高分子量谷蛋白单亚基缺失对软质小麦宁麦9号加工品质的影响
张平平, 马鸿翔*, 姚金保, 周淼平, 张鹏
江苏省农业科学院 / 江苏省农业生物学重点实验室 / 江苏省现代作物生产协同创新中心, 江苏南京 210014
* 通讯作者(Corresponding author): 马鸿翔, E-mail: hongxiangma@163.com, Tel: 025-84390300

第一作者联系方式: 张平平, E-mail: pp_zh@126.com, Tel: 025-84390257

摘要

基因敲除是研究高分子量谷蛋白(HMW-GS)亚基功能的重要方法。本研究以软质小麦宁麦9号野生型及其单亚基缺失系为材料, 探讨了HMW-GS缺失对籽粒品质性状、谷蛋白组分含量和加工品质的影响。在29份参试品系中, 野生型有3个穗系, Glu-A1x、Glu-B1x、Glu-B1y、Glu-D1x和Glu-D1y缺失型分别有5、7、5、5和4份。野生型与缺失型, 以及缺失型之间的蛋白质含量、湿面筋含量、籽粒硬度和溶剂保持力无显著差异。缺失型的谷蛋白/醇溶蛋白、高分量谷蛋白/低分子量谷蛋白含量比值低于野生型, 其中Glu-B1x和Glu-D1x缺失型的比值显著低于野生型( P<0.05)。缺失型的揉面仪峰值时间和8 min带宽变异范围分别为1.38~1.64 min和3.38%~3.98%, 显著低于野生型的2.00 min和4.57% ( P<0.05), 以Glu-B1x和Glu-D1x缺失型表现最低。与野生型相比, 缺失型的糖酥饼干直径均有增加, 其中Glu-B1x、Glu-B1y和Glu-D1y缺失型饼干直径的增加达显著水平( P<0.05), 而缺失型之间的差异不显著。在宁麦9号背景下, 高分子量麦谷蛋白单亚基缺失弱化了面筋强度, 改善了糖酥饼干加工品质, 亚基敲除可能是进一步提高软质小麦加工品质的有效途径。

关键词: HMW-GS缺失; 宁麦9号; 加工品质
Effect of HMW-GS Deletion on Processing Quality of Soft Wheat Ningmai 9
ZHANG Ping-Ping, MA Hong-Xiang*, YAO Jin-Bao, ZHOU Miao-Ping, ZHANG Peng
Jiangsu Academy of Agricultural Sciences / Jiangsu Provincial Key Laboratory for Agrobiology / Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing 210014, China
Abstract

Gene knockout is an effective approach to investigate gene function of high-molecular-weight glutenin subunit (HMW-GS). In this study, we developed a set of single HMW-GS deletion lines of Ningmai 9 (soft wheat) to understand the effects of HMW-GS deletion on kernel quality, quantity of gluten protein fractions and processing quality. Among the 29 lines tested, three lines were wild type and the remaining were knockout mutants including Glu-A1x, Glu-B1x, Glu-B1y, Glu-D1x, and Glu-D1y deletion types of five, seven, five, five, and four lines, respectively. Compared with the wild type, the HMW-GS deletion lines had no significant difference in flour protein content, wet gluten content, kernel hardness, and solvent retention capacity. All the single HMW-GS deletion types decreased the ratios of glutenin-to-gliadin and HMW-GS-to-LMW-GS in quantity, and the decreases in Glu-B1x and Glu-D1x deletion types were significant at P < 0.05. In the wild type, mixograph peak time was 2.00 min and TIMEX width was 4.57%; whereas, those in the HMW-GS deletion lines were significantly lower ( P < 0.05), varying from 1.38 min to 1.64 min and from 3.38% to 3.98%, respectively. Particularly, the Glu-B1x and Glu-D1x deletion lines showed the lowest mixograph peak time and TIMEX width. Although similar sugar snap cookie diameter was observed among the five deletion types, Glu-B1x, Glu-B1y, and Glu-D1y deletion types showed significantly higher cookie diameter than the wild type ( P < 0.05). In this study, single deletion of HMW-GS weakened gluten strength and improved sugar snap cookie processing quality, indicating that HMW-GS knockout can be used to improve soft wheat quality.

