苏云金芽孢杆菌肠毒素基因entFM的定位与序列分析

摘要：为确定Bt WB9的entFM基因是位于染色体或位于质粒上,以及明确entFM基因的特性,以entFM基因片段制备探针,通过Southern 杂交,分别对染色体DNA和质粒DNA进行杂交定位。结果表明,Bt WB9的entFM基因位于染色体上;克隆了Bt WB9、1072、TS16及Bc6A1 entFM基因ORF序列,序列分析表明,WB9 entFM基因的ORF序列共有个1281个核苷酸组成,可编码426个氨基酸。将WB9、TS16、1072 和Bc6A1 entFM基因编码的氨基酸序列与Bc Cx5相应氨基酸序列进行比较,结果显示WB9、TS16、1072与Bc Cx5之间有很高的相似性,主要差异在于Bc Cx5的EntFM氨基酸序列在39-42位置多出QTQT（谷氨酰胺-苏氨酸-谷氨酰胺-苏氨酸）四个氨基酸,在96-99位置还有SWDK四个氨基酸的差异。     

Glu-1和Glu-3等位变异对小麦加工品质的影响

Glu-1位点等位基因编码的高分子量麦谷蛋白亚基（HMW-GS）和Glu-3位点等位基因编码的低分子量麦谷蛋白亚基（LMW-GS）是决定小麦加工品质的重要因素。用优质面包小麦中优9507的两个杂交组合，即中优9507/鲁麦5号和中优9507/晋麦45 F5代，澳大利亚优质面包小麦Sunstate的两个杂交组合，即Sunstate/济南16和Sunstate/鲁麦21 F2     

郑丰5号α-醇溶蛋白基因的克隆与序列分析

α-醇溶蛋白是小麦籽粒贮藏蛋白的重要组分，其组成与含量对小麦加工品质具有重要影响。利用PCR从郑丰5号基因组中克隆α-醇溶蛋白基因，并对其序列进行分析。经克隆共获得32个α-醇溶蛋白新基因（ ZF5 A-1～ZF5A-32，GenBank注册序列号为JX828280～JX828311），其中15个为假基因，17个（ZF5A-1～ZF5A-17）具有完整开放阅读框。17个α-醇溶蛋白新基因中，除ZF5A-1、ZF5A-3、ZF5A-6、ZF5A-9、ZF5A-10、ZF5A-11、ZF5A-15编码的蛋白在特征Ⅱ区含有1个额外的半胱氨酸（ C）外，其他10个基因编码的蛋白均具有α-醇溶蛋白的典型结构。根据推断氨基酸序列中4种主要T细胞优势多肽的分布及多聚谷氨酰胺区的长度，推测ZF5A-7和ZF5A-12可能定位于6A染色体，ZF5A-4、ZF5A-13、ZF5A-14和ZF5A-17可能定位于6B染色体，而ZF5A-1～ZF5A-3、ZF5A-5、ZF5A-6、ZF5A-8～ZF5A-11、ZF5A-15和ZF5A-16可能定位于6D染色体。17个新克隆α-醇溶蛋白基因及4个已知α-醇溶蛋白基因编码的蛋白的二级结构预测结果表明：α-螺旋、β-折叠的位置和核心序列是相对保守的，但不同蛋白α-螺旋和β-折叠的数量以及参与形成同一保守区域α-螺旋和β-折叠的氨基酸残基数却并不相同。克隆的17个α-醇溶蛋白基因中，除ZF5A-17编码的蛋白缺少α-螺旋（ H2）、ZF5A-2、ZF5A-8编码的蛋白在特征区Ⅰ均存在1个额外的α-螺旋（ HE1）、GQ891685和ZF5A-15编码的蛋白在多聚谷氨酰胺Ⅱ区存在1个额外的α-螺旋（ HE2）外，5个保守的α-螺旋（ H1～H5）恒定出现在其他基因的2个谷氨酰胺重复区和特征区中；此外，在C-末端特征区大部分基因（61．11％）还形成1个β-折叠结构（ E）。郑丰5号中具有较多额外半胱氨酸、α-螺旋和β-折叠的α-醇溶蛋白基因，可能与其良好的加工品质密切相关。     

小麦品种“陕253”低分子量谷蛋白亚基基因的克隆及原核表达

利用LMW-GS特异引物,从强筋小麦品种陕253中克隆了1个1 498 bp的片段(GenBank登录号为FJ172533),该片段包含全长为912 bp的低分子量谷蛋白亚基的完整编码序列.经比较推导氨基酸序列的同源性,发现该基因属于Glu-D3位点编码低分子量谷蛋白亚基的基因,编码产物N-端具有LMW-m型低分子量谷蛋白亚基的典型特征,系统演化分析也支持这一结果.构建了该基因的表达载体pET32a-GluD3-S253,在宿主菌E. coll Rosetta-gami B(DE3)中经IPTG诱导表达融合蛋白.SDS-PAGE和Western blot检测表达产物,证实融合蛋白表达成功.     

