絮凝基因(FLOIG)的序列测定及分析

虽然酵母细胞絮凝的确切机制至今尚无定论,但已克隆了多个与絮凝相关的基因,如FLOI、FLO5、FLO11等[1-3].这些基因的表达可以赋予非絮凝酵母细胞以絮凝能力.酵母细胞的絮凝特性在酿造工业、固定化酶、精细化工和物生制药等领域具有广泛的应用价值[4,5].从一株强絮凝酿酒酵母菌株中克隆到一个约4.3kb的NDA片段,酵母转化实验证明该DNA片段能够赋予非絮凝酵母菌株以絮凝能力[6].本文简要报道对该DNA片段进行序列测定和分析的结果.     

FLO1基因内重复序列对酿酒酵母絮凝能力及稳定性的影响

[目的]了解絮凝基因FLO1中重复DNA序列A对酵母菌絮凝能力及其遗传稳定性的影响,为构建遗传性能稳定、工业应用前景优良的最小絮凝功能基因奠定理论基础.[方法]通过融合PCR方法构建了FLO1中全部重复DNA序列A发生缺失的衍生基因FLO1a,以含有FLO1基因的大肠杆菌为筛选模型,通过连续传代培养及质粒快速分析获得FLO1内重复DNA序列A不同程度、不同位点缺失的系列衍生基因FLO1a1 -FL01a5.完整FLO1基因和上述衍生基因转化非絮凝型酵母YS58,得到重组菌株YSF1、YSF1a及YSF1a1 -YSF1a5,分析了上述不同酵母菌株絮凝特性及其遗传稳定性.[结果]絮凝基因FLO1中重复DNA序列A完全缺失使酵母细胞完全失去絮凝能力,部分重复DNA序列A发生缺失导致絮凝能力降低,絮凝基因中重复DNA序列A的个数与细胞絮凝能力成正相关,但不是简单的比例关系.其中衍生基因FLO1a3含有的重复DNA序列A是FLO1基因的33.3％,但菌株YSF1 a3的絮凝能力可达YSF1絮凝能力的71.4％.而且菌株YSF1 a3的絮凝特性比菌株YSF1的絮凝特性具有更好的环境适应性和遗传稳定性.[结论]重复DNA序列A是絮凝基因中非常活跃的序列,是导致絮凝特性遗传不稳定的关键因素,该序列的部分缺失不但可以使酵母细胞呈现适度的絮凝能力,而且使絮凝特性具有更好的环境适应性和遗传稳定性.该研究为通过对絮凝基因内衔接重复序列的合理调控,促进酵母絮凝特性在发酵工业及其他生物化工过程和环境修复中的广泛应用提供了重要的理论依据和解决策略.     

A组轮状平素我国地方株VP6基因的序列测定及其在杆状病毒系统中的表达

通过反转录－聚合酶链反应获得了轮状病毒地方株Ｔ１１４ ＶＰ６全基因的ｃＤＮＡ片段,将其克隆入转移载体质粒ｐＶ Ｌ１３９３中,构建成重组质粒ｐＶＬ１３９３－ＶＰ６。对克隆的ＶＰ６基因进行序列测定,并且它和杆状病毒野毒株和ＤＮＡ共转染Ｓｆ９细胞,筛选纯化得到含ＶＰ６基因插入片段的重组杆状病毒,并进行了表达重组蛋白ＶＰ６的检测。     

酵母CFD1过表达对细胞寿命的影响

[目的]测定CFD1基因过表达对酿酒酵母复制寿命的影响。[方法]运用PCR介导的基因同源重组技术构建单基因过表达酵母菌株,利用光学显微镜检测酵母细胞的复制寿命,并检测其在氯化钠、过氧化氢异丙苯和维生素K3处理条件下的克隆形成能力。[结果]在氯化钠、过氧化氢异丙苯和维生素K3的培养条件下,CFD1基因过表达酵母菌株的克隆形成能力显著低于野生型酵母菌株。与野生型酵母菌株对比,CFD1基因过表达酵母菌株复制寿命变短,差异有统计学意义。[结论]CFD1基因过表达影响酿酒酵母的复制寿命,参与了酵母盐胁迫反应,并对强氧化剂的敏感性增强,这为全面研究CFD1的功能奠定基础。     

