Stacking HMW-GS transgenes in bread wheat: Combining subunit 1Dy10 gives improved mixing properties and dough functionality

We have determined the technological properties of four lines containing combinations of three HMW-GS transgenes, encoding HMW-GS 1Ax1, 1Dx5 and 1Dy10. These lines were produced by conventional crossing of three single transgenic lines of the bread wheat cultivar Anza that contains the endogenous HMW-GS pairs 1Dx2 + 1Dy12 and 1Bx7* + 1By8 and is null for the Glu-A1 locus. Consequently, the total number of HMW-GS ranged from 4 in the control line Anza to 7 in line T618 which contains all three HMW-GS transgenes. The lines were studied over two years using a range of widely used grain and dough testing methods. All lines with transgenic subunits showed higher levels of glutenin proteins than the Anza control, and these differences were highly significant for lines T616, T617 and T618, containing, respectively, the transgenes encoding HMW-GS 1Ax1 and 1Dy10, 1Dx5 and 1Dy10 and 1Ax1, 1Dx5 and 1Dy10. These increases in glutenin levels are compensated by lower levels of gliadins present in transgenic lines. These changes affected the ratio of polymeric to monomeric gluten proteins (poly:mono), the ratio of HMW-GS to LMW-GS (HMW:LMW) and the contents of individual 1Ax, 1Bx, 1By, 1Dx and 1Dy subunits. Transgenic lines expressing subunit 1Dy10 together with x-type subunits (T616, T617 and T618) were superior to line T606, which had only increases in x-type subunits. In particular, the combination of transgenic subunits 1Dx5 and 1Dy10 (line T617) gave better dough rheological properties than the other combinations of transgenic subunits. For example, dough development time and stability were increased by 3.5-fold and 8.5-fold, respectively, while the mixing tolerance index (MTI) was decreased by 3.3-fold in line T617 with respect to the control line. Alveograph analyses showed that all four transgenic combinations had increased P values compared to the Anza control but subunit 1Dx5 greatly reduced the extensibility (L). These results show that stacking HMW-GS transgenes by conventional crossing is a valid strategy for the improvement of wheat quality, with different effects being related to the different HMW-GS combinations.     

57份陕西新育成小麦品种（系）HMW-GS组成及品质分析

为探究陕西关中地区小麦HMW-GS亚基与品质性状间的关系,采用SDS-PAGE法对57份陕西关中地区小麦品种（系）HMW-GS亚基组成及相关品质性状进行了分析。结果表明,供试品种（系）中共检测出7种HMW-GS亚基类型和8种HMW-GS亚基组合;Glu-A1位点上有3种亚基类型,分别为1、2^*和Null,以1亚基为主（78.95%）;Glu-B1位点上检测到7+8（61.40%）与7+9（38.60%）两个类型;Glu-D1位点上检测到5+10（70.18%）和2+12（29.82%）两个类型。3个HMW-GS基因位点编码亚基共组成8种亚基组合,品质得分6～10分,其中1/7+8/5+10组合品质得分10分,出现频率最高。就HMW-GS不同位点对品质性状效应进行分析发现,Glu-D1位点对b^*值、形成时间、稳定时间、弱化度和粉质质量指数的影响达到极显著水平（P＜0.01）;对面团流变学特性的影响,Glu-D1>Glu-B1。不同类型亚基对小麦品质的效应存在差异,7+8亚基对蛋白质含量、湿面筋含量和容重具有正效应,7+9和5+10亚基对形成时间和稳定时间的影响显著高于其他亚基（P＜0.05）;携带1/7+8/5+10亚基组合小麦的蛋白质、湿面筋含量和容重最高;携带1/7+9/5+10亚基组合具有较高面粉L^*值和面团流变学特性指标值。     

