小麦类黄酮的遗传基础与功能性小麦育种应用

陈杰，陈伟

小麦类黄酮的遗传基础与功能性小麦育种应用

陈杰，陈伟

华中农业大学植物科学技术学院/作物遗传改良全国重点实验室，武汉 430070

随着人们生活水平的提高，对食物的要求逐渐由“吃饱”向“吃好”以及“吃入营养”“吃出健康”等方向转变。小麦是我国以及世界最重要的粮食作物之一，育种家们认为小麦育种也要从“产量育种”向“品质育种”转变，即产量基本不变的前提下使得小麦籽粒具有特定有益人体健康的“功能性”成分，这些成分一般是小分子代谢物。与之相对应，还进一步提出了“功能性小麦品种”的概念。黄酮类代谢物是目前受到广泛关注的一类物质，由于它能够影响植株表型以及人类健康，该类物质含量也是“功能性小麦”育种的范畴之一。为了更好地促进“功能性小麦”育种，需要使用多种手段解析影响特定“功能性”代谢物含量的分子机理和遗传基础。代谢组学手段与遗传学设计相结合能够高效鉴定影响代谢物含量的基因，然而由于小麦参考基因组信息公布较晚，小麦中这类研究进展相对滞后，导致对于代谢物的遗传基础揭示不足，从而限制其在“功能性小麦”育种中的应用。本文以黄酮类物质为例，概述了解析这类代谢物遗传基础的研究进展，相关研究结果将为以提高黄酮类物质含量为主要目标的“功能性小麦”育种提供分子资源和理论基础，以及为研究其他“功能性”代谢物提供借鉴。与此同时，还初步构想了在相关基础研究积累不足的前提下直接使用代谢组学手段辅助育种的方式，有望在获得育种中间材料的同时“顺便”解析关键遗传因子或者候选基因，从而有效推动“功能性小麦”育种。

黄酮；功能性小麦；遗传基础；育种

0 引言

“功能性小麦品种”是最近提出的新名词，广义上来说，主要包含“加工功能性品种”和“营养功能性品种”两类[1]。其中“加工功能性品种”主要指能改善食品结构及品质；“营养功能性品种”的基本概念为“含有对人体健康有益活性成分，可调节人体有益代谢，能给人体健康带来某种益处或满足特定人群的特殊需求，同时，可以作为日常食物的口感正常、无毒副作用的小麦品种类型”[1]。以上两类功能性小麦品种中，营养功能性品种主要由于小麦籽粒中有益健康的活性成分能够调节人体代谢或者满足特定人群的需求，从而符合当下人群对于“吃得饱”向“吃得好”以及“吃得健康”的观念转变。与此同时，积极开展功能性小麦育种也符合“健康中国2030”规划等国家战略。

营养功能性小麦品种的主要育种目标包含高黄酮[2]、高抗性淀粉[3–5]、高微量元素[6–8]、低植酸[9–11]、低醇溶蛋白[12]等方面。其中，黄酮类物质因其化学结构简单、易于检测鉴定、代谢通路清晰等原因，相关研究报道较多，针对这类物质代谢通路解析也是作物代谢组学中主要研究方向之一。本文将结合黄酮类物质的相关研究进展以及代谢组学手段在小麦研究中的应用，初步探讨通过解析小麦黄酮代谢通路助力功能性小麦育种的理论基础、分子资源和研究前景。