FEN—1基因反义阻断细胞株（FL—FEN—1^—）的建立

ＦＥＮ－１是一咱结构特异性核酸酶,它在ＤＮＡ复制和修复过程中都起着重要的作用。将ＦＥＮ－１基因的ＮｃｏＩ－ＢａｍＨＩ片段反向克隆到哺乳动物细胞重组表达质粒ｐＭＡＭｎｅｏＡｍｐ＾－中,得到ＦＥＮ－１反义表达质粒ｐＭＡＭｎｅｏＡｍｐ＾－ＦＮＢ＾－,并转染ＦＬ细胞,经Ｇ４１８筛选后,获得ＦＥＮ－１基因表达被阻断的哺乳动物细胞ＦＬ－ＦＥＮ－１＾－。对细胞生长曲线的测定发现,ＦＬ－ＦＥＮ－１＾－在地塞米松的     

人工合成表皮生长因子基因在甲基营养型酵母中的高效表达

选用酵母菌偏爱密码子人工合成了编码５１个氨基酸的人表皮生长因子（ｈＥＧＦ）基因．将合成基因与编码酵母α因子前导肽８５个氨基酸的ＤＮＡ片段融合后克隆到醇氧化酶基因启动子下游,并构建出多拷贝表达载体．此载体转化甲基营养型酵母株ＧＳ１１５后筛选出整合型ＭｕｔＳＨｉｓ＋基因型菌株．高密度培养及诱导表达后该株可分泌具完好生物活性和正确物理化学性质的人表皮生长因子,产量达１００ｍｇ／Ｌ,经３次柱层析纯度达９５％以上,为观察其生物学作用打下了良好基础     

大肠杆菌海藻糖合成酶基因的克隆和表达

利用Ｍｕ转座子细胞内克隆了大肠杆菌海藻糖合成酶 ｏｔｓＢＡ基因,克隆频率为１．４５ ｘ １０(－３)／ Ｋａｎ(ｒ)转导子。经遗传互补、酶切和部分序列分析表明ｏｔｓＢＡ基因位于克隆质粒。亚克隆 ２．８７ｋｂ ＤＮＡ片段至不同拷贝数表达质粒并分别转化大肠杆菌ｏｔｓＢＡ基因缺失株,转化株恢复 在０．５ｍｏｌ／Ｌ ＮａＣｌ培养基上生长的功能,高渗透压诱导实验表明,转化株能够合成克隆基因 产物海藻糖,但合成量不受克隆质粒拷贝数影响。海藻糖良好的抗高渗能力可能在农作物育 种方面发挥重要作用。为构建含有海藻糖合成酶基因的植物表达载体,并在农杆菌的介导下 转入植物,赋予其抗高渗、耐干旱能力奠定了重要的研究基础。     

λRed重组系统用于沙门菌质粒毒力基因spvC敲除株的构建

[目的]利用λRed重组系统敲除沙门菌质粒毒力基因spvC。[方法]首先以质粒p KD4为模板,扩增得到两侧含spvC同源臂、中间为卡那霉素抗性基因的线性DNA片段。再将此线性片段电转入具重组功能的感受态沙门菌菌株,发生重组后,卡那霉素平板筛选阳性转化子。最后利用表达FLP重组酶的质粒p CP20,将FRT位点之间的卡那霉素抗性基因消除,用PCR鉴定。Western Blot检测野生沙门菌和spvC敲除株感染的He La细胞ERK磷酸化水平。[结果]沙门菌质粒毒力基因spvC敲除株构建成功,spvC敲除株感染的He La细胞内ERK磷酸化水平升高。[结论]成功构建沙门菌质粒毒力基因spvC敲除株,验证了spvC基因的功能。     

