木糖发酵酵母Pichia stipitis木糖还原酶基因XYL1在酿酒酵母中的表达

通过PCR方法克隆得到树干毕赤氏酵母木糖还原酶 (XR)基因XYL1。将该基因连入酵母表达载体pYX2 12的强启动子磷酸丙糖异构酶 (TPI)启动子下 ,得到融合表达载体pYX XYL1。通过电转化方法将 pYX XYL1转入酿酒酵母SaccharonmycescerevisiaeW 30 3-1A中 ,酶活测定表明 ,在酿酒酵母中树干毕赤氏酵母木糖还原酶 (XR)基因XYL1得到活性表达 ,2酿酒酵母转化子粗酶液中木糖还原酶活分别为 0 .89U/mg(蛋白 )和 0 .83U/mg(蛋白 ) ,为供体菌的 1 5倍多。与基因供体菌不同 ,木糖还原酶基因在酿酒酵母中表达不需木糖诱导 ,为组成型表达。树干毕赤氏酵母木糖还原酶 (XR)基因XYL1的成功表达为后续的利用木糖的酿酒酵母菌株的构建奠定了基础     

蔗糖利用型毕赤酵母工程菌的构建

巴斯德毕赤酵母(Pichia pastoris)表达系统是近20年来应用最广泛的真核表达系统之一,已有500多种蛋白在该系统得到了成功表达,然而在毕赤酵母基因组中却缺少蔗糖酶基因,因此无法利用蔗糖。本研究从酿酒酵母基因组中克隆得到蔗糖酶基因SUC2。将SUC2亚克隆后得到表达载体pPICZαA-SUC2。经电转化后,表达载体整合进入毕赤酵母KM71H基因组,得到能够利用蔗糖的毕赤酵母工程菌。     

展示在毕赤酵母表面稻米脂肪酶的性质研究

人工合成酵母菌壁蛋白(GCW14)与稻米脂肪酶(RL)组合基因(GCW14-RL),将其亚克隆到pPIC9k载体后转化到GS115酵母菌中,成功构建了pPIC9k-GCW14-Rlipase载体,并将RL展示在毕赤酵母GS115表面;同时对其酶学性质进行研究,发现其在30~45℃的温度范围都能发挥作用,最适pH为8.0,重复使用5次依然可以保持80%以上的活性。     

产甘油假丝酵母烯醇化酶基因的克隆及序列分析

从产甘油假丝酵母(Candida glycerinogenes)基因文库中分离了一个含烯醇化酶基因(CgENO1)的克隆子.插入片段全长2 456 bp,包含一段1 314 bp的没有内含子的蛋白质编码区,编码一个含438个氨基酸残基的多肽链,其氨基酸序列与来源于酿酒酵母的烯醇化酶相似性为74.3%.多重序列比对显示,产甘油假丝酵母烯醇化酶(CgENO1)含有酵母烯醇化酶全部保守区.对编码区上下游序列分析显示,克隆片段含有典型的酵母基因上下游调控区,是一个完整的酵母新基因.     

甜味蛋白Brazzein在毕赤氏酵母中表达的初步研究

目的:采用pGAP ZαA为表达载体,SMD1168毕赤酵母为受体系统,构建分泌型的甜味蛋白Brazzein毕赤酵母重组菌.方法:按照毕赤酵母偏爱密码子设计Brazzein基因,共设计4对引物.在上、下游引物分别引入Xba Ⅰ与Xho Ⅰ酶切识别位点,采用SOE-PCR法合成Brazzein基因,构建克隆载体pUC57-Bra与酵母表达载体pGAPZαA-Bra.将重组质粒线性化,电转导入毕赤酵母(Pichia. pastoris SMD1168)中,用高浓度的抗生素Zeocin筛选高拷贝转化子.将重组菌株接种于YPD培养基诱导表达,进行SDS-PAGE分析.结果:成功构建了分泌型的Brazzein毕赤酵母重组菌.SDS-PAGE表明,目标蛋白的相对分子质量与理论值一致,在诱导72h后目的蛋白表达量达0.12g/L.对纯化后的蛋白进行生物活性测定,经鉴定具有一定的甜味,表达的Brazzein蛋白生物学活性良好.     

