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1.
扩增出广西本地沼泽型水牛（Bubalus bubalis）催乳素受体（PRLR）基因部分保守序列,用于下一步实验检测体外培养的水牛乳腺上皮细胞中催乳素受体基因的表达水平。根据已报道的奶牛催乳素受体基因cDNA序列的保守区,设计一对特异性引物。提取广西本地水牛的乳腺组织总RNA,并以其为模板,以下游特异性引物为反转录引物,在反转录酶（AMV）作用下合成cDNA;用Ex-Taq酶进行PCR扩增,得到催乳素受体基因的保守序列,长度为571bp。该扩增产物经纯化后,连接到pMD18-T载体上扩增。经序列分析结果表明：序列较保守,与GenBank上发表的奶牛催乳素受体基因的同源性达到98．9％,与绵羊的同源性达到96．5％,与猪、小鼠、人的同源性分别为86．5％、78．9%、77．1％。催乳素受体基因与奶牛相比,共有6个碱基发生了突变,其中1个为同义突变,其他5个均为错义突变,形成4个氨基酸发生变异。 相似文献
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尿卟啉原Ⅲ脱羧酶是生物卟啉类化合物分支合成的关键酶。从GenBank中搜寻到4种玉米尿卟啉原Ⅲ脱羧酶：Les22、UROD1、UROD2和截短UROD,氨基酸序列比对显示N端的同源性较差;玉米les22基因比urod1基因在开放阅读框的上游少3个碱基,导致阅读框移码。植物除玉米外存在高度同源的UROD1和UROD2,具有结构完整性。本文以玉米幼叶总RNA为模板,通过RT-PCR克隆了玉米les22和urod2基因,并对突变位点进行校正,同时通过定点突变获得urod1基因,它们都编码去除叶绿体导肽的尿卟啉原Ⅲ脱羧酶。再分别将les22、urod2和urod1基因插入大肠杆菌的不同表达载体,转化表达菌株BL21（DE3）,16℃诱导表达18h,SDS-PAGE分析显示,Les22的N端含有组氨酸标签或SUMO蛋白,重组蛋白表达为包涵体,UROD2的N端含有组氨酸标签或SUMO、TRX、GST或MBP蛋白,未检测到目的蛋白在上清液的特异表达,而UROD1和MBP融合为可溶性表达,表明玉米UROD的N端氨基酸残基可能参与蛋白在大肠杆菌的折叠。这为研究玉米UROD的种类和功能奠定基础。 相似文献
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本实验主要是从人乳腺癌细胞中克隆Rab7基因并进行基因序列比对和分析。通过培养乳腺癌细胞MCF-7和MDA-MB231,提取细胞总RNA,逆转录为cDNA,以此cDNA为模板,根据GenBank公布的人Rab7全长序列设计引物,扩增Rab7基因的开放阅读框。将其与真核表达载体pc-DNA3.1连接,构建pcDNARab7重组质粒。测序后发现MCF-7细胞中克隆的Rab7序列与GenBank序列的同源性为91%,发生了多处突变,其中94.4%为同义突变,5.5%为错义突变。但是MDA-MB231细胞中克隆的Rab7基因序列与GenBank中的Rab7序列的同源性为100%。由于发现Rab7在MCF-7乳腺癌细胞中发生突变,而在MDA-MB-231乳腺癌细胞中基因未发生突变,我们推测Rab7基因的突变是否与乳腺癌的产生有关系,这个发现为以后研究Rab7与乳腺癌的关系奠定了基础。 相似文献
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根据Genbank中挪威鼠（Rattus norvegicus）Hspa4基因序列(登录号：NM 153629)和玉米Ubi-1启动子序列(登录号：DQ141598 )设计引物,采用RT-PCR和PCR技术分别进行克隆。测序及同源性分析结果表明：Hspa4基因片段含有2530bp,编码840个氨基酸;获得的启动子Ubi-1序列大小为1992bp;克隆的序列与相应原序列同源性分别为99.96％和99.55%。以植物表达载体pCAMBIA1301质粒为基础,成功构建了植物表达载体pCAMBIA1301-Ubi-Hspa4,为利用Hspa4基因改良植物抗逆性打下了基础。 相似文献
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采用比较基因组学和RT-PCR方法，根据人和小鼠SMAF1基因的保守序列，设计简并引物，克隆了猪SMAF1基因的cDNA序列。所克隆片断全长256bp（GeneBank登录号 DQ191892），包括一个编码了81个氨基酸的完整编码区，该序列与人和小鼠的SMAF1基因的相似性分别为86%和78％，预测的氨基酸序列与人、小鼠、大鼠和牛的相似性分别为81％、67％、70%和84%。猪SMAF1基因在脂肪组织中高丰度表达，在4月龄瘦肉型猪（大白猪）脂肪组织中的表达量显著低于脂肪型猪（梅山猪）（P＜0.05）。本研究结果表明，猪SMAF1基因可能具有和其它物种SMAF1基因相似的功能，推测其在脂肪细胞发生和/或脂肪细胞功能中具有重要的调控作用。 相似文献