新OPG／OCIF变体基因的克隆及其表达

从正常中国人肝细胞株Ｌ０２中抽提总ＲＮＡ,利用ＲＴ－ＰＣＲ技术克隆了亲破骨细胞抑制因子（ＯＰＧ／ＯＣＩＦ）ｃＤＮＡ,同时从人胎肾细胞株２９３细胞中克隆了ＯＰＧ／ＯＣＩＦ基因３′端基因组序列,序列分析结果表明,与国外文献报道相比较,中国人肝细胞株中该ｃＤＮＡ３′端存在一个赭石型终止码突变,从而使这一蛋白质所报道的在Ｃ端少８个氨基酸残基。从２９３细胞克隆的基因组序列也同样存在这一突变。将ＯＰＧ／ＯＣＩ     

基因诱变、重组、克隆

921244导人架凝甚因FLOI培育酿酒醉母工业架凝菌株〔英〕/Watari,J.…子Agrie.Biol.Chem一1991,55(6)一15透7~1552〔译自DBA,1991,10(18),91一10238〕 以前的文章己介绍了FLOI在酿酒酵母中的克隆。现己证明含此基因的质粒整合在酵母第一染色体上。克隆于YCp型质粒(单拷贝的YCp HF19)的絮凝基因,其功能在MATa/MAT一2细胞中大大减弱,表明MAT对推测的FLOI基因的表达有抑制作用。将含有该片段的YRp型质粒(多拷贝的YRp GLF14)克隆到几种非絮凝工业酿酒酵母,包括底层、顶层发醉型菌株以及酿造威士忌、葡萄酒、日本烧酒等的醉母菌…     

Deletion of tandem repeats causes flocculation phenotype conversion from Flo1 to NewFlo in Saccharomyces cerevisiae

A gene, FLONS, conferring NewFlo-type flocculation ability in yeast was cloned. The 3,396-bp ORF encoded a peptide of 1,132 amino acids with high identity to Flo1 protein. Aligned with the FLO1 gene, two repeated regions (675 and 540 bp) were lost in the middle of FLONS, revealing that this gene was a derived form of the FLO1 gene. The missing repeated sequence contained three highly homologous repeat units. Although the flocculation phenotype of the transformant YTS-S with the FLONS gene was inhibited by both mannose and glucose, it exhibited some distinguished physiological characteristics from the reported typical NewFlo-type flocculation during detailed investigation. The deletion of repeats was suspected to cause conversion of the flocculation phenotype from Flo1 to NewFlo, suggesting that intragenic tandem repeats generated functional variability in Flo1 protein.     

Isolation and expression analysis of a LEAFY/FLORICAULA homolog and its promoter from London plane (Platanus acerifolia Willd.)

The LEAFY/FLORICAULA (LFY/FLO) homologous genes are necessary for normal flower development in diverse angiosperm species. To understand the genetic and molecular mechanisms underlying floral initiation and development in Platanaceae, an early divergent eudicot family consisting of large monoecious trees, we isolated a homolog of LFY/FLO, PlacLFY, and its promoter from London plane (Platanus acerifolia). PlacLFY is 1,419?bp in length, with an ORF of 1,122?bp encoding a predicted polypeptide of 374 amino acids and 5'/3'-UTR of 54 and 213?bp, respectively. The putative PlacLFY protein showed a high degree of identity (56-84?%) with LFY/FLO homologs from other species, including two highly conserved regions, the N and C domains, and a less conserved amino-terminal proline-rich region. Real-time PCR analysis showed that PlacLFY was expressed mainly in male inflorescences from May of the first year to March of next year, with the highest expression level in December, and in female inflorescences from June to April of next year. PlacLFY mRNA was also detected strongly in subpetiolar buds of December from 4-year-old and adult trees, and slightly in stem of young seedling and young leaf of adult plant. Additionally, we cloned 1,138?bp promoter sequence of PlacLFY and we drove GUS expression in transgenic tobacco by the chimerical pPlacLFY::GUS construction. Histological GUS staining analysis indicated that PlacLFY promoter can drive GUS gene expression in shoot apex, stem, young leaf and petiole, flower stalk, petal tip, and young/semi-mature fruits of transgenic tobacco, which is almost identical to the expression pattern of PlacLFY in London plane. The results revealed that the PlacLFY gene isolated from London plane is expressed not only in reproductive organ but also in vegetative organs. Moreover, this expression pattern is consistent with the expression pattern in tobacco of a GUS reporter gene under the control of the potential promoter region of PlacLFY.     

