High-resolution melting-based TILLING of γ ray-induced mutations in rice / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M2 plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M2 rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.

Treatment of early and mid-term primary biliary cirrhosis by Qingying Huoxue Decoction Combined ursodeoxycholic acid: a clinical observation / 中国中西医结合杂志

<p><b>UNLABELLED</b>OBJECTIVE To observe the clinical efficacy by Qingying Huoxue Decoction (QHD) combined ursodeoxycholic acid (UDCA) in treating patients with early and mid-term primary biliary cirrhosis (PBC). METHODS Totally 78 patients were randomly assigned to the treatment group and the control group, 39 in each group. All patients received basic treatment and took UDCA (at the daily dose of 13-15 mg/kg). Patients in the treatment group took QHD, one dose per day. The treatment course for all was 6 weeks. Clinical efficacy, gamma-glutamyl transferase (γ-GGT), alkaline phospatase (ALP), TBIL, alanine aminotransferase (ALT), and aspartate transaminase (AST) were observed before and after treatment. RESULTS Totally 21 (53. 8%) patients obtained complete response in the treatment group, with statistical difference when compared with that of the control group (11 cases, 30. 8%). Levels of GGT, ALP, ALT, AST, and TBIL decreased in the two groups after treatment (P < 0.01). Levels of ALP, GGT, and TBIL were obviously lower in the treatment group than in the control group (P < 0.05).</p><p><b>CONCLUSIONS</b>QHD combined UDCA in treating early and mid-term PBC patients was superior to the effect of using UDCA alone. It also could improve patients' liver function.</p>

Mechanisms for Solvent Tolerance and Application of Extremophile with Organic Solvent Tolerance / 微生物学通报

Organic solvent tolerant microorganism(OSTM) is a novel extremophile and it hasn't been systematically studied until 1980s.Relying on certain mechanisms,the OSTM is able to effectively de-fend and decrease the toxicity from organic solvents,which enable the OSTM to be potentially applied in the industrial fields such as whole-cell catalysis and environmental treatment,etc.The comprehen-sively understanding of the mechanisms involved in organic solvent tolerance of OSTM could be com-bined with genetic engineering in order to modify and optimize the various specifications of OSTM,and further broaden its application in other industrial areas.Latest studies on the tolerant mechanisms of OSTM,in this paper,will be reviewed from four aspects such as vesicle exocytosis and changes of phos-pholipid composition in membrane,etc.Besides,the application of OSTM in whole-cell catalysis and other fields will be introduced.

Protein Engineering of Microbial Lipases / 中国生物工程杂志

Microbial lipases are important industrial biocatalysts with the character of stereoselectivity,site selectivity and high catalytic activity with few side effects.They have been used widely in many industrial and agricultural fields.The technology of protein engineering has been successfully applied to improve the activity and stability of microbial lipases,which will raise the competitive capacity of microbial lipase preparations and enlarge theirs application fields.The strategies,the problems and the prospects of protein engineering technology which have been applied to modify the microbial lipases was surveied.

Development of Novel Microbial Lipase Resources / 微生物学通报

Microbial lipase,one of important industrial biocatalysts,has been used widely in many industrial and agricultural fields.It is always the research focus to screen,mine and develop the microbial lipases with novel catalytic activity and high stability.This paper introduces briefly the pathways and methods to mine novel microbial lipase resources from six aspects,including extremophile,metagenome,genome database,protein engineering,immobilization,chemical modification,etc.

Cloning of Endoglucanase I Gene from Trichoderma longi-brachiatum and Its Expression in Pichia pastoris / 微生物学通报

A cellulase high-yield strain was identified and named as Trichoderma longibrachiatum SSL by ITS sequence identification. The endoglucanase1 gene (eg1) encoding endo-l,4-?-D-glucanase I was ampli-fied by RT-PCR method, which including 1386 bp and encoding 461 amino acid. Sequence analysis showed that: This gene has a more 90% homology with the T. longibrachiatum eg1 gene. The eg1 gene encoding the mature peptide was inserted into the Pichia pastoris expression vector pPIC9K, which resulted in construc-tion of the recombinant expression plasmid, pPIC9k-eg1. The pPIC9k-eg1 was then introduced into the host Pichia pastoris GS115. After the induction of methanol, extracellular recombinant endoglucanase I from the supernatant of the recombinant Pichia pastoris strain reached 73 U/mL. A clear strengthening of the protein bands, whose molecular weight is about 58 kD, appeared in the SDS-PAGE.