毕赤酵母展示海藻糖合成酶及其性质研究

利用毕赤酵母表面展示天蓝色链霉菌(Streptomyces coelicolor)海藻糖合成酶(Trehalose synthesase,TS,EC 5.4.99.16)并研究其酶学性质。人工合成酵母内源壁蛋白(GPI-modified wall proteins,GCW14)与天蓝色链霉菌海藻糖合成酶组合DNA(GCW14-TS),将其亚克隆到pPIC9k载体后转化到GS115酵母菌中,成功构建了pPIC9k-GCW14-TS载体,并将TS展示在毕赤酵母GS115表面,然后对其酶学性质进行研究。发现其在30~45℃温度范围都能发挥作用,最适pH为8.5,K~+、Mg~(2+)等金属离子对其有促进作用且重复使用5次后仍然可以保持75%以上的酶活性。该基因工程菌具有海藻糖合成酶固定化酶性质,可用于将麦芽糖转化为海藻糖的产业化生产。     

粗糙脉孢菌漆酶基因的克隆及在毕赤酵母中的初步表达

采用连续延伸PCR方法克隆到粗糙脉孢菌（Neurospora crassa）漆酶基因，并将其克隆到表达载体pPIC9k，重组质粒经线性化、电激转化Pichia pastoris KM71，部分阳性克隆的PCR结果表明：漆酶基因已整合到巴斯德毕赤酵母染色体上，重组菌经甲醇诱导后3～5d产漆酶量最高，为2-3U／mL。     

康氏木霉纤维素酶CBHI基因克隆及在毕赤酵母中的表达研究

在毕赤酵母中表达康氏木霉(Trichoderma koningii)纤维素酶基因cbhI。提取经诱导的康氏木霉mRNA,通过反转录及PCR反应扩增纤维素酶cbhI基因cDNA。将cbhI基因克隆到毕赤酵母表达载体pPICZαA上,采用电激法将经SacⅠ线性化的重组质粒pPICZαA-cbhI转化毕赤酵母GS115,MD及G418抗性平板筛选cbhI基因高拷贝转化子,并用PCR鉴定转化子。BMMY培养基中加入0.5%甲醇对毕赤酵母进行纤维素酶CBHI诱导表达。SDS-PAGE电泳结果显示,表达的重组蛋白相对分子质量约67 kD,pNPC酶活为24.1 U/L,酶蛋白量为0.22 mg/mL,表明,康氏木霉cbhI可以在毕赤酵母中表达,并具有较高活性。     

Pichia stipitis木糖醇脱氢酶基因XYL2在酿酒酵母中的表达

通过PCR方法克隆得到树干毕赤氏酵母木糖醇脱氢酶(XDH)基因XYL2.将该基因连入酵母表达载体pYX212的强启动子磷酸丙糖异构酶(TPI)启动子下,得到融合表达载体pYX-XYL2.通过电转化方法将pYX-XYL2转入酿酒酵母Saccharomyces cerevisiae W303-1A中,酶活测定表明在酿酒酵母中树干毕赤氏酵母木糖醇脱氢酶基因XYL2得到活性表达,酿酒酵母转化子粗酶液中木糖醇脱氢酶比活为每毫克蛋白0.6 U左右,约为供体菌的2.4倍.与基因供体菌不同,木糖醇脱氢酶基因在酿酒酵母中表达不需木糖诱导,为组成型表达.     

谷胱甘肽生产菌重组巴斯德毕赤氏酵母的构建

目的：构建一株新型的产谷胱甘肽的重组巴斯德毕赤氏酵母，并研究该重组菌合成谷胱甘肽的能力；方法：利用PCR技术克隆来源于酿酒酵母的1-L-谷氨酰-L-半胱氨酸合成酶编码基因GSHI和谷胱甘肽合成酶的基因GSH2，串联插入表达载体pGAPZA，转化Pichia pastoris GS115，在Zeocin平板上挑选阳性转化子，并对阳性转化重组菌进行摇瓶培养筛选。结果：得到一株GSH的产量高达162．2mg／L重组菌，说明串联表达酿酒酵母GSH1和GSH2基因，重组甲醇酵母生产谷胱甘肽可以达到较高的水平。     