高分子量麦谷蛋白亚基组成及其与小麦品质性状的关系分析

为进一步明确小麦高分子量麦谷蛋白亚基(HMW-GS)与小麦品质性状的关系,以黄淮麦区的127份小麦品种(系)为材料,利用SDS-PAGE技术、近红外谷物分析仪、粉质仪和拉伸仪等对其进行HMW-GS鉴定和品质检测。结果表明,参试材料在 Glu-A1、 Glu-B1和 Glu-D1 3个位点上分别检测到2(x1、x-null)、4(x7+y8、x7+y9、x14+y15、x17+y18)、2(x5+y10、x2+y12)种不同的亚基类型,其中x1、x7+y9、x5+y10在各自位点上出现的频率均最高,分别为70.1%、42.5%和51.2%;共发现有14种HMW-GS组合类型,其中1Ax1/1Bx7+1By8/1Dx5+1Dy10和1Ax1/1Bx7+1By9/1Dx2+1Dy12出现的频率较高,分别为18.9%和17.3%。1Ax1、1Bx7+1By8、1Bx17+1By18、Dx5+1Dy10亚基对蛋白质、沉降值、稳定时间、最大抗延阻力和拉伸面积等品质性状有显著的正向效应,而1Bx14+1By15亚基对除蛋白质和湿面筋以外的其他品质性状有负向效应。携带1Ax1/1Bx7+1By8/1Dx5+1Dy10品种(系)的被测品质性状显著高于携带其他组合类型的品种(系),其次是携带1Ax1/1Bx17+1By18/1Dx5+1Dy10的品种(系),而携带1Ax-null/1Bx7+1By9/1Dx2+1Dy12和1Ax-null/1Bx14+1By15/1Dx5+1Dy10品种(系)的各个品质性状显著低于携带其他组合类型的品种(系)。该结果可为进一步提高优质亚基的育种利用率和我国小麦品质的遗传改良提供参考依据。     

Mixing properties and dough functionality of transgenic lines of a commercial wheat cultivar expressing the 1Ax1, 1Dx5 and 1Dy10 HMW glutenin subunit genes

In this work we report the effects of the HMW-GS 1Ax1, 1Dx5 and 1Dy10 on the breadmaking quality of the bread wheat cultivar Anza that contains the HMW-GS pairs 1Dx2 + 1Dy12 and 1Bx7* + 1By8, and is null for the Glu-A1 locus. This allows the characterization of individual subunits 1Dx5 and 1Dy10 in the absence of subunit 1Dx5, and the interactions between these subunits and subunits 1Dx2 and 1Dy12 to be determined. Three transgenic lines termed T580, T581 and T590, containing, respectively, the HMW-GS 1Ax1, 1Dx5 and 1Dy10 were characterized over 3 years using a range of widely-used grain and dough testing methods. The transgenic subunits 1Ax1, 1Dx5 and 1Dy10 accounted for 25.2%, 20.3% and 17.9%, respectively, of the total HMW-GS in the three transgenic lines. Although lines T581 and T590 expressed similar levels of subunits 1Dx5 and 1Dy10 they had different effects on other aspects of protein composition, including changes in the ratios of glutenin/gliadin, of HMW/LMW-GS, the 1Dx2/1Dy12, the x-type/y-type HMW-GS and the proportions of high molecular mass glutenin polymers. In contrast, lines transformed to express subunits 1Ax1 and 1Dx5 showed similar changes in protein composition, with higher protein contents and decreased ratios of glutenin/gliadin and 1Dx2/1Dy12. In addition, both transgenic lines showed similar increases in the ratio of x-type/y-type subunits compared to the control line. The transgenic lines were analysed using Farinograph, Mixograph and Alveograph. This confirmed that the expression of all three subunits resulted in increased dough strength (and hence breadmaking quality) of the cultivar Anza. A beneficial effect of subunit 1Dx5 has not been reported previously, transgenic wheat lines expressing this subunit giving overstrong dough unsuitable for breadmaking. However, the expression of subunit 1Dy10 had a greater effect on breadmaking quality than subunits 1Ax1 and 1Dx5. The Farinograph parameters such as dough stability and peak time were increased by 9.2-fold and 2.4-fold, respectively, in line T590 (expressing 1Dy10) with respect to the control line. Similarly, the Mixograph mixing time was increased by four-fold and the resistance breakdown decreased by two-fold in line T590 compared with the control line. The Alveograph W value was also increased by 2.7-fold in line T590 compared to the control line. These transgenic lines are of value for studying the contribution of specific HMW-GS to wheat flour functional properties.     