1 黄酮类代谢物简介

黄酮类代谢物（flavonoids）作为一类物质的总称（为避免混淆下文使用“类黄酮”指代），是植物产生的一种多酚类化合物，基本骨架为C6-C3-C6，其中2个苯环（A环和B环）由一个三碳杂环吡喃环（C环）互连（图1-A）[13]。根据C环3个碳原子的成环情况和氧化程度，以及B环的连接位置等，可将类黄酮大致分为黄酮（flavone）、黄酮醇（flavonol）、黄烷酮（flavanone）、黄烷酮醇（flavanonol）黄烷醇（flavanol）、花色素（anthocyanin）和异黄酮（isoflavone）等七大类（图1-A，其中异黄酮仅存在于豆类植物等少数植物种类中[14]）。在每一种类黄酮中，其B环上羟基的数量差异也进一步增加了该类代谢物骨架的多样性：例如在黄酮醇骨架下，当其B环上有1—3个羟基时，分别为山柰酚（kaempferol）、槲皮素（quercetin）和杨梅素（myricetin），而在黄烷酮醇中，3种对应的物质则分别为香橙素（aromadendrin）、花旗松素（taxifolin）和白蔹素（ampelopsin，图1-B）。

类黄酮的生物合成被认为起始于苯丙烷途径[15]，首先通过来自于苯丙氨酸的对香豆酰辅酶A（-Coumaroyl-CoA）与丙二酰辅酶A（malonyl-CoA）经由查儿酮合酶（chalcone synthase，CHS）的催化生成一种C环开环状态的类黄酮前体物质：柚皮素查儿酮（naringenin chalcone）[16]，该物质可以被查儿酮异构酶（chalcone isomerase，CHI）转化形成柚皮素（naringenin）[17]。自此之后，以柚皮素为代表的黄烷酮可以经由黄酮合酶（flavone synthase，FNS）生成黄酮骨架[18]并进一步经由类黄酮3-羟化酶（flavonoid 3-hydroxylase，F3H）生成对应的黄酮醇（图1-B）[19]。与此同时，黄烷酮可以由F3H催化生成黄烷酮醇[20]，进一步通过二氢黄酮醇还原酶（dihydroflavonol reductase，DFR）和花青素合酶（anthocyanidin synthase，ANS）生成花色素[21]，最后通过花青素还原酶（anthocyanidin reductase，ANR）获得黄烷醇（图1-B）[22]。除了以上代谢路径以外，黄酮醇还可以从黄烷酮醇经由黄酮醇合酶（flavonol synthase，FLS）催化生成[23]，黄烷醇则可以自黄烷酮醇经过DFR和无色花青素还原酶（leucoanthocyanidin reductase，LAR）催化生成[24]。在这些连续线性催化关系的同时，每一种骨架内B环上羟基数量的增加一般是由2种羟化酶（flavonoid 3′-hydroxylase，F3′H；flavonoid 3′, 5′-hydroxylase，F3′ 5′H）催化实现（图1-B）[25]。由此可见，不同类黄酮骨架之间呈现清晰且复杂的网状生物合成路径。

2 类黄酮的多样性与功能性小麦育种

代谢物的多样性很大程度上是因为修饰的多样性导致[26]。就类黄酮而言，最常见的修饰发生在羟基上。通过甲基转移酶或者糖基转移酶的催化，不同位置的羟基可能发生多种甲基化以及糖基化修饰组合，糖基所具有的羟基基团上还可以继续发生糖基化或者酰基化等修饰，从而进一步丰富了类黄酮的物质多样性以及生物活性范畴。如在玉米[27]、水稻[28]、大麦[29]和小麦[30]等作物中，病菌侵染与甲基化修饰类黄酮的含量变化相关；其中，7号位甲基化修饰的黄酮（芫花素：7--甲基芹菜素）[31]和黄烷酮（樱花素：7--甲基柚皮素）[32]均表现出良好的抗植物真菌病害的活性。糖基化修饰的黄酮能够帮助植株抵抗紫外线辐射逆境，其中5号位糖基化修饰的黄酮相较于7号位修饰的黄酮具有更好的紫外线抗性表型[13]。以山柰酚为代表的黄酮醇，其3号位发生糖基化修饰后，对于粉虱类昆虫具有毒性，而粉虱在取食植物汁液后进一步在糖基上进行丙二酰化修饰，从而消除该物质对于自身的毒性[33]。