Construction of Saccharomyces cerevisiae Mutant Deficient in adh2 and ald6 Genes / 微生物学通报

The purpose of this investigation is to improve ethanol production and decrease acetate formation in Saccharomyces cerevisiae strain YS2-?adh2.The strain YS2-?adh2 with deleted alcohol dehydrogenase Ⅱ(adh2) gene was isolated in our lab with higher ethanol production than that of the strain YS2.The ace-taldehyde dehydrogenase Ⅵ(ald6) gene encoded a cytosolic acetaldehyde dehydrogenase,a key enzyme of the pyruvate dehydrogenase(PDH) bypass,transfers acetaldehyde to acetate.To disrupt ald6 gene of the strain YS2-?adh2,ald6 gene targeting cassettes were synthesized by long flanking homology PCR(LFH-PCR) and then were transformed into YS2-?adh2 mutants by LiAc/SS Carrier DNA/PEG method.Positive transformants were selected with G418 and further confirmed by PCR.Once correctly integrated into the genome,the selective marker was rescued by transforming the plasmid pSH65 into the positive transformants and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure.We named the ald6 gene knocked-out strain as YS2-?adh2-?ald6 which has a 12.5% higher ethanol production and a 18% lower acetate formation compared to the strain YS2.

Cloning and Expression of Acyl Carrier Protein Gene from Schizochytrium / 微生物学通报

Acyl carrier protein is an essential component involved in the biosynthesis of DHA(Docosahexaenoic Acid) via PKS(Polyketide synthase) pathway,which takes the growing acyl chain from one enzyme to another.One cDNA clone,with high homology of ACP,was isolated from Schizochytrium sp.FJU-512 cDNA library.The deduced amino acid sequence contained 142 residues with isoelectric point of 5.04 and had the 4'-phosphopantetheine prosthetic(4'-PP) binding site.The target fragment was digested with BamHⅠ/HindⅢand inserted into the expression vector pET-30a resulting in the plasmid pET-30a/acp.The recombinant vector was transformed into E.coli BL21(DE3) and induced by IPTG.SDS-PAGE analysis demonstrated that ACP was effectively expressed.

Overexpression of Alcohol DehydrogenaseⅠ in Saccharomyces cerevisiae / 微生物学通报

To improve ethanol production in Saccharomyces cerevisiae,an integration plasmid pUPGKAT with PGK promoter(phosphoglycerate kinase promoter),adh1 gene(the coding sequences of alcohol dehydrogenaseⅠ) and CYC1 terminator(Cytochrome c transcription terminator) was constructed.Firstly,a fusion fragment composed of PGK promoter and adh1 gene was generated by over lap extension PCR and ligated into pUG6 resulting in plasmid pUPGKA.Subsequently,CYC1 termi nator was amplified from pSH65 by PCR and ligated to the SpeⅠand SacⅡrestriction site of pUPGKA.To integrate PGK-adh1-CYC1 into S.cerevisiae genome,pUPGKAT was digested by TthⅢⅠand the lin-earized plasmid was used to transform S.cerevisiae YS2-△adh2(adh2 disrupted strain) by lithium acetate method.The yeast mutant YS2-△adh2-adh1 which had the adh1 gene placed under the PGK promoter and harbored the adh2 deletion was constructed.Anaerobic fermentation showed overexpression of adh1 by PGK promoter resulted in a 8.84% higher ethanol production compared to YS2-△adh2.

Expression of Thraustochytrium sp.FJN-10 ?~4-Desaturase Gene in Saccharomyces cerevisiae / 微生物学通报

1.6 kb ?4-desaturase gene(FAD4)was amplified by PCR using plasmid pGEM-TFAD4 as template.The fragment was subcloned into the HindⅢ/XbaⅠrestriction site of pYES2.0 vector.Recombinant plasmid pYFAD4 was transformed into Saccharomyces cerevisiae strain INVScl for expression.It was found to exhibit ?4-fatty acid desaturase activity in the recombinant S.cerevisiae YFAD4 in the presence of exogenous fatty acid substrate docosapentaenoic acid(100?mol/L)under introduction of GAL1.Expression of the FAD4 under appropriate media and temperature conditions led to the production of DHA and it reached 41.13% of the total yeast fatty acid by GC detection.It was suggested that the protein encoded by FAD4 could specifically catalyze DPA into DHA.

Disrupting sfa1 Gene to Enhance Biosynthesis of Ethanol in Saccharomyces cerevisiae / 微生物学通报

The sfa1 gene encoded a bifunctional enzyme with the activities of both alcohol dehydrogenase and glutathione-dependent formaldehyde dehydrogenase in Saccharomyces cerevisiae.The gene disruption cassette produced by PCR using the same long oligonucleotides which comprise 19 or 22 nucleotides complementary to sequences in the templates(pUG6 and pUG66 marker plasmid)at 3' end and 45 nucleotides at 5' end that annealed to sites upstream or downstream of the genomic target sequence to be deleted.After two linear disruption cassettes with a Cre/loxP mediated marker were transformed into the cells of Saccharomyces cerevisiae YS-1,the positive transformants were checked by PCR to correct the integration of the cassette and concurrent deletion of the chromosomal target sequence.Once correctly integrated into the genome,the select marker can be efficiently rescued by transformating the plasmid pSH47 into YS-1 and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure.The expression of the Cre recombinase finally resulted in the removal of the marker gene,leaving behind a single loxP site at the chromosomal locus.The diploid mutant YS-1-sfa1 was generated,which could enhance the output of ethanol with 8.0% by shaking culture in flask compared with the original strain YS-1.