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玉米大斑病菌G蛋白β亚基编码基因的克隆与表达 总被引：1，自引：0，他引：1
玉米大斑病菌（Setosphearia turcica）属半知菌亚门丝状真菌，是严重威胁玉米生产的重要植物病原真菌。本研究克隆了玉米大斑病菌G蛋白β亚基编码基因Stgb-1。Stgb-1基因全长1296bp，由5个外显子和4个内含子组成；开放阅读框（ORF）为1056bp，编码351个氨基酸，蛋白质计算分子量为39.081kDa。STGB1蛋白推导氨基酸序列与Cochliobolus heterostrophus（100%）、Cryphonectria parasitica（80%）、Aspergillus fumigatus（81%）等丝状真菌的G蛋白β亚基编码基因均具有较高的同源性。同时，利用RACE技术和Genomic Walking技术获得了Stgb-1基因3’-末端cDNA和DNA的3’端侧翼序列，BlastN结果表明其cDNA 3’-UTR为136bp，poly(A)由9个A构成。此外，构建了pET28a-Stgb-1原核表达载体，SDS-PAGE检测和Western blot鉴定结果表明，目的蛋白在E.coli BL21菌株中已成功实现了诱导表达，为进一步研究蛋白结构与功能的关系奠定了基础。 相似文献
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表达鸡Ⅱ型干扰素基因的重组鸡痘病毒的构建 总被引：5，自引：0，他引：5
以插入鸡Ⅱ型干扰素（IFN－Ⅱ）cDNA序列的重组质粒AIFN为模板，根据鸡痘病毒早晚期启动子PE／L的序列设计上上游引物，鸡IFN－Ⅱ基因的cDNA3'端序列设计了下游引物，采用PCR法扩增包括启动子PE／L和鸡IFN－Ⅱ全长cDNA序列的基因片段。将该扩增片段定向插入鸡痘病毒转移载体1175中，获重组转移载体1175IFN，用以转染已感染鸡痘病毒282E4疫菌株（wt-FPV)的鸡（Gallus gallus)胚成纤维细胞（CEF）。1175IFN与wt-FPV基因组DNA之间的同源重组产生了表达IFN－Ⅱ的重组鸡痘病毒rFPV-IFN-Ⅱ。通过在含X－gal的营养琼脂上连续挑选篮色病毒斑获得并纯化rFPV-IFN-Ⅱ。以细胞病变抑制实验证实，rFPV-IFN-Ⅱ感染的CEF表达了具有抗水泡性口炎病毒（VSV）活性的鸡IFN－Ⅱ。rFPV-IFN-Ⅱ(M.O.I=0.01)感染CEF72h后，培养上清中IFN－Ⅱ的表达量为1280U／mL。动物实验表明，rFPV-IFN-Ⅱ接种雏鸡7d或14d后，其在体内表达的IFN－Ⅱ可显著减轻载体鸡痘病毒对1日龄SPF雏鸡体重增长的抑制作用。 相似文献
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根据GenBank上猪TNNC2（Fast skeletal muscle troponin C2）基因序列（GenBank accession No. DQ629177）设计一对引物,采用RT-PCR方法克隆得到617 bp TNNC2 cDNA片段（GenBank accession No. EF673726）,包括完整的开放阅读框（ORF）,与GenBank上公布的猪TNNC2基因（GenBank accession No. AY575058）的ORF核苷酸序列同源性达99 %,并发现开放阅读框内的5个点突变,319位点T→C,320 位点G→A,321位点C→T,导致氨基酸107位Ala（丙氨酸）→Met（蛋氨酸）,322位点A→G为同义突变,433位点A→T,导致氨基酸144位Glu（谷氨酸）→Asp（天冬氨酸）。根据已获得的TNNC2基因开放阅读框序列,重新设计引物扩增得到包含BamH I和EcoR I酶切位点的完整阅读框,将其首先克隆到pMD18-T载体中,经菌液PCR筛选和酶切鉴定后,用BamH I和EcoR I将目的片段切下,再克隆到原核表达载体pRSET A中构建重组表达质粒pRSET A-TNNC2。将重组质粒转化大肠杆菌BL21（DE3）,IPTG不同诱导条件诱导表达,经SDS-PAGE电泳和Western blot分析证实重组表达质粒pRSET A-TNNC2表达出24 kD左右的融合蛋白,最佳诱导时间为4 h,最佳的IPTG诱导浓度为0.6 mmol/L,表达产物以可溶性蛋白的形式存在。 相似文献
