一种检测转1Dx5基因小麦植株的新方法

目的:建立一种有效区分Glu-1D等位基因的Multiplex-PCR体系,快速检测转1Dx5小麦植株。方法:根据1Dx5和1Dx2亚基基因的区别设计特异的PCR引物,通过多重PCR技术检测花粉管通道法转化1Dx5核心片段的转基因T1代植株。结果:Multiplex-PCR能够在转基因T1代材料中扩增Glu-1D等位基因的特征条带,分别为343bp、320bp的1Dx5基因特异片段以及361bp的1Dx2基因特异片段,与预期结果一致。结论:该技术能够检测多个靶基因,有效地区分转基因Glu-1D上的等位基因,证实外源1Dx5基因已整合到受体基因组中,对检测基因组庞大、外源基因序列GC含量高且与内源基因同源性高的转基因小麦十分有效。     

基因枪法获得逆境诱导转录因子DREB1A转基因小麦的研究

以小麦品种H6756和藁城8901作为基因枪转化的靶材料,取其护颖至雌雄蕊原基形成期的幼穗,用含逆境诱导转录因子DREB1A和bar基因的质粒pAHC25轰击胚性愈伤组织,在分别含有5mgL和10mgLBasta溶液的培养基上进行筛选。得到的抗性愈伤组织在不含Basta溶液的培养基上再生培养,获得218棵再生植株。田间涂抹浓度为100mgL的Basta溶液检测后,对抗性植株作PCR检测,获得54棵再生植株。通过对其中20株T1代的PCR和Southern杂交分析,已获得14株含DREB1A和bar基因的转基因小麦植株,其中H675613株,藁城89011株。     

小麦HMW-GS1Dx5基因的克隆及其特异性表达

显微切割了普通小麦钢８２－１２２（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍ２ｎ＝４２）具有１Ｄｘ５＋１Ｄｙ１０亚基的１Ｄ染色体长臂端,利用ＰＣＲ扩增得到了ＨＭＷ－ＧＳ１Ｄｘ５亚基的５（端４００ｂｐ序列片段．以此作为探针从基因的组织特异性和特定发育阶段的表达两个方面研究了ＨＭＷ－ＧＳ１Ｄｘ５基因表达的规律．结果表明,干种子及萌发种子中存在此基因,而在发育的幼苗中此基因未表达．ＨＭＷ－ＧＳ１Ｄｘ５基因可能从开花初期开始表达．ＨＭＷ－ＧＳ１Ｄｘ５基因在籽粒成熟期表达,然而在营养器官如叶片中未表达,其表达存在组织特异性．ＨＭＷ－ＧＳ１Ｄｘ５基因在蜡熟期籽粒表达水平最高,其次是乳熟期籽粒．从开花１５ｄ至蜡熟期籽粒,表达趋于增加．开花１５ｄ其ｍＲＮＡ水平是蜡熟期籽粒ｍＲＮＡ的２８％,灌浆期为４０％、乳熟期为７２％、完熟期为５４％．这为进一步研究其表达调控和改善小麦品质打下基础     

外源1Dx5基因导致小麦高分子量麦谷蛋白亚基表达变异

目的:为了结合基因枪转化和传统杂交方法培育优质小麦品种,对转基因小麦和国内主栽小麦品种杂交后代外源基因遗传表达行为进行了研究。方法:采用SDS-PAGE对2个小麦杂交组合川89-107×B72-8-11b和鄂麦18×B72-8-11b的杂交及回交后代籽粒进行高分子量麦谷蛋白亚基遗传表达分析。结果:在亲本中能够稳定超量表达的外源基因1Dx5在杂交后代中出现了不同的表达量,而且在外源基因的影响下,杂交后代出现了新的、杂交亲本并不表达的高分子量麦谷蛋白亚基。结论:多拷贝的外源基因在不同于受体环境的细胞质中的表达发生了变化,且由于外源基因的插入引起了内源高分子量麦谷蛋白亚基组成的变异。     

小麦染色体显微切割及HMW-GS1Dx5亚基基因克隆

宏伟的人类基因组计划以及随之而来的动物、植物（如水稻等）基因组计划为克隆有益基因提供了新方法和思路,其中一个令人注目的领域是染色体显微切割。该技术起始于８０年代早期,首次应用显微切割和微克隆的是Ｓｃａｌｅｎｇｈｅ等在果蝇多线染色体上进行的。随后在哺乳...     