新疆雪莲rbcs基因的克隆及序列分析

为获得新疆雪莲Rubisco（1,5-二磷酸核酮糖羧化酶/加氧酶）小亚基基因,并对其序列进行生物信息学分析;从已建立的新疆雪莲cDNA文库中,随机挑取单克隆,采用通用引物M13扩增并测序,测序结果在NCBI网址进行Blast比对,结果发现了2个与rbcs基因同源的序列并命名为sikrbcs1和sikrbcs2;生物信息学分析表明：sikrbcs1基因包含l个540 bp的完整开放阅读框,编码179个氨基酸,sikrbcs2基因包含l个420 bp的完整开放阅读框,编码139个氨基酸。sikrbcs1基因编码的蛋白质的理论等电点是8.90,分子量为20.1042 kD,具有2个跨膜区域,sikrbcs2基因编码的蛋白质理论等电点是7.58,分子量为15.1093 kD,不具有跨膜区域。氨基酸多序列比对表明,Sikrbcs1与黄顶菊和向日葵的相似性最高,达到83.89%,Sikrbcs2与向日葵的相似性最高达到57.54%;系统进化树显示表明,sikrbcs1和sikrbcs2都与菊花亲缘关系最近。     

棘孢木霉ACCC30536葡聚糖酶Glu41基因克隆及分析

克隆获得生物防治菌棘孢木霉（T. asperellum）ACCC30536 菌株葡聚糖酶基因Glu41 的cDNA序列和DNA序列,cDNA和DNA的GenBank接受号分别为KJ541741 和KJ541742,编码区长度为1134bp,编码378 个氨基酸。克隆获得的启动子序列长度1500bp,含有6 个逆境响应元件。BlastP 相似性分析表明该基因与Glyco-hydrolase-16 家族中棘孢木霉CBS433.97 基因同源性最高相似度是60%,SignalP 信号肽分析显示N端第25 个和第26 个氨基酸中间存在一个信号肽剪切位点（AFA||AP）。Pfam 蛋白家族分析显示它属于Glyco-hydrolase-16 家族的葡聚糖酶基因。将棘孢木霉ACCC30536 菌株葡聚糖酶基因Glu41 基因对Glyco-hydrolase-16 家族中木霉属的CBS433.97 基因组进行同源性搜索,在基因组上获得4个同源序列。4 个同源基因表达中仅有Glu41 在杨树根茎叶粉的诱导条件下表达。表明棘孢木霉葡聚糖酶基因Glu41 与生物防治相关。     

稗草磷酸烯醇式丙酮酸羧化酶(PEPCase)基因的克隆与分析

为揭示C₄野生植物稗草（Echinochloa crusgalli）PEPCase的结构和功能特点,探索改善作物高光效新途径,本研究首次克隆了稗草（E. crusgalli）磷酸烯醇式丙酮酸羧化酶基因(ppc)的cDNA全长,测序及同源性比较分析结果证实,稗草ppc的cDNA全长为2 886 bp,编码961个氨基酸（GenBank登录号：AY251482）;核苷酸序列与谷子(Setaria italica)C₃型、高粱(Sorghum bicolor) C_3-2型、玉米(Zea mays) C_3-2型、水稻(Oryza sativa)C₃型磷酸烯醇式丙酮酸羧化酶基因的同源率分别达94.8%、93.2%、93.0%和89.7%。推导的氨基酸序列中C末端第771位氨基酸是丙氨酸（A）,表明是一个C₃型基因。与其他植物几种不同形式PEPCase进行的多重序列比对与系统进化分析结果也证实,此基因的核苷酸序列及其编码的氨基酸序列与所有参与对比的C₃型序列的同源性均远远高于与C₄型的同源性。与玉米、高粱C_3-2型PEPCase的同源性分别高达到96.5%、96.4%,与高粱、玉米的C_3-1型PEPCase的同源性分别为84.3%、83.8%,而与谷子、玉米、甘蔗和高粱的C₄型PEPCase的一致性相对较低,分别为82.2%、79.1%、77.1%、76.6%。因此进一步推论新克隆的稗草ppc基因属于C_3-2型。对其编码的蛋白序列进行了结构域、活性位点和功能位点预测。     