9.
为了探明胚乳特异启动子的调控活性，对小麦醇溶蛋白盒结合因子基因（pbf）的启动子序列进行了5´、3´和中间缺失片段-GUS基因嵌合体载体（pbf.a-d）的构建，并在小麦离体胚乳中转化。GUS瞬间表达活性检测和功能缺失试验表明：pbf 的-2510- -1bp全长序列能够在小麦离体胚乳中表现活性，而5´ 端-2510bp- -670bp以及3´端-1288bp- -1bp序列的缺失则使启动子在胚乳中丧失了活性；中间序列-2160bp- -689bp的缺失使GUS的表达强度明显降低。文中对pbf启动子序列中存在的重要基序种类和数量以及基序对启动子表达调控活性的作用也给予了初步分析。本文对小麦胚乳发育状态及GUS瞬间表达体系等对pbf启动子活性检测的影响也作了探讨。 相似文献
10.
水稻胚乳突变体的诱发及其微卫星分子标记鉴定 总被引：4，自引：1，他引：4
用 3 50Gy6 0 Coγ射线辐照高表观直链淀粉含量 (apparentamylosecontent,AAC)早籼稻新品种金早 97 47,筛选鉴定出 4个暗胚乳突变体和 2个云雾状胚乳突变体。AAC测定表明 ,暗胚乳和云雾状胚乳突变的AAC明显比原亲本低。以Wxup2 485为引物 ,发现两类胚乳突变的Wx基因微卫星分子标记相同 ,与原亲本显著不同 ,前者为 (CT) 1 8 (CT) 1 8,后者为 (CT) 1 1 (CT) 1 1。 相似文献
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Starch suspensions (0.25%) were gelatinized to 70 and 100°C, and starch ghosts (defined as gelatinized starch granule envelopes after the majority of internal starch polymers have been released) and remnants were collected by centrifugation and washed with water. Protein was revealed in isolated gelatinized normal starch ghosts using confocal laser scanning microscopy and a protein‐specific dye that fluoresces only after reaction with primary amines in protein. This technique eliminates background interference from residual dye. Observation of fluorescent‐labeled protein in the starch ghosts at different optical depths of field revealed that protein was concentrated in the envelopes of swollen, gelatinized potato, maize, and wheat starch ghosts. Only traces of protein were found in gelatinized starch granule remnants of waxy maize and amylose‐free potato starches after they were heated to 100°C, indicating that the proteins observed in gelatinized normal maize starch were largely granule‐bound starch synthase (GBSS). Moreover, fragility of the gelatinized waxy and amylose‐free starch granule remnants might be caused in part by the lack of GBSS. Gel electrophoresis of proteins in starch ghosts confirmed that GBSS in potato and maize was tightly associated with the starch ghosts. The study provides a structural explanation for a role of granule‐associated proteins in maintaining the integrity of starch ghosts and remnant structures, and their consequent effect on paste rheology. 相似文献