Keyword: HMW-GS deletion; Ningmai 9; Processing quality

小麦贮藏蛋白由醇溶蛋白和麦谷蛋白组成, 分别约占贮藏蛋白总量的50%[1], 是小麦加工品质的基础。醇溶蛋白为单体蛋白, 对面筋质量的贡献取决于总量及与谷蛋白含量的比[2, 3]。麦谷蛋白亚基具有半胱氨酸残基, 可以通过二硫键形成复杂的网络结构, 与小麦面筋质量密切相关[4, 5, 6]。麦谷蛋白包括高分子量谷蛋白亚基(HMW-GS)和低分子量谷蛋白亚基(LMW-GS), 分别约为谷蛋白总量的20%和80%, HMW-GS负责构建面筋网络的骨架而尤为重要[1, 7]。HMW-GS编码基因定位于第一同源群1A、1B和1D染色体长臂近着丝点处的Glu-A1Glu-B1Glu-D1位点(统称Glu-1位点), 每个Glu-1位点编码x型和y型两种亚基, 两个编码基因紧密连锁, 共6个HMW-GS基因, 通常表现为共分离[1, 8, 9]。普通小麦中, 由于Glu-A1y亚基沉默, 多数品种有5个HMW-GS[10]。发掘和聚合优质HMW-GS一直是小麦品质育种最有效的途径[11, 12, 13]

自从Payne等首次发现HWM-GS与面筋强度的关系以来, 谷蛋白位点、位点等位变异及互作对加工品质的影响是主要研究内容[14, 15, 16, 17], 通过反向遗传学方法研究HMW-GS缺失或敲除对面筋品质影响的报道亦较多。如Lawrence等[18]利用一套在3个Glu-1位点分别缺失的品系, 发现不同位点缺失均显著降低了面筋强度和面包加工品质; 张莉丽等[19]利用近等基因系研究了Glu-A1位点1亚基表达与否对面筋品质的影响; 武茹等[13]发现Glu-A1Glu-D1双缺失位点的杂交转育后代的面粉沉降值显著降低; Yang等[20]试验表明, 小偃81中Glu-1位点缺失显著降低了面粉的谷蛋白大聚体含量及其面团弹性, 且位点效应为Glu-D1 > Glu-B1 > Glu-A1; Jondiko等[21]和Zhang等[22]研究了HMW-GS沉默表达与墨西哥卷饼和馒头加工品质的关系, 提出操控Glu-1位点可能是特定食品加工品质提高的有效途径; Li等[23]不仅验证了谷蛋白亚基缺失显著降低面筋强度和面包加工品质, 而且发现不同亚基对面筋弹性和延展性的贡献不同。可见, HMW-GS缺失可降低面筋强度, 有可能利于中弱筋及特殊面制品加工小麦品种的培育, 但亚基缺失对软质小麦加工品质的影响仍未见报道。不同研究材料由于具有的亚基组合不同, 其所揭示的相同类型的亚基功能或对面筋质量的贡献也可能有所不同。

宁麦9号是江苏省农业科学院育成的软红冬小麦, 该品种正常大田栽培条件下籽粒蛋白质含量12.5%左右, 籽粒质地软质到中硬, 面筋强度较弱[24, 25], 野生型的高低分子麦谷蛋白亚基5个编码基因均正常表达[26]。本研究拟在前期研究的基础上[27], 通过多份Glu-1位点单亚基缺失突变的品系比较试验, 初步探讨在遗传背景相似条件下高分子量谷蛋白单亚基对软质小麦面筋质量和加工品质的影响, 为通过操纵HMW-GS改良小麦加工品质提供理论依据。

