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。     

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。     

杂交后代中转基因小麦外源1Ax1基因的遗传

高分子量麦谷蛋白亚基1Ax1基因是决定小麦加工品质的主效基因之一,在小麦胚乳中增加1Ax1基因的表达量可以提高其加工品质,这对于小麦的品质改良具有重要意义。采用外源1Ax1基因超量表达的转基因小麦‘B102-1-2’为父本,常规小麦品种‘鄂麦12’和‘川89-107’为母本进行杂交试验。采用SDS-PAGE技术检测并分析各组合亲本、F1代、F2代的HMW-GS组成,从而研究转基因小麦‘B102-1-2’中外源品质基因1Ax1表达的遗传规律。研究结果表明:外源基因有效地整合进入主栽小麦的基因组中,并且正确表达,在F2代中表现出15∶1的分离比,遵循孟德尔遗传模式,这对于杂交育种策略的选择制订具有指导意义。     

无载体框架序列转基因小麦中外源1Ax1基因表达框的遗传分析

为了研究无载体框架序列转基因小麦中转基因表达框的遗传规律,选育稳定表达的转基因株系,利用基因枪介导1Ax1基因的最小表达框转化得到了转基因小麦,对其后代转基因植株中1Ax1基因表达框的遗传进行了分析,结果表明：无载体框架结构的1Ax1基因在转基因后代中稳定遗传,在T1代中呈现3:1的分离,遵从孟德尔遗传模式;SDS－PAGE分析表明其中部分转基因后代分离出新的高分子量蛋白亚基分子量略低于1Dx5亚基。     

杂交后代中转基因小麦外源1Ax1基因的遗传

高分子量麦谷蛋白亚基1Ax1基因是决定小麦加工品质的主效基因之一,在小麦胚乳中增加1Ax1基因的表达量可以提高其加工品质,这对于小麦的品质改良具有重要意义。采用外源1Ax1基因超量表达的转基因小麦‘B102-1-2’为父本,常规小麦品种‘鄂麦12’和‘川89-107’为母本进行杂交试验。采用SDS-PAGE技术检测并分析各组合亲本、F₁代、F₂代的HMW-GS组成,从而研究转基因小麦‘B102-1-2’中外源品质基因1Ax1表达的遗传规律。研究结果表明:外源基因有效地整合进入主栽小麦的基因组中,并且正确表达,在F₂代中表现出15:1的分离比,遵循孟德尔遗传模式,这对于杂交育种策略的选择制订具有指导意义。     

多种优质高分子量谷蛋白亚基的聚合育种研究

用携带HMW-GS14 15的小偃6号作为轮回亲本,携带HMW-GS5 10的法国优质面包小麦品系707作为供体亲本,在回交后代的BC1、BC2、BC3、BC3F1、BC3F2代其它农艺性状选择的基础上,利用1对特异引物逐代检测出携带优质,Dx5基因的单株进行回交和自交。BC1代中随机检测的58个单株的Dx5基因分布符合1对等位基因的遗传分离比例1：1;BC1代小麦相同3个单株3个不同生长季节Dx5基因检测的结果完全一致,检测结果非常稳定;已将优质Dx5基因导入BC3F2后代的部分单株内;携带Dx5基因的株系XN89-7-3微量SDS沉淀值为18．8mL,比小偃6号提高了23．68％;蛋白质电泳筛选出了6个聚合多种优质亚基且编码基因纯合的单株,微量SDS沉淀值为19．9mL,比小偃6号提高了30．92％;选择农艺性状与轮回亲本相似并具有Dx5基因特异扩增产物的单株进行回交或自交,可加快回交转育的进度。实践证明,回交转育与分子标记辅助选育相结合的育种方法是快速定向聚合多种优质HMW-GS基因的有效方法之一。     

小麦花药培养后代和杂交后代中高分子量麦谷蛋白亚基变异分析

通过对Alondra、Orofen等5个小麦品种进行花药培养,同时以新春9号、京771、CB037等9个高分子量麦谷蛋白亚基组成不同的小麦品种相互间配制24个正、反交组合,分析小麦加倍单倍体无性系和品种间杂交后代中高分子量麦谷蛋白亚基变异,探讨利用花药培养和杂交手段改良HMW-GS组成的可能性。SDS-PAGE电泳分析发现,小麦加倍单倍体无性系中HMW-GS发生了频繁变异,Alondra加倍单倍体中变异率最高(61.8%),Verry加倍单倍体次之(16.7%),均出现了原始材料中所不具备的亚基类型;HMW-GS在部分F1杂种中呈现不完全共显性、亚基表达沉默和正、反交亚基表达不一致现象,宁春4号/CB037、京771/宁春4号2个组合中出现了双亲所不含有的亚基;通过连续自交和对新出现亚基的跟踪选择,获得了表达新亚基的高代株系。研究结果对于改良小麦加工品质,加深了解小麦HMW-GS编码基因的遗传特性、结构特性等具有一定理论意义和实践价值。     

