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.     

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.     

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.     

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.     

Introgression of transgenes into a commercial cultivar confirms differential effects of HMW subunits 1Ax1 and 1Dx5 on gluten properties

Transgenes encoding the HMW subunits 1Ax1 and 1Dx5 have been transferred from “model” wheat lines into the commercial French bread wheat cultivar Soissons, using three backcrosses. Five pairs of BC3 expressing and null lines were isolated from each cross and multiplied to provide grain for functionality studies. Analysis of white flour samples confirmed the expression of the transgenes. SE-HPLC and Reomixer studies showed that the two transgenes had differential effects on dough functional properties. Thus, subunit 1Dx5 resulted in detrimental effects on dough development which were associated with decreased extractability of large glutenin polymers. In contrast, lines expressing subunit 1Ax1 contained increased proportions of extractable large glutenin polymers with three lines showing higher torque at similar mixing times (i.e. increased dough strength). This confirms the results obtained with the model wheat lines and shows that the 1Ax1 transgene can be used to increase dough strength in commercial cultivars.     

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.     

转基因小麦外源基因在主栽小麦种质中的遗传规律

为了研究转基因小麦中外源品质基因1D x 5在我国主栽小麦种质中的遗传规律,以转基因小麦B 72-8-11b为父本,主栽品种川89-107和鄂麦18为母本进行杂交,采用SDS-PAGE技术检测并分析各组合亲本、F1、F2、BC1F2、BC2F1、BC2F2代的HMW-GS组成。结果表明,外源基因有效地整合到主栽小麦的基因组中,并能够遵循孟德尔遗传模式稳定地遗传给下一代。     

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.     

Genetic Transformation of a High Molecular Weight Glutenin (Glu-1Dx5) to Rice cv. Fatmawati

Abstract

In order to improve rice dough functionality, we co-transformed the Glu-1Dx5 gene encoding a high molecular weight (HMW) glutenin subunit Dx5 from bread wheat, Triticum aestivum L. and either bar gene conferring resistance to herbicide bialaphos or hpt gene conferring resistance to hygromycin B to rice callus cells of cv. Fatmawati. We molecularly characterized 9 plants regenerated from bialaphos-containing medium and 63 plants from hygromycin-containing medium. The Glu-1Dx5 gene was detected by PCR analysis in 15 transgenic T₀ plants. Further analysis of T₁ and T₂ plants revealed that some transgenic plants carried the Glu-1Dx5 gene. Analysis of the endosperm extracts of T₂ plants by SDS-PAGE revealed the existence of a protein similar in size to the wheat Glu-1Dx5 gene product, suggesting successful expression of the transgene. These plants will be incorporated into breeding program for further assessment of their benefits.     

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.     

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.     

Overexpression of a Gluten Protein in Transgenic Wheat Results in Greatly Increased Dough Strength

The transgenic wheat line B73-6-1 contains additional genes encoding a gluten protein called HMW subunit 1Dx5, resulting in a four-fold increase in the proportion of this component in the seed proteins and corresponding increases in the proportions of total HMW subunits and total glutenins. This is associated with a dramatic increase in dough strength, as measured using a small scale Mixograph, with dough produced from B73-6-1 being too strong to be used in conventional breadmaking.     

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。     

TaNAC14转基因小麦外源目标基因拷贝数及遗传稳定性分析

外源基因拷贝数是影响转基因植物遗传稳定性及其自身表达水平的重要因素。为了探析TaNAC14基因在小麦生长发育中的生物学功能,本研究通过农杆菌介导的遗传转化法获得了14个T0代TaNAC14转基因小麦植株,采用BASTA溶液涂抹和目标序列PCR检测相结合的方法,从14个转基因小麦植株中鉴定出9个阳性植株。通过TaqMan实时定量PCR方法,以小麦内源单拷贝基因Pinb为内参基因,对9个T0代转基因小麦阳性植株中外源目标基因拷贝数进行检测,结果表明,T0代转基因小麦再生植株中有5株为单拷贝,4株为双拷贝,其中单拷贝插入整合的比率接近55.6%。选取单拷贝转基因植株收获的种子进行种植,根据BASTA溶液涂抹鉴定对T0:1代小麦株系遗传情况进行分析,结果表明目标基因TaNAC14是可遗传的。此外,还对T1代转基因小麦目标基因表达水平进行测定,结果表明,与野生型JW1相比,各转基因小麦植株目的基因TaNAC14表达量均极显著增加。该研究结果为后续TaNAC14基因的功能研究奠定了基础。     