1 材料与方法
1.1 试验材料及其田间种植

试验材料为宁麦9号野生型的3个穗系及26份高分子量谷蛋白单亚基缺失系(表1)。该套缺失系(M6)由本实验室利用甲基磺酸乙酯(EMS)处理宁麦9号, 并通过大规模贮藏蛋白和农艺性状筛选鉴定而获得[27]。2013— 2014年度种植于江苏省农业科学院六合试验基地(南京市六合区竹镇镇)。田间试验采用随机区组设计, 2行区, 行长1.5 m, 3次重复。播种方式为条播, 采用1.3倍播量, 齐苗后间苗固定基本苗, 基本苗2.1× 106株hm-2。基施浓度为45%的三元复合肥(含N、P2O5和K2O各15%) 225 kg hm-2, 全生育期施纯氮总量为225 kg hm-2, 按基肥、分蘖肥、拔节肥5︰2︰3比例分配。试验地0~20 cm土壤中有机质含量为13.2 g kg-1, 速效氮、速效磷、速效钾含量分别为87.1、48.3和85.2 mg kg-1。其他管理措施同大田生产。

表1 参试品系的高低分子量麦谷蛋白亚基组成 Table 1 High-molecular weight glutenin subunits (HMW-GS) composition of wheat lines
1.2 品质测试

使用单籽粒谷物分析仪(Perten Instruments North America Inc., Springfield, IL, USA), 按AACC方法[28]测定籽粒硬度(AACC 55-31.01)。每样品取250 g籽粒, 使用Brabender Quadrumat Junior Laboratory按照标准程序制粉, 出粉率约60%。按AACC方法[28]测定蛋白质含量(AACC 39-11.01)、4种溶剂保持力(AACC 56-11.02)、湿面筋含量(AACC 38-12.02)、面团的揉面特性(AACC 54-40.02)及饼干直径(AACC 10-52.02)。其中, 蛋白质含量用Perten DA7000近红外分析仪(Perten Instruments North America Inc., Springfield, IL, USA)测定, 4种溶剂保持力分别为水溶剂保持力(water solvent retention capacity, WSRC)、碳酸钠溶剂保持力(sodium carbonate solvent retention capacity, SCSRC)、乳酸溶剂保持力(lactic acid solvent retention capacity, LASRC)和蔗糖溶剂保持力(sucrose solvent retention capacity, SUSRC), 揉面特性用10 g微量揉面仪(National Manufacturing, Lincoln, NE, USA)测定。

1.3 贮藏蛋白亚基分离、鉴定和量化

参考前人描述的SDS-PAGE方法[17, 29, 30]分离和鉴定高低分子量麦谷蛋白亚基, 其中Glu-B3鉴定依据其与醇溶蛋白的连锁关系。参考Zhang等[22]的反相高效液相色谱法(reversed-phase high-performance liquid chromatography, RP-HPLC)和凝胶色谱法(size-exclusion high-performance liquid chromatography, SE-HPLC)分别测定和计算高分子量谷蛋白/低分子量谷蛋白含量比值(HMW/LMW)和谷蛋白/醇溶蛋白含量比值(Glu/Gli)。参考张平平等[31]的RP-HPLC法分离醇溶蛋白。

1.4 统计分析

用Statistical Analysis System 9.0统计分析软件处理数据。以缺失类型为变异因素, 同一缺失类型的不同品系作为重复观察值进行相关性状的方差分析, 并在野生型以及缺失类型间进行显著性比较。

2 结果与分析
2.1 参试品系的贮藏蛋白组成分析

宁麦9号野生型高低分子量谷蛋白亚基Glu-A1、Glu-B1、Glu-D1、Glu-A3、Glu-B3和Glu-D3组成分别为1、7+8、2+12、c、f和c。3个Glu-1位点的1、7、8、2和12的5种单亚基缺失系分别有5份、5份、5份、7份和4份, 这些缺失系的LMW-GS组成与野生型相同(表1图1)。对参试品系的醇溶蛋白进行RP-HPLC分离, 野生型以及各缺失品系也显示出相同的亚基分离图谱(图2)。

图1 亚基缺失品系的麦谷蛋白(A)和醇溶蛋白亚基(B) SDS-PAGE分离图谱
1: 野生型; 2: Glu-A1x(1)缺失; 3: Glu-D1x(2)缺失; 4: Glu-B1x(7)缺失; 5: Glu-B1y(8)缺失; 6: Glu-D1y(12)缺失; 7: Glu-A1x和Glu-B1y双缺失。
Fig. 1 SDS-PAGE profiles of HMW-GS (A) and LMW-GS (B) in the deletion lines tested
1: Wild type; 2: Glu-A1x(1) deletion; 3: Glu-D1x(2) deletion; 4: Glu-B1x(7) deletion; 5: Glu-B1y(8) deletion; 6: Glu-D1y(12) deletion; 7: double deletion of Glu-A1x and Glu-B1y.