叶籽银杏GbMADS5基因的克隆与序列分析

MADS-box基因是一类编码转录因子的基因大家族,在植物的发育过程中具有重要作用。本研究采用RT-PCR技术从叶籽银杏（Ginkgo biloba var.epiphylla Mak.）中克隆了MADS-box基因GbMADS5。序列分析表明,该基因编码区全长为666bp,含有一个完整的开放阅读框,编码221个氨基酸残基组成的蛋白质,具有典型的植物MADS-box基因结构,编码肽链包含了MADS区、I区、K区和C末端,但该基因不具有拟南芥（Arabidopsis thaliana）AG基因的N端区域。GbMADS5基因编码的蛋白序列与其它植物的MADS-box蛋白序列有着较高的一致性,与裸子植物（Gymnospermae）的AG基因相似性最高,其中与苏铁（Cycas revolute）的同源性高达91.07%。系统发育树分析显示,GbMADS5基因属于AG亚家族基因。     

大肠杆菌Top10甘露醇-1-磷酸脱氢酶基因克隆及生物信息学分析

根据细菌中存在的甘露醇-1-磷酸脱氢酶（mannitol-1-phosphate dehydrogenase,mtlD）的基因序列,采用PCR扩增的方法从大肠杆菌（Escherichia coli）Top10中克隆到了mt／D基因（Acession numeber：EU433565）。mtlD开放阅读框为1149bp,编码含有382个氨基酸残基,分子量为41．2kD的蛋白,预测等电点为5．37。mtlD含有38个碱性氨基酸,50个酸性氨基酸,158个疏水氨基酸及72个极性氨基酸。二级结构预测表明该蛋白含约60．47％的α-螺旋、4．71％的β-转角、10．73％的延伸链和20．48％的不规则卷曲。亲疏水性分析显示,mtlD是亲水性蛋白。亚细胞定位分析表明mtlD主要定位于细胞质中。序列分析表明大肠杆菌Top10 mtlD基因与大肠杆菌W3350、大肠杆菌DEC12a和盐生盐杆菌（Halobacterium salinarum）的mtlD基因同源性分别为99％、97％和93％。大肠杆菌Top10mtlD基因的克隆及生物信息学分析为今后对mtlD的进一步深入研究奠定了基础。     

甘薯番茄红素β-环化酶基因的克隆与转化烟草的研究

番茄红素β-环化酶（LYC-B）催化番茄红素形成β-胡萝卜素，是β-胡萝卜素生物合成的关键酶之一。从甘薯块根总RNA逆转录的cDNA中扩增出lyc-b的保守序列后，用cDNA末端快速扩增方法（RACE）获得了该基因全长cDNA，共1 844 bp，开放阅读框1 503 bp，编码501个氨基酸。构建了lyc-b植物表达载体p23-lyc-b，通过农杆菌介导法转化烟草，经PCR及Southern杂交表明该基因已整合到烟草的基因组中。     

Identification of low-molecular weight glutenin subunits of wheat associated with bread-making quality

Although it is known that the compositions of low‐molecular weight glutenin subunits (LMW‐GSs) are important factors for bread‐making quality of wheat, it is still not clear which LMW‐GSs confer improved bread‐making quality and how those LMW‐GSs interact with high‐molecular weight (HMW) GSs. Using a hard red winter wheat line with good bread‐making quality and a Japanese wheat cultivar with poor quality as well as their progeny we identified LMW‐GSs associated with the bread‐making quality. One such LMW‐GS, KS2, which had a molecular weight of 42 kDa and was allelic to HS1, was associated with bread‐making quality. Furthermore, by using four recombinant inbred lines with different HMW‐GS and LMW‐GS combinations, KS2 and HMW‐GS 5+10 showed interaction effects on the bread‐making quality. Two‐dimensional polyacrylamide gel electrophoresis (Page) analysis showed that KS2 consists of two protein components and that HS1 is composed of three components. The N‐terminal amino acid sequences of the five components were identical to the most frequently analysed sequence of LMW glutenin components.     