基因枪法转化小麦的金粉用量及转基因植株表型特征分析

研究了不同金粉用量对小麦幼胚瞬间及稳定转化频率的影响,结果表明此实验系统的金粉用量以每枪500μg金粉为佳。对获得的T     

基因枪法获得GAI转基因橡胶植株的研究

将含有Camv35S启动子、卡那霉素抗性基因和GUS报告基因,目的基因为GAI基因的转化载体质粒pBI121,通过基因枪轰击巴西橡胶树(Hevea brasiliensis Muell-Arg.)花药愈伤组织,50 mg L~(-1)卡那霉素的继代培养基进行抗性筛选.获得了抗性再生植株,经过PCR、Southern检测结果表明:GAI基因已经成功转入橡胶树基因组中.     

基因枪法获得GAI转基因橡胶树植株的研究

将含有Camv35S启动子、卡那霉素抗性基因和GUS报告基因,目的基因为GAI基因的转化载体质粒pBI121,通过基因枪轰击巴西橡胶树(Hevea brasiliensis Muell-Arg.)花药愈伤组织,50 mg L~(-1)卡那霉素的继代培养基进行抗性筛选.获得了抗性再生植株,经过PCR、Southern检测结果表明:GAI基因已经成功转入橡胶树基因组中.     

基因枪法转化小麦的金粉用量及转基因植株表型特征分析

研究了不同金粉和量对小麦幼胚瞬间及稳定转化频率的影响,结果表明此实验系统的金粉用量以每枪500μg金粉为佳。对获得的T0及T1代植株的PCR及Southern分析证实了外源bar基因片段已经稳定整合至小麦基因组中;对T1代植株的L－PPT涂抹实验表明转化植株对该除草剂的抗性水平较低;分析了T0、T1及T2代植株的表型特征,结果表明T0代植株生长矮小、结实充低等表型方面的异常很可能只是一种由于生理及栽培原因所导致的暂时现象。     

小麦HMW-GSIDx5基因在小麦不同发育阶段表达的研究(英文)

小麦面粉的烘烤品质与其高分子量谷蛋白5亚基（HMW－GSIDx5）基因的表达有关。本文从该基因的组织、器官特异性和特定发育阶段的表达特性等方面,对其在小麦发育过程中表达的变化规律进行了研究,从分子水平为小麦的栽培和育种提供依据。显微切割普通小麦钢82－122的ID染色体长臂为模板,扩增该基因400bp的片段为探针（Fig．1）,并经重组后测序（Fig．2）确认扩增无误。小麦苗期、生长期、开花期及成熟期分别取样、分离总RNA、做斑点（Fig．3,4＆5）或Northern杂交（Fig．6）、并扫描足量（Fig．7）。结果表明：该基因只在籽粒中表达（Fig．3,4＆5）,从开花15天起（Fig．4）,逐渐增加,至腊熟期达最高,以后渐次降低（Fig．6＆7）,有趣的是：在干种子和萌发种子亦有少量表达（Fig．1）。Cressey和陈和等方曾报告在开花15天可检测到HMW－GS蛋白。     

小麦HMW-GS1D×5基因时间特异性表达的研究

利用小麦高分子量谷蛋白１Ｄ×５亚基探针,研究ＨＭＷ－ＧＳ１Ｄ×５基因表达的规律。结果表明,ＨＭＷ－ＧＳ１Ｄ×５基因从开花初期开始表达。这为从生理、营养、施肥等诸多方面人为地对ＨＭＷ谷蛋白基因的表达加以调控,使之在特定时期能够充分表达该基因从而改善小麦品质打下基础。     

Inheritance and Expression of Copies of Transgenes 1Dx5 and 1Ax1 in Elite Wheat （Triticum aestivum L.） Varieties Transferred from Transgenic Wheat through Conventional Crossing

To study the inheritance and expression of multiple copies of transgenes from transgenic wheat lines, three crosses between transgenic wheat lines B72-8-11b and B102-1-2 and Chinese elite wheat varieties Chuan89-107 and Email 8 were carried out. Chuan89-107×B72-8-11b, Chuan89-107×B102-1-2 and Email 8×B72-8-11b, and F_1 plants were selfed or backcrossed to obtain different generation populations. Protein analysis in grains of F_1 and F_2 and backcross progenies of BC_1F_1, BC_1F_2, BC_1F_3, BC_2F_1, BC_2F_2 and BC_2F_3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the transgenes lDx5 and lAx1 were expressed and segregated in the target wheat according to Mendelian laws. A range of lDx5 expression levels were observed in the progenies of Chuan89-107×B72-8-11b and Emai 18×B72-8-11b, but the expression levels of lAx1 in progenies of Chuan89-107×B102-1-2 rarely changed. It suggested that the two foreign genes had different mechanisms of expression in the cross progeny, even though they were produced in the same way and the foreign lDx5 gene of 5-10 copies had the more complicated expression mechanism than the lAx1 gene of 4-5 copies.     