SPECIFIC DETECTION OF AMANITA PHALLOIDES MYCELIUM AND SPORES BY PCR AMPLIFICATION OF THE GPD (GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE) GENE FRAGMENT

Oligonucleotide primers designed to flank a 635 bp fragment of the gene encoding glyceraldehyde‐3‐phosphate dehydrogenase (gpd) from Araanita muscaria were used to amplify the corresponding gpd fragment from Amanita phalloides. The A. phalloides PCR product was cloned, sequenced and found to be 70 ‐ 77% similar to the known basidiomycetes gpd genes within the exon part and 25 ‐ 52% within the intron part. Based on these data, species‐specific amplification was achieved using a pair of oligonucleotide primers complementary to the A. phalloides gpd intron sequences. These primers allowed the amplification of the corresponding gpd fragment from the A. phalloides but not from various other basidiomycetes, ascomycetes and human matrices. PCR amplification of the A. phalloides DNA gave the predicted PCR product of 284 bp. The created PCR system is an efficient tool for the specific, rapid and sensitive detection of A. phalloides mycelium and spores.     

甘蓝型油菜KCS13基因克隆与组织表达特异性分析

通过巢式PCR和基因组步移方法,从油菜的基因组中获得一段长度为3 356bp的序列。分析显示,该序列包含了KCS13基因的编码序列和启动子,命名为甘蓝型油菜KCS13基因,其转录区全长为1 587bp,编码区长1 389bp,无内含子,编码一条长462个氨基酸的多肽链。在甘蓝型油菜A、C基因组中都有KCS13基因存在。该基因主要在花蕾中表达,茎、叶和种子中表达稍弱,根中未检测到该基因表达。     

Rapid DNA purification for Hal gene PCR diagnosis in porcine tissues and extension to other meat species

Two different halothane (Hal) gene polymerase chain reaction (PCR) tests were applied to genomic DNA extracted from porcine blood, semen, muscle and fat tissues by a rapid and simple Chelex-100 based method. One of the PCR procedure is designed from the ryanodine receptor coding sequence to produce a 81 base pair (bp) fragment, while the other is designed from pig intron sequences to produce a 659 bp fragment. Oligonucleotide primers derived from the coding sequence were also used for other meat species. Amplification products obtained from porcine, bovine, ovine, equine and deer genomic DNA were successfully digested with Hha I restriction enzyme to produce the same electrophoretic pattern as in the normal homozygous (NN) pig. No PCR products could be amplified from chicken and turkey DNA.     

基于物种特异性PCR技术检测鱼糜中白鲢成分

目的建立一种基于物种特异性引物PCR测定混合鱼糜中白鲢鱼糜成分的快速检测方法。方法根据NCBI数据库中白鲢小清蛋白特异性较强的DNA序列位置设计引物进行PCR实验,通过测序得到了白鲢小清蛋白DNA的一段内含子序列,在此基础上设计了白鲢的特异性引物。提取样品DNA后进行PCR实验,产物经2%琼脂糖电泳分析进行引物特异性验证。结果在白鲢小清蛋白内含子位置设计的白鲢特异性引物,对巴沙鱼、铜盆鱼等8种鱼具有很强的物种特异性,可以实现对这9种鱼的混合鱼糜中白鲢成分的定性检测,且方法灵敏度为1%。结论本方法无需测序,能够快速、准确检测鱼糜中白鲢成分。     

Differentiation of fish species by PCR-based DNA analysis of nuclear genes

Food control laboratories are confronted with new challenges in respect to fishery product authentication; examples are identification of hybrids (e.g. in case of catfish, tilapia, sturgeon, snapper) and assignment of fish to certified stocks. Against this background, differentiation of fish species and populations by polymerase chain reaction (PCR)–based DNA analysis of nuclear genes has become of considerable importance as a tool for the completion of mitochondrial gene analysis. Four applications of nuclear gene analysis are presented: (1) fish species identification using the intronless rhodopsin RH1 gene; (2) detection of “fish” as an allergenic foodstuff by means of universal primers amplifying a segment of a parvalbumin gene; (3) differentiation of cod (Gadus morhua) from various fishing grounds by exon-primed intron-crossing PCR of a parvalbumin gene intron; (4) detection of hybridization between North Atlantic redfish species (genus: Sebastes) by restriction fragment length polymorphism analysis of amplicons obtained from the second intron of the 7S ribosomal protein gene.     