高温胁迫下黄嘌呤脱氢酶基因超表达对水稻幼苗的保护作用

【目的】黄嘌呤脱氢酶(xanthine dehydrogenase, XDH)是嘌呤代谢的关键酶。通过分析高温胁迫对OsXDH超表达转基因水稻株系幼苗叶片生理指标的影响,探究XDH缓解水稻高温胁迫的生理机制。【方法】以OsXDH超表达转基因株系(xdh1和xdh5)及受体品种日本晴(WT)为材料,研究了高温胁迫对水稻幼苗叶片叶绿素含量、相对含水量、可溶性蛋白含量、活性氧代谢、抗氧化酶活性、XDH活性及嘌呤代谢产物尿囊素(allantoin)和尿囊酸(allantoate)含量等生理指标的影响。【结果】高温胁迫处理前,超表达株系的叶绿素含量、相对含水量、可溶性蛋白含量、活性氧代谢及抗氧化酶活性等生理指标均与野生型无显著差异;高温胁迫5 d,超表达株系的叶绿素、相对含水量、可溶性蛋白含量及抗氧化酶活性均高于野生型,而过氧化氢(H₂O₂)及丙二醛(MDA)的含量显著低于野生型;适温恢复生长5 d,野生型和超表达株系的叶绿素含量、相对含水量及可溶性蛋白含量均有所提高,超表达株系均高于野生型,H₂O₂ 和MDA的含量降低,超表达株系均低于野生型;受高温诱导超表达株系与野生型中XDH酶活性及尿囊素和尿囊酸的含量均提高,且在整个处理过程中超表达转基因株系均高于野生型。【结论】XDH通过调控酰脲类物质的合成,补偿自身的抗氧化能力并增强抗氧化酶系统的活性,从而有效提高水稻幼苗对高温胁迫的耐受能力。     

盐生草HgNHX1基因在大麦株系中的功能验证

为了探讨盐和干旱环境胁迫对转盐生草液泡膜型Na⁺/H⁺逆向转运蛋白基因HgNHX1大麦幼苗生长及抗逆性生理指标的影响,以转HgNHX1基因大麦及野生型株系为材料,分析盐(150 mmol· L^-1 NaCl)和自然干旱环境下转基因大麦幼苗的生长特性及生理指标的变化。结果表明,在盐胁迫条件下,随着胁迫时间的延长,转基因株系中HgNHX1 基因相对表达量逐渐上升;干旱胁迫条件下,转基因株系中HgNHX1 基因相对表达量呈先上升后下降的趋势,在处理3 d时达到最高。且在胁迫处理的三个不同时间点,转基因株系的相对含水量和游离脯胺酸含量均高于野生型株系,而相对电导率和丙二醛含量均低于野生型株系。这些结果说明,转基因株系中HgNHX1基因能够应答盐和干旱胁迫,具有提高大麦耐盐、抗旱性的功能。     

叶肉细胞特异表达焦磷酸化酶基因OsIP1在不同源库类型水稻品种中的效应

 将水稻自身焦磷酸化酶基因OsIP1置于叶肉细胞特异表达启动子下,利用农杆菌介导法将嵌合基因cyFBPase:OsIP1分别导入“源限制型” 水稻品种中超123和“库限制型” 品种农垦57。 根据荧光定量PCR结果和农艺性状,筛选高表达且农艺性状没有明显改变的T2转基因纯合株系,用以检测和分析在水稻叶肉细胞中特异性过表达水稻焦磷酸化酶基因的效应。初步的研究结果显示：1）在营养生长期,转基因株系的最高茎蘖数和穗数比各受体亲本均有不同程度的提高,尤其是在分蘖性较强的品种上;2）在籽粒灌浆结实期,叶片、叶鞘中可溶性总糖、蔗糖和淀粉含量在整个灌浆期的变化趋势与对照相似,且多数转基因植株叶片和叶鞘中的蔗糖含量（除了灌浆高峰期时的叶鞘）均显著高于对照;3）转基因株系单株干物质量较野生型对照有显著提高。单株产量呈现不同程度的增加,其中“源限制型”品种中超123转基因株系单株产量较对照增幅达显著水平。     

Technological quality of transgenic wheat expressing an increased amount of a HMW glutenin subunit

The transgenic line B73-6-1 (developed and characterized by UK scientists) contains 10–15 extra copies of a native HMW glutenin gene (1Dx5) with an approximately four-fold increase in the amount of the encoded protein. The technological and rheological properties of this line and the non-transformed control line were studied in a continental type, arid climatic environment (Martonvásár, Hungary, 2000–2002). The results show that in the three years of the field experiment, the transgene and associated functional properties were stably inherited over several generations. No difference was found in yield between the transgenic line and the original genotype, but clear genotypic differences were found in kernel hardness and kernel size. The transgenic line had an increased kernel hardness and a smaller kernel size. The transgenic line B73-6-1 tended to have a higher protein content than the control L88-6, but this difference was not statistically significant. In contrast there was a significant, 400%, increase in the amount of 1Dx5 HMW glutenin subunits, Dx/Dy-, HMW/LMW- and glutenin/gliadin ratio. At the same time there was a decrease in wet gluten content and SDS sedimentation. Thus, significant changes in the structure of the protein matrix caused by the altered x to y ratio of HMW-GS and as a result of this alteration, flour functional properties changed. Parameters that characterize the stability and strength of the dough confirmed that B73-6-1 was stronger, but the extensibility was reduced. This flour would be suitable for blending with flour of lower grade.     

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。