小麦高分子量谷蛋白亚基基因分子育种研究进展

为给小麦品质改良工作者提供通过分子生物技术优化HMW-GS组成方面的全面信息,综述了HMW-GS的基因克隆、分子标记以及基因工程改良三个方面近年来的国内外研究进展.迄今为止, 被克隆和测序的HMW-GS基因已有20多个,即1Ax1、1Ax2*、1Ax2*B、1Ay1、1Bx7、1Bx9、1Bx17、1Dx2、1Dx5、1Bx20、1By8、1By9、1Dy10、1Dy12、1AxNull、1Bx14、 1Bx23、1Dx2.2、1Dx2.1、1Dy10.1、1Dy12t等,还不断有新的基因被发现和克隆.克隆方法可概括为两种:一种是以HMW-GS克隆作为探针筛选cDNA或基因组DNA文库, 从而获得所需的靶基因序列, 然后再选择合适的载体进行克隆测序;另一种则是采用PCR技术.HMW-GS基因的分子标记方法主要有RFLP法、PCR法和SNP(单核苷酸多态性)法.目前已有研究者通过基因工程方法将部分外源HMW-GS基因导入小麦,有效地改善了受体品种的加工品质.     

Pasting properties of transgenic lines of a commercial bread wheat expressing combinations of HMW glutenin subunit genes

Seven transgenic lines of a commercial wheat (Triticum aestivum L.) cultivar expressing transgenic subunits 1Ax1, 1Dx5 and 1Dy10, alone or in combination have been developed. Pasting properties were determined in these transgenic lines using a Rapid Visco Analyser (RVA) in order to determine the possible impact of HMW-GS transgene expression on the starch properties. Expression of the HMW-GS transgenes increased the proportions of the corresponding 1Ax, 1Dx and 1Dy subunits affecting significantly the ratios of HMW-GS:LMW-GS and x-type:y-type HMW-GS. Starch granule size distribution varied significantly among all transgenic lines, with the Anza control and transgenic line T616 (expressing subunits 1Ax1 and 1Dy10) showing the highest and the lowest percentage of B granules, respectively. All transgenic lines increased the water-binding capacities (WBC) at 25 °C and 90 °C. Line T606 (expressing subunits 1Ax1 and 1Dx5) and line T590 (expressing subunit 1Dy10) showed the lowest and the highest values for peak viscosity, respectively. Notably, lines expressing only transgenic x-type subunits (T580, T581 and T606), with high ratios of x-type:y-type HMW-GS, had low peak viscosities, final viscosities and breakdown viscosities. Line T590 had the highest breakdown viscosity while lines T606 and T581 had the lowest.     

Molar fractions of high-molecular-weight glutenin subunits are stable when wheat is grown under various mineral nutrition and temperature regimens

Molar fractions of the high-molecular-weight glutenin subunits (HMW-GS) were determined for flour from bread wheat (Triticum aestivum L. cv Butte86) produced under 13 different combinations of temperature, water and mineral nutrition. Albumins, globulins and gliadins were removed from the flour by extraction with 0.3 M NaI in 7.5% 1-propanol. Total HMW-GS were recovered by extracting the remaining protein with 2% SDS and 25 mM DTT. Individual HMW-GS were then separated and quantified by RP-HPLC. Constant molar fractions for the five HMW-GS were maintained under all environmental conditions, despite large differences in duration of grain fill, total protein per grain, flour protein percentage, and total HMW-GS per grain. Similar molar fractions were found for five other US wheat varieties. The Bx7 subunit accumulated to the highest level at 30% of total HMW-GS. The Dx and Dy subunits were present in smaller but nearly equal proportions, 22% and 23%, respectively, and the Ax and By subunits were the least abundant, 14% and 12%, respectively. Although the amounts of HMW-GS per unit of flour are strongly affected by environment, the different subunits respond so similarly to external conditions that their final proportions appear to be determined mainly by genetic factors.     