Technical note: A simplified PCR-based assay for the characterization of two prolactin variants that affect milk traits in sheep breeds

In the present study, a rapid and cost-effective PCR-based assay was developed for the genetic identification of 2 different variants within intron 2 of the prolactin gene. This polymorphism has previously been associated with milk traits in some ovine breeds and was recently proposed as a potential marker for future breeding schemes in dairy sheep. Until now, 2 alleles (A and B) have been identified by PCR-RFLP that included HaeIII digestion of a 2.5-kb PCR fragment. By partial sequencing of the prolactin gene intron 2, it was found that the B variant results from a 23-bp deletion of the A variant of the prolactin gene and not from an extra HaeIII digestion site, as had been reported. This finding assisted the design of new primers for analysis of prolactin intron 2 variants based on the size of an easily amplified short PCR product, thereby avoiding the need and cost for additional digestions. The method was validated by genotyping 80 animals from 2 breeds and showed 100% sensitivity and specificity compared with the PCR-RFLP assay. The established simplified PCR assay was then successfully used to genotype 356 Chios sheep.     

转基因水稻潮霉素标记基因PCR检测方法的建立及其在基因水平转移研究中的应用

为建立用于基因水平转移研究, 尤其是DNA经加工和消化后稳定性研究的针对转基因水稻潮霉素标记基因hpt（hygromycin phosphotransferase）的定性和实时定量PCR体系,设计针对hpt的上游通用引物多个片段定性PCR扩增体系,以植物叶绿体基因rbcl为内对照,PCR扩增产物经测序验证.将定性PCR中最小片段（236 bp）连接到质粒载体pUC18-pMD T载体上,提取质粒经验证后做外标.应用TaqMan-MGB荧光探针和引物,建立定量的外标校正曲线法,并评价方法的精密度.建立的定性PCR体系能稳定扩增出236 bp～910 bp不同大小的5个hpt片段,并经测序验证.实时定量PCR的线性范围为105～10拷贝（R^2=0.998）,最低能检出10拷贝,重复性好.本研究已成功建立了用于转基因水稻标记基因hpt基因水平转移研究的定性和定量PCR系统。     

Association of polymorphisms in the calpain I, calpain II and growth hormone genes with tenderness in bovine M. longissimus dorsi

The objective of this study was to determine if there is an association between tenderness in bovine M. longisimus dorsi (LD) and polymorphisms in the bovine calpain I (exons 9 & 14), calpain II (regulatory subunit) or growth hormone (intron 3) genes. Genomic DNA was isolated from bovine LD (n = 281) on which quality attributes (Warner Bratzler shear force (WBSF), sarcomere length and composition) were also characterised. DNA polymorphisms were analysed using polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis. Association analyses were performed between genotypes at the four polymorphic loci and day 14 WBSF values. It was found that the calpain 1 exon 9 genotypes had an association with WBSF such that animals with the GA genotype exhibited decreased WBSF and increased tenderness when compared to animals with the GG genotype (P < 0.05). This observation concurs with that of earlier studies, suggesting that this polymorphism is a functional marker for beef tenderness.     

PCR法检测肉制品中鹅源性成分

目的 建立一种PCR法检测肉制品中鹅源性成分的方法。方法 根据鹅的线粒体DNA(mtDNA)序列设计引物, 以9种动物的DNA为模板, 利用新设计的鹅的特异性引物进行PCR反应, 并用琼脂糖凝胶电泳检测引物的特异性和敏感性。结果 通过PCR反应, 仅鹅的DNA扩增得到了194 bp的目的片段, 其余物种DNA和空白对照均无目的片段。扩增产物的核苷酸序列与GENBANK中检索到的相应序列基本相符合。 结论 该方法特异性强、灵敏度高, 适合检测肉制品中的鹅源性成分。