PCR-based assay for detecting IB-genes for low molecular weight glutenin subunits related to gluten quality properties in durum wheat

A PCR assay has been developed for selecting low molecular weight (LMW) glutenin genes, located on chromosome 1B, and related to durum wheat quality characteristics. Most durum wheat lines possessing good technological properties can be identified on the basis of the presence of specific 1B LMW glutenin components that have been designated LMW-2 glutenin subunits. On the basis of nucleotide sequences corresponding to LMW glutenin genes, a series of specific oligonucleotide primers were prepared and used in PCR analysis. A pair of these primers gave a single amplification product which can distinguish between durum wheat lines possessing LMW-2 glutenin subunits and lines possessing other 1B-LMW allelic variants. Because the proposed PCR analysis can be carried out using part of the endosperm of a single seed, it represents a helpful approach for speeding up the selection of genotypes possessing LMW-2 glutenin subunits. This part of primers could also be very useful in genome mapping analysis and for testing the purity of wheat flour stock.     

3个新疆地方小麦品种Glu-A3位点低分子量谷蛋白亚基新基因的序列分析

麦谷蛋白可分为高分子量麦谷蛋白亚基(HMW-GS)和低分子量麦谷蛋白亚基(LMW-GS),其中低分子量麦谷蛋白亚基对小麦品质具有重要影响.本文采用PCR方法从中国小麦微核心种质中的3个新疆小麦品种红春麦(新疆玛纳斯)、红春麦(新疆昌吉)和红金包银(新疆伊吾)中分别得到了3个低分子量麦谷蛋白亚基(LMW-CS)新基因(GenBank:DQ519084、DQ519085和DQ517534).它们具有LMW-i型基因的典型结构特征,与已报道的Glu-A3位点编码的LMW-GS基因序列有很高的一致性,高达79.06%～94.24%.DQ519084和DQ517534在C末端保守区有9个半胱氨酸残基,DQ519085其编码区内存在1个提前终止密码子,推测其为假基因.     

不同品质类型春小麦HWM—GS形成时间和积累强度及与品质的关系

供试材料籽粒形成过程中，麦谷蛋白低分子量亚基部分首先出现，随籽粒发育，麦谷蛋白亚基类型逐渐增多，HMW－GS各个亚基在开花后10天内没有形成，开花15天左右，出现Glu-1位点上编码的高分子量X亚基，25天时，所具有的HMW－GS全部形成，随灌浆成熟，HMW－GS各亚基积累呈递增趋势，积累高峰出现在开花20天至成熟，5＋10亚基与具有该亚基供试品种的优良品质之间存在密切关系，但具有2＋12亚基的东农7742的亚基形成早，2与12亚基的高积累量及1和7＋8亚基的高积累使其同样具有优质特性，Glu-1品质评分与沉淀值之间呈显著正相关，能够反映小麦品质，但并不绝对，小麦高分子量麦谷蛋白亚基总积累与沉淀值呈显著正相关，各高分子量亚基与沉淀值之间的相关未达显著水平，但亚基2与12的积累量与沉淀值的相关性强，尤其是12亚基的积累。     

不同品质类型春小麦HMW-GS形成时间和积累强度及与品质的关系

供试材料籽粒形成过程中, 麦谷蛋白低分子量亚基部分首先出现. 随籽粒发育, 麦谷蛋白亚基类型逐渐增多. HMW-GS各个亚基在开花后10天内没有形成, 开花15天左右, 出现Glu-1位点上编码的高分子量X型亚基. 25天时, 所具有的HMW-GS全部形成. 随灌浆成熟, HMW-GS各亚基积累呈递增趋势, 积累高峰出现在开花20天至成熟. 5+10亚基与具     

Molecular structure of a novel y-type HMW glutenin subunit gene present in Triticum tauschii

An unusually small y-type high molecular weight (HMW) glutenin subunit gene from Triticum tauschii was sequenced. This gene, encoded at the Glu-D^t1 locus was designated 12.4^t and is the smallest HMW glutenin subunit gene described so far in Triticum species. Oligonucleotide primers based on published sequences of HMW glutenin genes were designed to amplify the encoding region and the central repetitive domain of the gene, which produced fragments of 1.4 and 0.85 kb, respectively. PCR products were cloned and sequenced. The derived amino acid sequence was compared with the amino acid sequences of the HMW glutenin subunits Dy12^t, from T. tauschii, and subunits Dy10 and Dy12 of T. aestivum. The amino acid sequence deduced from the nucleotide sequence demonstrated that deletions of hexapeptides and nonapeptides were responsible for the reduction in the size of this HMW glutenin subunit. The estimated molecular weight of the Dy12.4^t subunit, calculated on the basis of the deduced amino acid sequence, was 45,228 Daltons. There were also single amino acid differences in the N-, C-terminal and central repetitive domains of this gene in comparison to the three other y-type subunits encoded at the Glu-D1 locus. The Dy12.4^t subunit showed the highest similarity to the Dy12 subunit present in the hexaploid wheat Chinese Spring.     