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不同类型玉米籽粒淀粉积累、相关酶活及基因表达差异分析 总被引：3，自引：0，他引：3
为探究不同类型玉米淀粉形成机理,对普通玉米、甜玉米、糯玉米淀粉积累、相关酶活及基因表达进行测定,分析不同类型玉米淀粉积累、相关酶活及基因表达之间的差异及相互关系。结果表明,不同类型玉米总淀粉和直链淀粉百分含量为:普通玉米>糯玉米>甜玉米,支链淀粉百分含量为:糯玉米>普通玉米>甜玉米,灌浆期间总淀粉和直链淀粉含量3个玉米类型间差异显著;灌浆期间,普通玉米各淀粉合成相关酶活性最高,甜玉米淀粉合成相关酶活性最低,糯玉米则介于普通玉米和甜玉米之间,但其GBSS酶活性很小。灌浆期间3个类型玉米除GBSS酶活性差异不显著外,其他淀粉合成相关酶活性差异显著;普通玉米淀粉合成相关基因表达量总体均高于甜玉米和糯玉米,甜玉米和糯玉米相关突变基因仍存在表达。表明不同类型玉米淀粉含量和组成上差异明显;普通玉米淀粉的形成需要淀粉合成相关酶相互作用,淀粉合成相关酶活性的缺失会改变淀粉组成;不同类型玉米淀粉合成相关基因表达差异显著,但都存在转录活性。对普通玉米进行相关性分析同时发现,淀粉的合成不仅受到转录调控,还受到转录后调控,淀粉的合成是淀粉合成各酶之间相互协调的结果。 相似文献
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Granule bound starch synthase1 (GBSS1) is a key enzyme in amylose biosynthesis and is encoded by the A, B and D GBSS1 wx loci in wheat. Wheat lines with mutations at the three GBSS1 loci have been identified. We have characterized and compared the grain starch of CDCW6 wheat line (null B and D for GBSS1) with PI235238 (null A and B for GBSS1), waxy (null A, B and D for GBSS1), and AC Reed (wild type wheat) grain starches. The grain starch of waxy, CDCW6, PI235238, and AC Reed lines contained ≈0, 12, 23, and 25% amylose (w/w), respectively. Waxy, partially waxy, and wild wheat grain starches showed significant differences in onset and peak transition temperatures as determined by differential scanning calorimetric analysis. Grain starches extracted from waxy, CDCW6, and PI235238 also had higher enthalpy of gelatinization values than did wild wheat starch. X-ray diffraction analysis revealed the highest crystallinity for starch extracted from waxy wheat, followed by CDCW6. The starch produced from the CDCW6 line may find special food and industrial applications because of its relatively low amylose concentration. 相似文献
16.