A：类黄酮的主要亚类；B：类黄酮B环不同羟基数量对应物质的信息

除了能够帮助植物适应复杂环境中的生物胁迫和非生物胁迫外，类黄酮的不同修饰产物还有利于人体健康。Montbretin A（MbA）是一种强效的特异性人胰腺α-淀粉酶抑制剂，从而对于治疗2型糖尿病具有良好的应用前景[34]。该物质在杨梅素黄酮醇骨架的4′和3号位羟基上具有包括葡萄糖、木糖和鼠李糖在内的多个糖基化修饰以及咖啡酸等酰基化修饰[35]，催化这些修饰的候选基因已被成功克隆[35-36]，从而使得MbA的异源生物合成成为可能。麦黄酮是五羟黄酮的3′和5′号位羟基分别有一个甲基化修饰的黄酮物质，该物质最早分离自二粒小麦叶片[37]，故而得名“麦黄酮”。麦黄酮具有潜在的食补和药用价值[38]，包括降脂[39]、消炎[40]、抗病毒[41]、抑制肿瘤生长[42]和抗癌[43]等活性。与此同时，麦黄酮还被认为是小麦、水稻、玉米等单子叶植物以及苜蓿等少数双子叶植物中木质素合成的起始位点[44–46]。除此以外，其他类黄酮物质也可能具有利于人体健康活性[47]以及参与木质素的合成[48-49]。多种类黄酮物质的存在有可能影响小麦的加工品质以及小麦制品的口感[50]。

在与类黄酮物质相关的功能性小麦育种范畴中，彩色小麦（即“彩麦”）是为人所熟知的领域之一。彩麦按照籽粒颜色区分主要有红色、蓝色和紫色等颜色类型，这些不同颜色主要是由于类黄酮途径中的花色素物质积累导致的[51-52]。相应地，彩麦籽粒的下游制品也能够具有相应的颜色[53]，从而极大地丰富了天然来源小麦制品的多样性。在控制彩麦颜色的候选基因和分子机理方面，控制红色的R基因被证实是一个MYB转录因子编码基因[54]，且该基因在调控花色素合成通路的同时还能促进种子中脱落酸的合成来抑制种子发芽[55]，从而解释了红皮小麦一般更抗穗发芽[56]的现象。然而，关于调控蓝色或者紫色的关键基因还在鉴定过程中[52]。因此，系统性解析小麦中的类黄酮代谢路径，不仅符合当下消费者对营养品质日益增长的需求[57]，还能为与木质素相关的基础研究提供新的视角[49, 58]，从而为功能性小麦育种提供分子资源和理论基础。

3 类黄酮代谢通路的功能基因鉴定

由于类黄酮骨架生物合成的代谢路径相对清晰（图1-B），即使在小麦中鲜有针对具体基因如何参与代谢通路的报道，研究者们依然可以使用基于序列比对的反向遗传学手段对同源基因进行相关研究。如通过与水稻中已经验证的类黄酮通路基因进行序列比对，可以得到小麦中的同源基因（表1）。然而，这些基因如何参与类黄酮通路还需要进一步的试验证实。如在水稻中麦黄酮合成路径并不是预期的先由芹菜素2次羟基化生成五羟黄酮后再发生2次甲基化，而是羟基化与甲基化交替进行生成麦黄酮[59]。在这个过程中，序列相似的同源基因可能同时具有新的酶活功能，从而影响代谢通路的走向[60]。基于此，一方面可以比较容易地探究小麦中类黄酮通路同源基因响应胁迫或者参与生长过程中的表达规律[61-64]；另一方面也需要具体研究这些基因如何参与类黄酮通路[65]。