基因枪法获得无选择标记的1Dx5基因表达的转基因小麦

利用基因枪将无选择标记的优质高分子量麦谷蛋白亚基基因1Dx5导入新疆耐盐小麦品种新冬26,为利用优质基因进行小麦品质改良奠定基础。构建无选择标记的线性1Dx5表达框。利用基因枪将其转入不含该亚基的小麦品种新冬26幼胚盾片中,经PCR二分法筛选,从转化的1 000块幼胚盾片中共获得3株转基因阳性植株,转化效率0.3%。利用SDS-PAGE分析目的基因在转基因后代籽粒中的表达。转基因植株后代种子分析表明,1Dx5在转基因后代部分种子中表达。本研究成功地将无选择标记的线性1Dx5片段导入普通小麦新冬26中,并在后代部分种子中得到了表达。为利用优质亚基基因改良小麦加工品质奠定基础。     

小麦HMW-GS转基因沉默效应的遗传分析

以主栽小麦品种'高原602'和'高原142'为母本,'QQ5'为父本进行杂交,采用SDS-PAGE检测亲本、杂种F1、F2及BC1F1、BC1F1′代的HMW-GS表达情况.结果表明:'QQ5'中HMW-GS的沉默效应在杂交后代中表现为显性,在杂种F1、F2及BC1F1、BC1F1′代中遵循孟德尔遗传规律;'QQ5'自身的沉默效应对育成品种中的其他HMW-GS也有作用;在'QQ5'和杂交后代中,对HMW-GS的沉默并不影响LMW-GS的表达;在杂交F2中,出现了一些原本只在'QQ5'的野生型(bobwhite)中表达的带型,这说明'QQ5'中HMW-GS的基因组DNA并没有被破坏,其沉默机制可能在mRNA水平.     

矮败小麦轮回选择群体品质性状的分子改良

利用HMW-GS优异种质和矮败小麦遗传改良工具,通过苗期HMW-GS基因PCR分子跟踪并结合籽粒SDS-PAGE检测,将优异HMW-GS导入并聚合于矮败小麦,构建矮败小麦优质轮回选择群体.结果表明,在开花期对轮回选择群体的287个单株进行1Dx5和1Bx14基因的PCR检测,有225个单株携带1Dx5基因,有120个单株携带1Bx14基因;其中,有58个单株同时携带1Dx5和1Bx14基因.对群体中287个单株籽粒的HMW-GS组成进行分析,有22%籽粒在Glu-B1、Glu-D1位点发生了亚基聚合:224个单株携带5+10亚基,126个单株携带14+15亚基,有63个单株同时携带5+10+14+15.     

The Interactive Effects of Transgenically Overexpressed 1Ax1 with Various HMW-GS Combinations on Dough Quality by Introgression of Exogenous Subunits into an Elite Chinese Wheat Variety

Seed storage proteins in wheat endosperm, particularly high-molecular-weight glutenin subunits (HMW-GS), are primary determinants of dough properties, and affect both end-use quality and grain utilization of wheat (Triticum aestivum L). In order to investigate the interactive effects between the transgenically overexpressed 1Ax1 subunit with different HMW-GS on dough quality traits, we developed a set of 8 introgression lines (ILs) overexpressing the transgenic HMW-glutenin subunit 1Ax1 by introgression of this transgene from transgenic line B102-1-2/1 into an elite Chinese wheat variety Chuanmai107 (C107), using conventional crossing and backcrossing breeding technique. The donor C107 strain lacks 1Ax1 but contains the HMW-GS pairs 1Dx2+1Dy12 and 1Bx7+1By9. The resultant ILs showed robust and stable expression of 1Ax1 even after five generations of self-pollination, and crossing/backcrossing three times. In addition, overexpression of 1Ax1 was compensated by the endogenous gluten proteins. All ILs exhibited superior agronomic performance when compared to the transgenic parent line, B102-1-2/1. Mixograph results demonstrated that overexpressed 1Ax1 significantly improved dough strength, resistance to extension and over-mixing tolerance, in the targeted wheat cultivar C107. Further, comparisons among the ILs showed the interactive effects of endogenous subunits on dough properties when 1Ax1 was overexpressed: subunit pair 17+18 contributed to increased over-mixing tolerance of the dough; expression of the Glu-D1 allele maintained an appropriate balance between x-type and y-type subunits and thereby improved dough quality. It is consistent with ILs C4 (HMW-GS are 1, 17+18, 2+12) had the highest gluten index and Zeleny sedimentation value. This study demonstrates that wheat quality could be improved by using transgenic wheat overexpressing HMW-GS and the feasibility of using such transgenic lines in wheat quality breeding programs.     