R. A. Graybosch C. J. Peterson L. E. Hansen S. Rahman A. Hill J. H. Skerritt 《Cereal Chemistry》1998,75(1):162-165
Granule-bound starch synthase (GBSS) is the primary enzyme responsible for the synthesis of amylose in amyloplasts of cereal endosperm cells. Bread wheats, due to their hexaploid genetic system, carry three genes (wx loci) encoding GBSS. Purification and separation of GBSS from more than 200 North American hexaploid wheats allowed the identification of genotypes that carry null alleles at either the wx-A1 and wx-B1 loci. In addition, the cultivar Ike carried both wx-A1 and wx-B1 null alleles. No wx-D1 nulls were detected. Null alleles were found in 10% of the hard winter wheats tested, but in only 2% of the sampled soft winter wheats. Amylose contents of wheats carrying single null alleles at either the wx-A1 or wx-B1 loci often were lower than those of wild type wheats, but greater reduction in amylose content was observed in Ike. Monoclonal antibodies were used to quantify water-extractable GBSS in both wild-type and null genotypes. Gene dosage compensation was evident, although GBSS content, as measured by ELISA, was significantly lower in Ike than in all other wheats. The identification of null alleles in adapted genotypes suggests the development of wheats with a wide range of amylose contents will be possible by simple genetic crossing and selection. 相似文献
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The Waxy (Wx) gene in hexaploid wheat (Triticum aestivum L.) encodes granule‐bound starch synthase (GBSS1), which is involved in the synthesis of amylose, a mostly linear glucan polymer that makes up ∼25% of wheat starch. A null mutation of the Wx gene in each of the three genomes is associated with starch almost entirely consisting of the branched glucan polymer amylopectin (waxy starch), with corresponding changes in functionality. However, the rheological behavior of partially waxy starch remains unclear. The objective of this study was to characterize flour and baking quality in 16 near‐isogenic lines, null at the Wx locus on zero, one, two, or all three genomes, grown in four different environments. Across allelic groups, significant variations in amylose concentrations, flour paste viscosity, loaf structure and texture, dough stability, and proximate variables were observed. Because waxy wheat starch has greater water absorbance and resistance to retrogradation than normal starch, its inclusion in flour blends has been suggested as a means of improving the texture and appearance of bakery products and noodles. The results indicate that wheat encoding <3 functional homeologs of GBSS1 produces starch that has potential in the production of certain food items, such as Asian noodles. However, further research is necessary to determine the optimal amylose‐to‐amylopectin ratio to improve baking quality. 相似文献
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Mengliang Tian Gongxie Tan Yongjian Liu Tingzhao Rong Yubi Huang 《Genetic Resources and Crop Evolution》2009,56(2):247-255
Waxy maize is a special type of cultivated maize and has grown in China for long history. However, the evolution and origin
of waxy maize still remain unknown. We analyzed the origin and evolution of waxy maize by sampling DNA sequences from four
taxa with eight populations: waxy maize and other maize cultivars from Southwest China or America (Zea mays L. ssp. mays), parviglumis (Z.
mays L. ssp.
parviglumis Iltis et Doebley), three more distant species within this genus (Z. luxurians (Durieu et Ascherson) Bird, Zea perennis (Hitchcock) Reeves et Mangelsdorf, and Zea diploperennis Iltis, Doebley et Guzman), and a representative of sister genus (Tripsacum dactyloides L.). We sequenced 20 sequences and downloaded 26 sequences from NCBI for the glb1 locus, which encodes a nonessential seed storage protein. Within the Zea genus samples, the waxy maize has the minimum sequence diversity, which contains 31.1% of the level of diversity of parviglumis
and 58.5% of the level of diversity of normal maize from Southwest China. Sequence variation within glb1 locus is consistent with neutral evolution in all four taxa according to Tajima’D test. From the NJ tree for glb1 sequences waxy maize formed two main groups which are intermixed with normal maize sequences. These results suggest that
the Chinese waxy maize originate from a single gene mutation from normal maize. Combined with the history of maize dispersal
in China we can even think that Chinese waxy maize was divergenced from Chinese flint maize. 相似文献
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适于玉米杂交种纯度鉴定的SSR核心引物的确定及纯度鉴定图谱绘制 总被引：2，自引：0，他引：2
为了确定一套适于玉米杂交种纯度鉴定的核心SSR引物，利用10个SSR引物对420份已知玉米杂交种进行DNA指纹分析。综合考虑多态性和杂合率两个指标，确定bnlg161和bnlg1450为玉米杂交种纯度鉴定的首选核心引物，利用这两个引物进行筛选，412个品种（占98%）能够找到具有杂合带型的鉴定引物；确定bnlg439、bnlg125、umc2105、umc1705、bnlg1792这5个引物为玉米杂交种纯度鉴定的备选核心引物；phi072、bnlg162和phi065因多态性或杂合率低，不推荐作为玉米纯度鉴定用引物。研究了纯度鉴定用特异引物的选择问题，并确定了部分品种的纯度鉴定用特异引物。基于以上研究结果，进一步筛选玉米杂交种的双亲互补型引物，建立已知玉米杂交种的纯度鉴定DNA指纹图谱。此外，对玉米杂交种纯度鉴定用核心引物及特异引物的选择标准进行了探讨。 相似文献