Isolation and characterization of EMS-induced Dy10 and Ax1 high molecular weight glutenin subunit deficient mutant lines of elite hexaploid wheat (Triticum aestivum L.) cv. Summit

The mixing properties of the dough are critical in the production of bread and other food products derived from wheat. The high molecular weight glutenin subunits (HMW-GS) are major determinants of wheat dough processing qualities. The different alleles of the HMW-GS genes in hexaploid wheat vary in their effect on dough quality. To determine the contribution of the individual HMW-GS alleles, lines deficient in HMW-GS proteins were generated by chemical mutagenesis in the elite bread wheat Triticum aestivum cv. Summit. In this report we describe the identification and characterization of Dy10 and Ax1 deficient lines. Examination of the effect of Dy10 and Ax1 deficiency on dough rheological properties by mixography showed shorter mixing time to reach peak resistance, and weaker and less extensible doughs relative to the wild type control. This is the first time that the role of Dy10 in vivo has been examined apart from the Dx5 + Dy10 allelic pair combination.     

Variant high-molecular-weight glutenin subunits arising from biolistic transformation of wheat

Genetic transformation via the biolistic method has been used to introduce genes encoding natural and novel high-molecular-weight glutenin subunits (HMW-GS) into wheat. The appearance of new seed proteins of sizes not predicted by the transgene coding sequences was noted in some experiments. In this report, the identities of thirteen of these novel proteins were determined by tandem mass spectrometry (MS/MS). Seven different proteins larger than and two proteins smaller than the native protein were shown to contain peptides from 1Dx5. A novel protein found in some progeny of crosses between a transgenic plant and Great Plains winter wheats was larger than but contained several peptides from 1Dy10. In one line, a protein larger than and a protein smaller than HMW-GS each contained peptides from the N- and C-terminus of 1Dx5 and from the repeat region of 1Dy10. In a sixth transgenic line, the native Bx7 gene was apparently replaced by a gene that encodes a larger version of 1Bx7. The variant proteins accumulate in the polymeric protein fraction, indicating that they can form inter-molecular disulfide bonds. These results show that novel proteins found in some transformants are encoded by altered versions of either the transforming or endogenous HMW-GS genes.     

Technological quality of field grown transgenic lines of commercial wheat cultivars expressing the 1Ax1 HMW glutenin subunit gene

Ten transgenic lines were studied which expressed a transgene encoding HMW subunit 1Ax1 in three elite spring wheat cultivars: Imp, Canon and Cadenza. These lines contained one to five copies of the transgene and the 1Ax1 subunit was expressed as 1–20% of the total glutenin protein. These lines were grown in field trials in a continental, arid climate (Martonvásár, Hungary) over two years (2004, 2005). The expression of the transgenes and their effects on the grain properties were stably inherited over the two years. Significant differences in yield were observed between three of the transgenic lines and the original genotypes, but no differences were found in their adaptiveness. Clear differences were found in the technological and rheological properties of four lines, with all the parameters characterising dough strength and extensibility (GI, W, G, Re, Ext, A) changing significantly. These differences were associated with increases in the ratio of HMW/LMW subunits and decreases in the ratios of 1Dx/1Dy and 1Bx/1By subunits. Two transgenic lines of cv Imp had high over-expression of the 1Ax1 subunit which in one line resulted in an overstrong type of dough, similar to that described previously for lines over-expressing HMW subunit 1Dx5. Transformation of cvs. Canon and Cadenza resulted in two lines with increased dough stability due to the significantly improved gluten quality. It is concluded that significant changes in the structure of the glutenin polymers caused by the altered ratio of x-type to y-type HMW subunits led to the changes in flour functional properties.     