除了使用序列比对这一反向遗传学方式外，还可以结合多种组学手段来鉴定参与小麦类黄酮通路的候选功能基因。如对紫色小麦材料ZNM168籽粒不同发育时期进行转录组和代谢组联合分析，成功检测到4种以矢车菊素为主要骨架的糖基化修饰代谢物与籽粒颜色形成有关，并且推测包括BZ1、CHS和ANS等基因在颜色相关代谢物生物合成路径中发挥关键作用[66]。另外，通过使用遗传群体设计（如使用自然群体材料的GWAS分析或者人工构建分离群体的QTL定位）对代谢组学手段检测到的多种类黄酮物质含量进行遗传定位也有助于快速鉴定候选基因。然而，根据所定位到的遗传位点推测候选基因需要借助参考基因组信息，因此，在小麦具备参考基因组之前的代谢组正向遗传学研究都未能提供可能的候选基因[67-68]。随着六倍体小麦高质量参考基因组的释放[69]，使得随后的小麦代谢物—GWAS（mGWAS）[70]或者代谢物—QTL（mQTL）[71]研究中批量鉴定候选基因成为可能，并以此解析代谢通路。如，通过鉴定并验证麦黄酮及其修饰代谢物在自然群体材料中mGWAS位点的候选基因，Chen等[70]首次解析了小麦中的一条黄酮代谢通路。类似地，Shi等[71]也使用人工构建的分离群体进行mQTL定位，对影响多种代谢物含量的候选基因进行鉴定并验证了其编码产物能够催化类黄酮物质酶活反应。以上研究表明，可以将代谢物相对含量作为“表型”数据，结合不同遗传群体的多态性标记信息，发挥代谢组学检测手段高通量的优势，从而快速鉴定小麦中影响类黄酮等代谢物含量的候选基因（图2），为以提高代谢物含量为目标的功能性小麦育种提供分子资源。

表1 水稻中已报道部分类黄酮代谢路径基因在小麦中的同源基因

表中小麦同源基因按照与已报道基因的序列相似度排列

The wheat homologs were ranked according to their sequence similarities against respective rice targets

4 代谢组学手段在功能性小麦育种中的应用

传统的育种流程一般使用农艺性状有差异的材料作为亲本进行杂交，通过该过程中的染色体重组来实现新的基因型组合，并在后代材料中对目标农艺性状进行选择。然而，由于小麦的连锁不平衡程度较强[79]，导致杂交过程中染色体重组率较低，从而需要花费大量的时间和构建较大规模的群体以获得所需基因型材料[80]。与此同时，可观测农艺性状数量较少以及变化幅度不大，并且从基因型到表型之间的调控关系较为复杂，进一步增加了育种的周期和难度。代谢物含量或者修饰状态的变化能够影响植株表型，因此，代谢组一直以来被认为是基因型和表型之间的桥梁[81]。其中，最广为人知的例子莫过于植物激素能够在植株生长发育以及逆境响应过程中发挥关键作用[82]，这些激素类代谢物的不同修饰形式往往对应激素分子的活性与否[83]。与此同时，不同类型的代谢物之间往往具有复杂的相关性，如糖基化修饰的类黄酮物质能够抑制内源生长素在植物体内的极性运输从而影响生长素含量及分布[84]，以及在小麦中一直以来观察到的红粒小麦更加抗穗发芽现象[56]是由于调控花色素的一个MYB转录因子也能影响小麦籽粒中的ABA含量，从而改变种子发芽性状[55]。以上例子表明农艺性状有可能受到复杂因素的影响，而通过对大量代谢物含量的数据进行建模预测等研究有可能解析代谢物含量与农艺性状之间的内在联系[85-86]，例如通过小麦叶片和小穗代谢组数据均能较好地预测小麦产量[87]。因此，通过对小麦不同组织及发育时期进行代谢组学研究，有助于更好地揭示功能性小麦育种过程中的相关分子机理以及育种目标的构成因素（如解析育种目标物质的代谢通路）。