Functional properties of flours from field grown transgenic wheat lines expressing the HMW glutenin subunit 1Ax1 and 1Dx5 genes

The high molecular weight glutenin subunits (HMW-GS) of wheat are major determinants of the viscoelastic properties of gluten and dough. The bread making quality of field grown transgenic lines of bread wheat expressing the HMW-GS 1Ax1 or 1Dx5 genes were evaluated over a two year period. Subunit 1Ax1 represented about 29% and 48% of the total HMW-GS in lines 1-2 and 2-2, respectively, while subunit 1Dx5 represented 65.4% and 62% of the total HMW-GS in transgenic lines 6-2 and 9, respectively. The expression of subunits 1Ax1 or 1Dx5 in transgenic wheat led to corresponding decreases in the proportions of endogenous HMW-GS. HMW-GS 1Ax1 and 1Dx5 had contrasting effects on dough quality determined by the Alveograph and sedimentation test. Subunit 1Ax1 increased the tenacity (P), extensibility (L), deformation work (W), and sedimentation value, with the increase being related to the level of expression. In contrast, subunit 1Dx5 led to a smaller increment in the tenacity (P), but to drastic decrease in both extensibility (L), deformation work (W), and the sedimentation value. Expression of subunit 1Ax1 in transgenic wheat resulted in lines with improved rheological properties whereas the lines expressing subunit 1Dx5 resulted in unsuitable breadmaking-related characteristics.     

Creating Wheat Germplasm for High Quality Breeding by Monosomic Backcrossing

By crossing bread wheat cultlvar GC8901 with the 1D monosonlc line of Xiaoyan No. 6 and backcrosslng the offsprlng with the Xlaoyan No. 6 1D monosonlc llne for 5 years, high-molecular-welght glutenin subunlts 1Dx5＋1Dy10 from GC8901 have been transferred Into wheat cultivar Xiaoyan No. 6. The BC5F1 offspring lines had been detected by using methods of cytology, marker, molecular marker and six elite single plants with high molecular-welght glutenin subunlts： lAx1, 1Bx14＋1 By15, 1Dx5＋1 Dy10 were Identified. Those lines have high-yleld potential with better agronomic characters and have been used In high quality wheat breeding processes as well.     

Transformation of pasta wheat (Triticum turgidum L. var. durum) with high-molecular-weight glutenin subunit genes and modification of dough functionality

Particle bombardment has been used to transform three cultivars (L35, Ofanto, Svevo) and one breeding line (Latino × Lira) of durum wheat (Triticum turgidum L. var. durum). These varieties were co-transformed with plasmids containing selectable and scorable marker genes (bar and uidA) and plasmids containing one of two high-molecular-weight (HMW) glutenin subunit genes (encoding subunits 1Ax1 or 1Dx5). Ten independent transgenic lines were recovered from 1683 bombarded scutella (transformation efficiency thus 0.6%). Five lines expressed either subunit 1Dx5 or 1Ax1 at levels similar to those of endogenous subunits encoded on chromosome 1B. To identify the effects of the transgenes on the functional properties of grain, three lines showing segregation for transgene expression were used to isolate sibling T₂ plants which were null or positive for the transgene product. Analysis of these plants using a small-scale mixograph showed that expression of the additional subunits resulted in increased dough strength and stability, demonstrating that transformation can be used to modify the quality of durum wheat for bread and pasta making.     

Silencing of HMW glutenins in transgenic wheat expressing extra HMW subunits

Wheat HMW glutenin subunit genes 1Ax1 and 1Dx5 were introduced, and either expressed or overexpressed, into a commercial wheat cultivar that already expresses five subunits. Six independent transgenic events were obtained and characterized by SDS-PAGE and Southern analysis. The 1Dx5 gene was overexpressed in two events without changes in the other endosperm proteins. Overexpression of 1Dx5 increased the contribution of HMW glutenin subunits to total protein up to 22%. Two events express the 1Ax1 subunit transgene with associated silencing of the 1Ax2* endogenous subunit. In the SDS-PAGE one of them shows a new HMW glutenin band of an apparent M_r lower than that of the 1Dx5 subunit. Southern analysis of the four events confirmed transformation and suggest that the transgenes are present in a low copy number. Silencing of all the HMW glutenin subunits was observed in two different events of transgenic wheat expressing the 1Ax1 subunit transgene and overexpressing the Dx5 gene. Transgenes and expression patterns were stably transmitted to the progenies in all the events except one where in some of the segregating T₂ seeds the silencing of all HMW glutenin subunits was reverted associated with a drastic lost of transgenes from a high to a low copy number. The revertant T₂ seeds expressed the five endogenous subunits plus the 1Ax1 transgene. Received: 16 June 1999 / Accepted: 29 July 1999     

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.     

Analysis of dough functionality of flours from transgenic wheat

The rheological properties of flours from five different lines of transgenic wheat that either express or over-express subunits 1Ax1 and 1Dx5 were analyzed by mixograph assays and SDS sedimentation tests. In one case, the over-expression of subunit 1Dx5 resulted in a ca. 2-fold increase in mixing time, associated with a significant improvement in dough strength, and a lower resistance breakdown, suggesting an important increase in dough stability. However, the flour failed to develop properly without mixing with control flour because the rate of mixing was insufficient to develop the dough, i.e., the flour was overstrong. In two wheat transgenic lines, the expression of 1Ax1 and 1Dx5 transgenes, associated with silencing of all the endogenous high-molecular-weight glutenin subunits, resulted in flours with lower mixing time, peak resistance and sedimentation volumes, suggesting a lower gluten strength.