Incorporation of high-molecular-weight glutenin subunits into doughs using 2 gram mixograph and extensigraphs

To study the contributions of high-molecular-weight glutenin subunits (HMW-GS) to the gluten macropolymer and dough properties, wheat HMW-GS (x- and y-types) are synthesized in a bacterial expression system. These subunits are then purified and used to supplement dough mixing and extensigraph experiments through dough partial reduction and reoxidation to allow these exogenously added HMW-GS to incorporate into gluten polymers. Detailed results are given for seven mixing and two extension parameters. HMW-GS synthesized in bacteria behaved similarly under these conditions to the same HMW-GS extracted from wheat flour. These experiments initially focused on the HMW-GS of the D-genome of hexaploid wheat encoded at the Glu-D1 locus; e.g. the Dx2, Dx5, Dy10, and Dy12 subunits. Experiments used five different flours and results are shown to be consistent when normalized to results from Dx5. The incorporation of Dx-type subunits into the gluten disulfide bonded network has greater effects on dough parameters than incorporation of Dy-type subunits. When Glu-D1 x- and y-type subunits are incorporated together, there are synergistic effects greater than those with either subunit type alone. This synergistic effect was greatest with approximately equal amounts of Dx- and Dy-type subunits - implying a 1:1 stoichiometric relationship.     

Transgenic wheats with elevated levels of Dx5 and/or Dy10 high-molecular-weight glutenin subunits yield doughs with increased mixing strength and tolerance

To test the effects of independently increasing the in vivo levels of high-molecular-weight glutenin subunits (HMW-GS) Dx5 and Dy10 on wheat flour properties, we increased the copy numbers of their corresponding genes by genetic transformation. Thirteen transformants with increases in one or both subunits were chosen for biochemical and functional characterization by solvent fractionation, RP-HPLC, SDS-sedimentation, and micro-mixing. Increases in Dx5 and Dy10 contents ranged from 1.4- to 3.5-fold and 1.2- to 5.4-fold, respectively, and generally resulted in increased polymeric protein, increased mixing times and tolerances, and lower peak resistances. Increases in Dx5 content had larger effects on most parameters than comparable increases in Dy10. Flours with more than 2.6-times the native levels of Dx5 could not be mixed under standard 2-g mixograph conditions, while flours with 5.4 times the native levels of Dy10 could be mixed if sufficient time was allowed. Increases in Dx5 and Dy10 had additive effects on mixing behavior. These experiments demonstrate that dough mixing strength and tolerance can be increased by raising the levels of native HMW-GS Dx5 or Dy10, but that the effects of the two subunits are quantitatively and qualitatively different.     

转基因小麦1Dx5基因沉默的遗传表达和品质效应分析

为了明确通过RNA干扰技术（RNAi）获得的转基因小麦品系“Glu1Dx5RNAi”中1Dx5基因的遗传规律,以“Glu1Dx5RNAi”为供体亲本,以弱筋品种扬麦18和扬麦13为轮回亲本进行回交, 采用半籽粒SDSPAGE法检测亲本、F₁、F₂和BC₁F₁籽粒的高分子量谷蛋白亚基（HMWGS）组成。结果表明,1Dx5基因的沉默效应在杂交后代中表现为显性遗传,在F₂和BC₁F₁世代1Dx5亚基不表达的种子数与1Dx5亚基表达的种子数的比例分别符合13∶3和3∶1的分离比例,说明转小RNA基因导致1Dx5基因沉默是单基因显性遗传。对转基因小麦“Glu1Dx5RNAi”及其受体品种Bobwhite进行品质测试表明,Glu1Dx5RNAi蛋白质含量（13.28%）比转化受体Bobwhite（12.83%）高0.45个百分点,硬度指数、微量SDS沉降值比受体品种分别降低3.13和3.7 mL。1Dx5基因沉默对弱筋小麦育种可能会有一定的利用价值。     