Cloning and characterization of a novel low molecular weight glutenin subunit gene at the Glu-A3 locus from wild emmer wheat (Triticum turgidum L. var. dicoccoides)

Amplification of the coding region, and upstream and downstream sequences of a low-molecular-weight glutenin subunits (LMW-GS) gene from wild emmer wheat (Triticum turgidum L. var. dicoccoides, 2n = 4x = 28, AABB) accession TD22 was carried out using designed allele-specific PCR (AS-PCR) primers. The complete 1,176 bp sequence of a novel LMW-i type subunit gene at the Glu-A3 locus, named LMW-TD22, is described. Analysis of the nucleotide and deduced amino acid sequences showed that this gene possessed striking characteristics although its molecular structure was generally similar to those of previously reported i-type LMW-GS genes that were isolated from common wheat and related species. The deduced amino acid sequence of LMW-TD22 gene contained 390 amino acid residues with the predicted molecular weight being 43,009.3 Da, which appeared to be the longest gene among the cloned LMW-i type genes from bread wheat and related species. The distinct feature of LMW-TD22 was two long polyglutamine stretches of 12 and 17 glutamines occurring in the repetitive and C-terminal domains as well as a cysteine residue present in the seventh amino acid residue of the signal peptide. These polyglutamine repeats are believed to improve the structure of gluten polymer and increase the strength of dough formed from the polymer. In addition, the putative 44 k subunit encoded by LMW-TD22 was verified by N-terminal microsequencing, gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. Certain types of post-translational modifications, such as phosphorylation and glycosylation, may be associated with this LMW-i type subunit. A. Wang and Y. Xiao made equal contribution to the research as the first author.     

小麦谷氨酰胺合成酶前体Ⅱ基因的克隆与分析

采用RACE方法,从小麦耐盐突变体RH8706-49的转录产物中获得谷氨酰胺合成酶Ⅱ(GS2)前体基因的全长cDNA序列,共包含1 690 bp,其中ORF区1 284 bp,编码427氨基酸。该基因已提交GenBank（登录号：DQ103756）。经Blast比对,该基因与大麦、水稻、玉米GS2的同源性分别达94.6%、87%和90.4%。经Northern Blottin     

高分子量麦谷蛋白亚基组成与小麦烘烤品质关系研究

用SDS-PAGE方法测定了全国9个小麦主产省、市163个小麦品种和11个引进品种的高分子量麦谷蛋白亚基（HMW-GS）组成及其含量、沉降值和面粉GMP含量。结果表明，不同HMW-GS及其组合形式对小麦品质的影响显著不同，某些HMW-GS对我国小麦烘烤品质的影响不同于其他国家，如4+12亚基影响较大，而7+8亚基影响偏小。但单个亚基品质评分     

Glu-1位点缺失对小麦麦谷蛋白聚合体粒度分布及面团特性的影响

以Glu-1位点正常和部分缺失的小麦品系为材料,探讨HMW-GS和LMW-GS组成与谷蛋白聚合体粒度分布和面团特性的关系,为利用HMW-GS缺失系改良小麦品质提供理论依据。在20个供试硬白冬麦品系中,1个品系为Glu-A1位点缺失,5个品系为Glu-D1缺失,3个品系为Glu-A1和Glu-D1双缺失。所有品系的蛋白质含量皆较高(13.39%~14.12%),品系间无显著差异,缺失系与非缺失系间也无显著差异。Glu-1位点缺失显著降低了高分子量谷蛋白/低分子量谷蛋白比(HMW/LMW)、不溶性谷蛋白大聚体的含量和百分比。谷蛋白/醇溶蛋白比(GLU/GLI)在基因型间变幅较小,且在缺失系和非缺失系间无显著差异。Glu-1位点缺失显著降低了面团弹性,但显著提高了面团的延展性。部分Glu-1位点缺失系仍具有较高的面团强度和突出的延展性,谷蛋白聚合体粒度分布和面团特性受谷蛋白亚基组成和表达量的共同影响。研究结果表明,利用Glu-1位点亚基缺失可能是改善面筋延展性,提高食品加工品质的方法之一。