Gene controlled by promoter--P_(TH4) depending on whiG of Streptomyces coelicolor

The downstream gene controlled by promoter--PTH4 which is related to Streptomycesdifferentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces.     

小麦尿卟啉原Ⅲ合成酶基因克隆及序列分析

根据水稻已公布的尿卟啉原Ⅲ合成酶(UROS)基因和小麦EST的保守序列,设计特异性引物对小麦尿卟啉原Ⅲ合成酶基因的部分片段进行克隆,得到了364 bp的cDNA(命名为UROS1)。以UROS1作为种子进行电子克隆,得到一段长为1210 bp的cDNA序列,并设计特异性引物克隆到1个1077 bp cDNA序列。对该片段分析结果表明,克隆得到的小麦UROS基因包含了信号肽区和全长的成熟肽区。小麦UROS基因与水稻UROS基因的同源性为86%左右,其推导氨基酸序列与水稻和拟南芥蛋白序列同源性分别约91%和79%。动物、植物以及微生物间核酸序列的保守性较低,氨基酸序列保守性也不高,但都存在UROS保守结构域(Hem D)。进化分析显示,该酶在不同物种间的进化速度差异较大。     

家蚕细胞色素P450基因Bmcyp6u1的克隆、序列分析与表达谱

细胞色素P450第6亚家族基因为昆虫所特有,与抗性相关。为了检测家蚕Bombyx mori cyp6u1基因是否与耐氟性相关,首先克隆了cyp6u1基因。采用生物信息学方法获得与黑腹果蝇Drosophila melanogaster cyp6u1基因同源的家蚕B. mori cyp6u1基因序列, 预测该序列的开放阅读框（ORF）为1 476 bp, 编码491个氨基酸, 推定的蛋白质分子质量为56.15 kD, 等电点为9.23。以家蚕5龄第3天幼虫精巢cDNA为模板, 设计特异引物PCR扩增出一条约1 500 bp的条带, 大小与家蚕cyp6u1序列的ORF预测值接近, 命名为Bmcyp6u1基因（GenBank登录号：HM130560）。同源性分析表明, Bmcyp6u1基因与蜜蜂Apis mellifera的同源基因cyp6AS13的相似性为56%, 与拟南芥Arabidopsis thaliana的cyp72A82的相似性为48%, 与人Homo sapiens的cyp3A7基因的相似性为50%。芯片数据分析表明, Bmcyp6u1基因在家蚕5龄第3天幼虫各组织表达量很低, 只在精巢组织(5龄第3天)稍有表达, 推测该基因具有组织特异性。     

结球甘蓝抗TuMV相关基因的克隆

以结球甘蓝高抗TuMV自交不亲和系84075为材料,构建了cDNA文库。根据抗病基因保守序列（NBS－LRR）设计一对简并引物,以84075的基因组DNA和cDNA为模板,进行PCR扩增,分别扩增出一条513bp片段,扩增片段进行克隆测序。选取两个与抗病基因同源性较高的克隆片段作探针（命名Borl,Bor2）,对构建的cDNA文库进行筛选,得到一个阳性克隆（命名TuR2）,测序及序列分析表明,该基因总长为762bp,编码226个氨基酸、包含681bp的开放阅读框。与已克隆的抗病基因有不同程度的同源性。利用TuR2作探针,进行了Southern杂交、Northern杂交以及抗病性的共分离检测分析。结果表明,TuR2可能吧单拷贝形式存在,其表达是组成成型的,且无组织特异性;初步确定是一个与结球甘蓝抗TuMV相关的基因。     