甘肃小麦品种（系）面筋强度和糯蛋白基因分布规律研究

高分子量麦谷蛋白、 1B/1R易位系和糯蛋白基因是影响小麦面包、面条和馒头加工品质的重要因素。为明确甘肃省小麦品种（系）品质状况,用 Dx 5、 By8、 By9、Bx7^OE、 1B/1R、Wx等基因的特异性分子标记鉴定其对应基因在甘肃4个麦区298份小麦材料中的存在状况。结果表明,甘肃省小麦品种（系）中被检测基因分布频率在不同生态区域存在一定差异,从西部到东南部, Dx 5、 By8、Bx7^OE、 Wx-B 1b基因的检出频率逐渐降低,而 By9基因和1B/1R易位系检出频率逐渐升高;春小麦中 Dx 5（46.4%）、 By8（26.1%）、Bx7^OE（1.4%）、 Wx-B 1b（14.4%）基因的检出频率明显高于冬小麦中 Dx 5（26.3%）、 By8（25.0%）、Bx7^OE（0）、 Wx-B 1b（7.5%）基因的检出频率;但春小麦 By9（34.8%）基因、 1B/1R易位系（27.5%）检出频率低于冬小麦 By9（41.3%）、 1B/1R（38.8%）的被检出频率。面筋强度和糯蛋白基因的分布频率表明甘肃省小麦品种（系）用于加工面包、面条和馒头的品质状况较差,且从西部到东南部品质逐渐变劣,春小麦品质优于冬小麦。甘肃省大部分小麦品种（系）仅适宜于加工普通馒头和面条。     

转基因小麦与普通小麦杂交后代中稳定株系的筛选

为了给小麦品质改良提供优异的种质,以小麦转1Dx5和1Ax1基因品系为父本,以长江中下游冬麦区小麦栽培品种为母本配制杂交组合,获得BC1F1、BC1F2、BC1F3和BC1F4代.在各杂交后代中,采用系谱选择法结合SDS-PAGE检测技术,鉴定各系的HMW-GS组成,获得了多个外源1Dx5或1Ax1基因稳定超表达的小麦新型纯系.     

甘肃冬小麦品种(系)面粉品质性状相关基因分析

为了解甘肃省近20年育成的106份冬小麦品种(系)中加工品质性状相关基因分布情况,用22个分子标记对供试材料的HMW-GS、LMW-GS、面粉色泽及籽粒硬度等品质性状相关基因进行了分析。结果发现,供试品种(系)的HMW-GS相关基因中,在Glu-A1位点检测到34份品种(系)含有AxNull,频率为32.08%;在Glu-B1位点检测到Bx7+By8和Bx14+By15共2种基因组合,分别占17.92%和25.47%;在Glu-D1位点检测到11份品种(系)含有Dx5+Dy10,占10.38%。对LMW-GS鉴定结果显示,29份品种(系)含Glu-A3d基因,分布频率为27.36%。HMW-GS和LMW-GS亚基组合中,含有4个、3个和2个位点优质亚基基因组合的品种(系)分别占0.94%、8.49%和3.77%。对面粉色泽相关基因Ppo-A1、Ppo-D1、Psy-A1、Lox-B1和TaPod-A1位点的检测发现,优异等位变异占比分别为39.62%、50.94%、31.13%、30.19%和38.68%。对籽粒硬度相关基因检测发现,在Pina、Pinb和Pinb-2等位变异位点的检测...     

Effects of specific domains of high-molecular-weight glutenin subunits on dough properties by an in vitro assay

An in vitro system for incorporating bacterially produced high-molecular-weight glutenin subunits (HMW-GS) into doughs was used to study the effects of specific domains of the HMW-GS. Synergistic effects of incorporating into doughs both the Dx5 and Dy10 subunits are localized to the N-terminal domains. All single and pair-wise combinations of original subunits and hybrid subunits with their N-terminal domains exchanged between Dx5 and Dy10 finds three classes of respondents: the greatest response is when the N-termini of both Dx5 and Dy10 are present, followed by presence of the Dx5 N-terminus alone, and the least response by the presence of the Dy10 N-terminus alone. In addition, studies of Dx5 variants possessing repetitive domains of different length and composition find evidence that the length of the HMW-GS repetitive domain is important for dough properties and that the exact composition of the repeat domain has a detectible, though lesser contribution. Finally, in this experimental system, the Glu-D1 x- and y-subunits function in the mixing experiments as if they were a fused dimer, although the exact molecular basis of the effect is not known.     

