α-secreting signal peptide. The plasmid was digested with SalI and transformed into HIS4 mutant Pischia pastoris GS115 strain by LiCl. The phenotype of colonies were identified by comparison of the growth performance on MM and MD plates. Two colonies T1, T2 were selected from 4.0 mg/mL G418 plates, followed by induction of pIGF-I expression with 1.5% methanol. SDS-PAGE analysis of supernatant of the P. pastoris GS115 strain revealed an exogenous protein with molecular weight of approximately 7.5 kDa. Dot blot hybridization assay indicated a specific reaction with rabbit polyclonal antibody against the IGF-I protein. The optimal culture condition of T2 was achieved, and 410 mg IGF-I per liter of fluid culture was produced in P. pastoris strain.

The prolactin (PRL) gene was studied as a candidate gene for the prolificacy of Small Tail Han sheep. Single nucleotide polymorphisms of exon 1 to exon 5 of PRL gene were detected in high fecundity breeds (Small Tail Han and Hu sheep) and low fecundity breeds (Dorset, Texel and Corriedale sheep) with five pairs of primers by PCR-SSCP. For the first time in sheep, the entire exon sequence of PRL gene was obtained by means of clone sequencing. In the exon 2, single nucleotide polymorphism (SNP) was detected in five sheep breeds. In the other exons (exon 1, exon 3, exon 4, exon 5), SNP was detected only in Hu sheep. For the exon 2 of PRL gene, the Small Tail Han ewes with genotype CC had 0.39 (P<0.05) or 0.98 (P<0.05) lambs more than those with genotype CD or DD; the Small Tail Han ewes with genotype CD had 0.59 (P>0.05) lambs more than those with genotype DD.

Intestinal epithelial cells of tilapias, Oreochromis nilotica, were isolated and cultivated by primary culture of cells as the cells model to research the effect of Photosynthetic Bacteria (PSB0201) treated widely with the probiotic on the epithelial morphologic characters, viability, livability, permeability, cytosolic free calcium and membranous phospholipase A2 (PLA2) activity of tilapias. However, there was no significance （P > 0.01）comparing with the control although there was little effect of definited concentration PSB0201 on the intestine epithelial morphology and biochemical indexes. The result showed no harm on the epithelial cells of tilapia intestine during definite time and provided new idea and partial academic elements to reappraise the safety of PSB0201 treated as probiotic and apply in agriculture including aquaculturet.

Due to the lack of polyadenylation in prokaryotic mRNA, the primer design of prokaryotic DDRT-PCR is different from eukaryocyte. Oligo(dT) primers are effectively replaced with primers designed according to highly iterated palindrome in complete genome. The primer design method can not only overcome shortcoming in prokaryotic mRNA to maximatily amplify whole cDNA, but aslo enhance the RNA finger-printing reproducibility and weaken false positive.It supples novel thought for prokaryotic differential display RT- PCR.

Antimicrobial peptides are relatively small molecules, less than 100 amino acids, which have a broad spectrum of antimicrobial activity. Avian antimicrobial peptides, classed as β-defensins has been identified from bloods of chicken, turkey, and ostrich; epithelial cells of chicken and turkey; and king penguin stomach contents. These peptides are active against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, fungi, and enveloped virus. The present paper describes molecules characteristic, antimicrobial activity, and potential application of these peptides, to provide a suggestion for research on this topic in the future.

By using of three basic elements, actin4 promoter of Bombyx mori, red fluorescent protein and the identified sequence of polyadenylation acid in SV40, the pBacA4Red1 transgene vector was constructed successfully. Red fluorescence was observed in the three-day preblastodermic eggs after the reconstructed piggyBac expression vector was microinjected. This indicated that the expression vector was correctly constructed and can express in the silkworm eggs. As the green fluorescent protein(GFP), the RFP can also be used as the reporter gene to research the functional gene of silkworm.

In order to increase the frequency of wheat haploid embryo formation through wheat × maize, we implied two method of hormone treated: Different volume concentrations of DMSO in 100mg/L 2, 4-D (treatment 1) and different hormone treatment combinations (treatment 2). The results showed that different volume concentrations of DMSO and different hormone treatment combinations had difference on the frequency of embryo formation. In treatment 1, the optimal volume concentration of DMSO in 2, 4-D is 3.0%(17.0%), moreover the frequencies of haploid embryo formation in other volume concentrations of DMSO are all higher than in control (9.7%).The frequency of embryo formation of different hormone treatment combinations was that 2,4-D+3.0% DMSO single treatment (7.3%)﹥2,4-D twice treatments (6.9%)﹥2,4-D and 2,4-D+3.0% DMSO combined treatment (5.5%)﹥2,4-D single treatment (2.9%).

A suppression subtractive hybridization (SSH) library was constructed from the tissue of Tamarix albif lonum M. T. Liu treated with PEG6000. The results showed that the rate of recombination in the library was 95% and the size of inserts was 100～1000 bp.100 ESTs drought stress-associated genes were obtained by sequencing the positive clones picked randomly. The EST expression profile showed that the resistance-related genes include osmotic regulator, signal transduction, transcriptional regulators, antioxidant enzyme, protein metabolism, transmembrane transport , ion homeostasis and photosynthesis. The results can contribute the further investigation of those genes in the future.

In this study, 11150 ESTs (including 10 cDNAs), about 8.1Mb in length, were downloaded from ESTs databases of Lentinula edodes in Fungal Genomics Project (FGP) website and NCBI website. By searching with SSRhunter 1.3 software and handwork, 469 SSRs were identified in 316 ESTs, approximately 2.83% of total ESTs. The average distance between SSRs was about 17.3kb. Trinucleotide and hexanucleotide ESR-SSRs were found to be the two most aboundent repeats, accounting for 38.00% and 20.00% of the total ESR-SSRs respectively, and (A)n, (T)n, (GA)n, (AG)n, (TGA)n, (GAT)n and (TCTTT)n to be the most familiar motifs, which comprised about 35.94% of all EST-SSRs. Agarose electrophoresis manifested that 39 out of 51 EST-SSRs primer pairs designed by Oligo 6.0 software possessed obvious PCR product in L. edodes, while there were no visible PCR product amplified by the selected primer pairs form other species. Some EST-SSR primer pairs revealed polymorphism in electrophorogram.

A selected strain of Bacillus subtilis A33 which can hydrolysis the glucomannan of Amorphophallus is used as material to isolate the full-length gene of β-mannanase by PCR. Sequence analysis show this DNA fragment encodes β-mannanase which belongs to the family of mannanase.This Sequence had was submitted in GenBank（GenBank Accession：DQ269473）.To express it in E. coli it is recombinant into the high efficiency expressive vector pET-32a and a recombinant plasmid pET-32a-ManA is constructed and transformed into strain E. coli BL21（DE3）.β-mannanase is expressed efficiently after inducing.