一色齿毛菌锰过氧化物酶2基因克隆及生物信息学分析

摘要 锰过氧化物酶（manganese peroxidase,MnP）是由一系列同功酶组成的木质素降解酶。我们前期工作克隆了一色齿毛菌（Cerrena unicolor) MnP1基因序列。在此基础上,本研究采用简并PCR、染色体步移和RACE等技术对C. unicolor mnp2基因(Cu-mnp2)序列进行克隆。同时,采用生物信息学软件对Cu-mnp2的基因结构、Cu-MnP2的蛋白质结构及多物种MnPs蛋白质序列的系统进化关系进行分析。克隆得到3 053 bp的Cu-mnp2 DNA序列(GenBank:JX270806.1)和1 429 bp的Cu-mnp2 cDNA序列(GenBank: JQ782580.1)。序列分析结果显示,Cu-mnp2 DNA序列包含14个外显子和13个内含子,启动子区域包含TATA-BOX、SP1和AP1等作用元件;Cu-mnp2 cDNA序列包含71 bp的5′UTR、230 bp的3′ UTR以及1 128 bp的开放阅读框(ORF)。Cu-mnp2 ORF序列的BLAST比对结果表明,Cu-mnp2与Trametes versicolor FP-101664 SS1 mnp序列覆盖度为53%,序列相似性为65%;与Heterobasidion irregulare mnp、C. unicolor mnp1等cDNA序列都有较高的序列相似性。Cu-mnp2的ORF编码(GenBank:AFK91530.1)由340个氨基酸残基组成的多肽链(Cu-MnP2)。Cu-MnP2蛋白质序列的BLAST比对和蛋白质三维结构均显示,Cu-MnP2包含Mn ²⁺ 、Ga ²⁺ 、血红素及芳香底物结合位点。对包含Cu-MnP1、Cu-MnP2蛋白质序列在内的多物种MnPs蛋白质序列的系统发育分析表明,多物种的MnPs分为两大类群,分别为包含4个二硫键的短MnPs和包含5个二硫键的长MnPs。其中,Cu-MnP1与Cu-MnP2均属于短MnPs,Cu-MnP2与Trametes versicolor MrP 的蛋白质序列亲缘关系最近。通过Cu-mnp2基因的克隆和序列分析,对继续研究C. uniclor的MnP同工酶基因结构和功能奠定基础。     

Gene controlled by promoter--PTH4 depending on whiG of Streptomyces coelicolor

The downstream gene controlled by promoter--P_TH4 which is related to Streptomyces differentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces.     

Genetic basis of flocculation phenotype conversion in Saccharomyces cerevisiae

Two NewFlo-type flocculent transformants Saccharomyces cerevisiae YTS-S and YTS-L were obtained from a partial yeast genomic library. Even though both of the transformants displayed the same flocculation phenotype, they represented different physiological characteristics during detailed investigation. Analysis of the two transformants YTS-L and YTS-S confirmed the presence of FLONL and FLONS genes, respectively. The 3396-bp ORF of FLONS encoded a protein of 1132 amino acids. Meanwhile, the presence of a 1686-bp ORF encoding a 562-amino acid protein was revealed in FLONL. Both FLONL and FLONS showed high identity to FLO1 gene. Aligned with the intact FLO1 gene, FLONS lost two internal repeated regions, whereas one repeated sequence was inserted into the middle of the FLONL gene. All of the altered regions could be found in the middle repetitive sequence of the FLO1 gene. The results indicate that FLONL and FLONS are both derived forms of the FLO1 gene. Genetic variability triggered by tandem repeats in FLO1 gene is believed to be responsible for the differential phenotypic properties of the yeast strains YTS-S and YTS-L.