图2 宁麦9号野生型(A)和缺失系T13 (B)的醇溶蛋白色谱分离图谱Fig. 2 Separation profiles of gliadin in Ningmai 9 wild type (A) and the deletion line T13 (B) by RP-HPLC

表2可以看出, 野生型及单亚基缺失类型间的蛋白质含量差异较小, 变异范围为9.6%~10.6%; 湿面筋含量亦无显著差异, 变异范围为21.7%~ 24.4%。贮藏蛋白组分含量的比较显示, 野生型、Glu-B1y(8)和Glu-D1y(12)缺失类型的Glu/Gli变异范围为1.39~1.44, 显著高于Glu-A1(1)、Glu-B1x(7)和Glu-D1(12)的缺失型(1.27~1.31)。野生型和Glu- D1y(12)缺失型的HMW/LMW分别为0.20和0.21, 显著高于其他亚基缺失型, Glu-D1x(2)和Glu-B1x(7)缺失类型的HMW/LMW则表现最低, 分别为0.16和0.15。

表2 不同亚基缺失类型的贮藏蛋白组成特点 Table 2 Kernel quality traits in wild-type and single HMW-GS deletion lines
2.2 HMW-GS单亚基缺失对籽粒品质性状的影响

野生型及各缺失类型间的籽粒硬度变异范围为43.4~50.7, Glu-A1x(1)和Glu-D1x(2)的缺失型的籽粒硬度显著高于其他3种单亚基缺失类型, 而野生型的籽粒硬度为46.0, 表现中等硬度。出粉率在野生型以及各缺失类型间也无显著差异(表3)。此外, 野生型及单亚基缺失类型间的吸水率差异不显著, 水溶剂保持力、碳酸钠溶剂保持力、乳酸溶剂保持力和蔗糖溶剂保持力的变异范围分别为58.4%~60.3%、71.3%~73.1%、81.1%~92.9%和104.6%~111.8%。

表3 不同亚基缺失类型的籽粒品质性状 Table 3 Kernel quality traits in wild-type and single HMW-GS deletion lines
2.3 HMW-GS单亚基缺失对面团流变学及加工品质的影响

除峰宽和衰减度外, 参试品系间的揉面仪参数显著不同(表4)。其中, 野生型的揉面仪形成时间为2.00 min, 显著高于高分子量谷蛋白的5种单亚基缺失类型(1.38~1.64 min), Glu-B1x缺失类型的形成时间最短。野生型的峰高最大, 为50.9%, Glu-D1x缺失类型最低, 为44.8%。8.00 min带宽也表现为野生型最高, 为4.57%, Glu-B1x缺失类型最低, 为3.38%, 缺失类型间的差异不显著, 变异范围为3.38%~ 3.98%。饼干直径则呈现出相反的变化趋势, 各缺失类型间差异不显著, 变异范围为16.2~16.7 cm, 且以Glu-D1y缺失类型最高, 野生型仅为15.7 cm。图3显示了野生型和HMW-GS单亚基缺失系对揉面特性和饼干加工品质的影响。

表4 不同亚基缺失类型的面团流变学特性 Table 4 Dough rheological properties in wild-type and single HMW-GS deletion lines

图3 宁麦9号野生型和代表性缺失系的面团揉混特性(A)和饼干加工品质(B)
CK2: 野生型; T5: Glu-A1x缺失系; T13: Glu-D1x缺失系; T23: Glu-B1x缺失系; T30: Glu-B1y缺失系; T34: Glu-D1y缺失系。
Fig. 3 Dough mixographic properties (A) and sugar-snap cookie quality (B) of wild-type and typical HMW-GS deletion lines
CK2: Wild type; T5: Glu-A1x deletion line; T13: Glu-D1x deletion line; T23: Glu-B1x deletion line; T30: Glu-B1y deletion line; T34: Glu-D1y deletion line.