Milling and baking properties of field grown wheat expressing HMW subunit transgenes

Milling and baking tests were carried out on three transgenic wheat lines and their parental varieties grown in the field at two UK sites. The transgenic and control lines were essentially similar in their milling properties but the subunit 1Ax1 and 1Dx5 transgenes had different effects on breadmaking. The subunit 1Dx5 transgene resulted in a low loaf volume and poor crumb structure when expressed in lines with two or five endogenous HMW subunits, and this was accompanied by a greatly increased elastic modulus of the gel protein fraction. In contrast, the 1Ax1 transgene resulted in improved breadmaking quality and a more modest increase in the gel protein elastic modulus when expressed in the two subunit background. Blending of flour from a line expressing the 1Dx5 transgene with flour from a normal breadmaking wheat variety resulted in decreased breadmaking quality, even at a ratio of 1:9. The difference in the results obtained with the 1Ax1 and 1Dx5 transgenes may relate to the presence of an additional cysteine residue in the protein encoded by the latter, which promotes a more highly cross-linked glutenin network.     

氮肥运筹对小麦HMW GS形成与积累的影响

为给小麦优质栽培提供理论依据,以多穗型品种031085和大穗型品种山农91为试验材料,利用SDSPAGE电泳等分析技术,研究了氮肥运筹对不同类型小麦品种高分子量谷蛋白亚基（HMWGS）形成的影响。结果表明,小麦HMWGS出现的时间多在花后10~15 d,且多穗品种相对早于大穗品种;氮肥运筹对HMWGS出现时间影响不大;亚基组合1Bx7+1By9出现时间早于亚基1Dx2+1Dy12;不同施氮处理下,开花后各小麦品种HMWGS亚基的积累过程均呈三次曲线增长;各亚基积累量所占百分含量随时间变化较为平稳,受施氮影响较小,y型亚基百分含量高于x型亚基。     

A transgenic durum wheat line that is free of marker genes and expresses 1Dy10

As currently practiced, genetic engineering of monocots requires the use of selective agents, such as herbicides and antibiotics, and marker genes for resistance to favor the multiplication of the initially transformed cells. In the present paper we have used “minimal gene cassettes” and positive selection to generate transgenic durum wheat lines free of herbicide and antibiotic resistance marker genes. Two biolistic transformation experiments were carried out using three “minimal gene cassettes” consisting of linear DNA fragments each excised from the source plasmids. The targeted trait genes were two bread wheat sequences encoding the Dx5 and Dy10 high-molecular-weight (HMW) glutenin subunits, which have been associated with superior bread-making quality and which are absent from durum wheats. The positive selectable marker was the Escherichia coli phosphomannose isomerase (pmi) gene, whose product catalyzes the reversible interconversion of mannose-6-phosphate and fructose-6-phosphate, allowing plant cells to utilize mannose as a carbon source. PCR assays of genomic DNA from regenerated plants identified 15 T₀ plants that contained the pmi marker gene for an overall transformation efficiency of 1.5%, which is similar to biolistic transformation efficiencies of durum wheat with intact circular plasmids. Line TC-52, which initially contained pmi, non-expressed 1Dx5, and expressed 1Dy10 HMW glutenin subunit transgenes, was further investigated. PCR was used to follow inheritance of the pmi marker gene and 1Dx5 from the T₁ to T₃ generations. Transgene expression was monitored by the chlorophenol-red assay for pmi and SDS-PAGE of seed proteins for 1Dy10. From these analyses, we observed that the 1Dy10, 1Dx5 and pmi transgenes were not linked, allowing us in the T₃ generation to identify 1Dy10 transgenic segregants that contained no marker or silent 1Dx5 transgenes. Homozygotes containing and expressing only the 1Dy10 transgene were identified in the T₄ generation. These experiments show that it is possible to combine biolistic transformation by minimal gene cassettes with genetic segregation to make marker-free transgenic wheat plants with new traits.