Study of high molecular weight wheat glutenin subunit 1Dx5 by 13C and 1H solid-state NMR spectroscopy. I. Role of covalent crosslinking

This work describes a carbon and proton solid-state NMR study of the hydration of a high molecular weight wheat glutenin subunit, 1Dx5. The effect of the presence of disulfide bonds on the hydration behavior of the subunit is investigated by a comparison of the unalkylated and alkylated forms of the protein. Hydration induces partial plasticization of the protein so that some segments become more mobile than others. The 13C cross-polarization and magic-angle spinning (MAS) spectra of the samples in the dry state and at two hydration levels (approximately 40 and approximately 65% D2O) were used to monitor the protein fraction resisting plasticization (trains). Conversely, 13C single pulse excitation and 1H-MAS experiments were used to gain information on the more plasticized segments (loops). The molecular motion of the two protein dynamic populations was further characterized by 13C T1 and 1H T(1rho), T2, and T1 relaxation times. The results suggest that hydration leads to the formation of a network held by a cooperative action of hydrogen bonded glutamines and some hydrophobic interactions. The looser protein segments are suggested to be glycine- and glutamine-rich segments. The primary structure is therefore expected to significantly determine the proportion of trains and loops in the network. The presence of disulfide bonds was observed to promote easier plasticization of the protein and the formation of a more mobile network, probably involving a higher number of loops and/or larger loops.     

簇毛麦5VS染色体臂特异分子标记开发与遗传效应分析

簇毛麦(Dasypyrum villosum(L.)P.Candargy 2n=14,VV)是小麦改良重要的三级基因源.簇毛麦5VS染色体臂上携带抗白粉病基因Pm55、抗条锈病基因Yr5V和籽粒硬度基因Dina/Dinb等优异基因.已创制的小麦-簇毛麦T5VS.5AL和T5VS.5DL易位系为小麦抗病和品质改良提供了优...     

Study of wheat high molecular weight 1Dx5 subunit by (13)C and (1)H solid-state NMR. II. Roles of nonrepetitive terminal domains and length of repetitive domain

This work follows a previous article that addressed the role of disulfide bonds in the behavior of the 1Dx5 subunit upon hydration. Here the roles of nonrepetitive terminal domains present and the length of the central repetitive domain in the hydration of 1Dx5 are investigated. This was achieved by comparing the hydration behavior of suitable model samples determined by (13)C- and (1)H-NMR: an alkylated 1Dx5 subunit (alk1Dx5), a recombinant 58-kDa peptide corresponding to the central repetitive domain of 1Dx5 (i.e., lacking the terminal domains), and two synthetic peptides (with 6 and 21 amino acid residues) based on the consensus repeat motifs of the central domain. The (13)C cross-polarization and magic angle spinning (MAS) experiments recorded as a function of hydration gave information about the protein or peptide fractions resisting plasticization. Conversely, (13)C single pulse excitation and (1)H-MAS gave information on the more plasticized segments. The results are consistent with the previous proposal of a hydrated network held by hydrogen-bonded glutamines and possibly hydrophobic interactions. The nonrepetitive terminal domains were found to induce water insolubility and a generally higher network hindrance. Shorter chain lengths were shown to increase plasticization and water solubility. However, at low water contents, the 21-mer peptide was characterized by higher hindrance in the megahertz and kilohertz frequency ranges compared to the longer peptide; and a tendency for a few hydrogen-bonded glutamines and hydrophobic residues to remain relatively hindered was still observed, as for the protein and large peptide. It is suggested that this ability is strongly dependent on the peptide primary structure.     

转基因小麦外源优质亚基基因的杂交转育研究

在获得外源品质基因1Dx5和1Ax1超量表达的转基因小麦的基础上,利用小麦转基因品系‘B72-8-11b’和‘B102-1-2’为父本,主要以湖北省栽培品种‘鄂麦12’为母本,配置杂交组合。杂交后代中采用系谱选择法,结合HMW-GS鉴定,研究了转基因小麦外源品质基因在F1、F2、F3、F4代的传递,并筛选出外源1Dx5或1Ax1基因保持超表达的2个新型转基因株系;同时证明了将外源品质基因向栽培品种转育,是提高小麦优质亚基含量和提高HMW-GS总量的有效方法之一。