3 讨论

研究表明, 多数基因型的HMW-GS中x型亚基的含量较多, y型亚基的含量则较少[32]。本文也有相似的结论, 具体表现为x型亚基缺失系的HMW/ LMW较低。HMW-GS缺失会直接导致面筋弹性降低, 但不同研究材料或不同遗传背下, HMW-GS的缺失效应不同。Jondiko等[21]观察了硬质高蛋白背景下HMW-GS缺失对面筋质量的影响, 个别优质亚基组合的HMW-GS单亚基缺失虽然降低了面团强度, 但显著提高了面团延展性, 利于增加墨西哥卷饼的直径和货架寿命。Zhang等[22]和张平平等[33]利用相似的研究方法(蛋白质含量> 13%)详细研究了单亚基和位点缺失对贮藏蛋白组成、面筋质量和北方馒头加工品质的影响, 表明亚基缺失类型对面筋质量的影响依赖于HMW-GS组成, 在某些HMW-GS组成背景下可协调面筋的弹性和延展性, 改善馒头的加工品质。有研究表明蛋白质含量较低时, HMW-GS等位变异对面筋质量的影响变小[34, 35]。但Huebner等[36]对一套美国软麦的比较则显示, 尽管蛋白质含量较低(< 10.5%), Glu-D1位点的亚基等位变异, 尤其是贮藏蛋白组分的含量对面筋质量和饼干加工品质亦具有显著影响。但这些研究材料的遗传背景差异较大, HMW-GS缺失效应的普遍性或特定遗传背景下的表现需要进一步探讨。

本研究材料遗传背景为软质小麦宁麦9号, 野生型和缺失系的蛋白质含量变异范围为9.6%~10.6%, 亚基缺失通过改变谷蛋白总量及组分的含量, 显著降低了面筋强度, 这与前人的研究相一致。但与Glu-D1Glu-B1位点为5+10、17+18或7OE+8相比, 本研究2+12和7+8的背景下, 亚基间的加性和互作效应较小, 这可能是不同单亚基缺失系间面筋强度差异较小的原因。不同的单亚基缺失, 尤其是Glu-B1x、Glu-B1y和Glu-D1y缺失显著提高了糖酥饼干直径, 表明在该背景下, 面筋强度和耐揉性是影响糖酥饼干加工品质的重要因素。Li等[23]同样通过EMS化学诱变法获得了硬质小麦小偃54 (1, 14+15, 2+12)的HMW-GS的单亚基和双亚基缺失近等基因系, 表明各HMW-GS均对面筋弹性、延展性、面包体积等品质性状存在加性和上位性效应, 且效应大小不同, HMW-GS缺失对面包加工品质不利。本研究还显示, 除蛋白质含量和籽粒硬度在个别类型间有显著差异外, 多数面粉相关的品质性状在不同组成类型间均无显著变化, 如影响软质小麦加工品质较重要的3个性状中, 蛋白质含量的变异范围小于1%, 溶剂保持力相似, 籽粒硬度皆为中硬偏软类型, 亚基缺失仅显著降低了面筋强度和耐揉性。结合前人研究和本试验结果, 认为HMW-GS敲除对面筋强度要求较高的硬质麦培育应用价值较差, 对面筋强度要求较低但延展性要求较高的硬质麦或低筋软质麦培育具有较好的应用前景, 但HMW-GS敲除在不同谷蛋白组成和蛋白质含量背景下的效用性还有待进一步研究。

4 结论

HMW-GS单亚基缺失未显著改变籽粒的品质性状, 但显著改变了Glu/Gli和HMW/LMW的比例, 显著降低了面筋强度和耐揉性, 缺失系间的面筋质量无显著差异, 部分亚基缺失系的糖酥饼干直径较野生型显著增加。HMW-GS敲除是提高软质小麦加工品质的途径之一。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。The authors have declared that no competing interests exist.

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