除了使用以代谢组学为主的手段解析代谢通路从而挖掘分子资源用于小麦育种以外，还可以充分发挥其具有高灵敏度和高通量的特点，对以具体代谢物（如麦黄酮）含量为目标的功能性小麦育种过程中相关物质含量进行高通量检测，有可能达到即使不解析代谢通路也能获得育种材料的目的，并且在获得目标材料的同时“顺便”鉴定关键调控基因[88]。如图3所示，通过对小麦种质资源进行代谢谱检测，能够快速获得目标代谢物在不同（籽粒）材料中的相对含量。从中选取具有优良性状的当地主推品种（目标代谢物含量相对较低）为受体，与具有高目标代谢物含量的品系为供体进行杂交。杂交后代不断与受体回交以维持优良农艺性状，在此过程中也可以使用育种芯片或者分子标记来检验回交后代基因型与受体的一致性[89]。为了更好地监测杂交后代代谢通路变化情况，可以在获得每一代种子时将其一分为二，通过代谢组学手段检测不包含胚的半粒种子中目标代谢物含量，具有高含量对应的半粒含胚种子则继续种植并进入下一轮回交流程。该育种过程可以在快速育种温室中进行，每一代材料生长周期最短（如春小麦）可在3个月内完成[90]，从而缩短育种周期。最后，由于所获得品系遗传背景与受体基本一致，保持了对应的优良农艺性状，而控制改良后性状的候选基因则由供体提供，因此，通过进一步分析基因组中供体区段所包含的基因编码序列，结合代谢物化学特性及其所在通路等信息，可以鉴定候选基因以及开发分子标记。

5 结论和展望

本文以类黄酮为例，概述了解析代谢物遗传基础对于功能性小麦育种的意义，以及代谢组学手段在育种过程中的应用方式。在与类黄酮通路代谢物含量相关的小麦育种中，“彩麦”因其表型可以仅凭肉眼容易分辨，目前已经育成了包括绿色（如“灵绿麦”和“秦绿”等系列）、蓝色（如农大5321蓝和山农蓝麦1号）、紫色（如紫麦4179和紫麦19）和黑色（如“灵黑麦”和“秦黑”等系列）在内的多个“彩麦”品种。其余类黄酮物质由于不具有明显的显色性，其在育种过程中的含量变化一般需要通过高效液相色谱或者质谱等仪器进行测定。因此，尽管目前与类黄酮通路相关的功能性小麦育种已经有相当的研究积累与尝试[52, 91-94]，除了“彩麦”外已经审定的高类黄酮小麦仅有山农101（鲁审麦20206035，总黄酮含量1.013 mg·g-1）。造成这种差异性现象除了检测成本较高以外，相关标准缺位也是重要原因之一。最近，由中国国际科技促进会发布了《高麦黄酮小麦籽粒中游离态麦黄酮及总麦黄酮含量指标和测定方法》行业标准（T/CI 004-2022），其中，约定了高麦黄酮小麦品种定义、麦黄酮含量指标，即非彩色小麦籽粒中自然产生和积累的游离态麦黄酮含量大于等于0.5 mg·kg-1或者总麦黄酮含量大于等于1 mg·kg-1以及检测方法。因此，通过加深对关键代谢物遗传基础的认识以及随着影响代谢物含量分子资源的丰富，能够更好地实现不同目标功能性的小麦育种，以便满足消费者的差异化需求以及符合“健康中国2030”规划等国家战略。在推动“功能性小麦”育种过程中将获得一系列小麦种质资源，从而掌握品种芯片并夯实功能农业基础。

图2 快速鉴定影响代谢物含量的候选基因并构建代谢物与表型之间的网络

以高麦黄酮育种目标为例，综合利用代谢组学检测手段、多代杂交回交流程以及快速育种温室体系，可以在关键基因未知的情况下快速获得育种中间材料，并且帮助筛选控制麦黄酮含量的候选基因

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The genetic basis of flavonoid contents in wheat and its application in functional wheat variety breeding

CHEN Jie, CHEN Wei

College of Plant Science and Technology, Huazhong Agricultural University/National Key Laboratory of Crop Genetics and Improvement, Wuhan 430070

Accompanying the elevated expenses on consumption, people’s urge upon food has been gradually changed from “eat to be fed” to “eat to be satisfied” and further to “eat to gain nutrition” and “eat to be healthy”. Accordingly, breeders considered the wheat breeding goals should be set as breeding wheat with better quality along with higher yield, wherein the phrase “functional wheat variety” was recently raised. Flavonoids comprise one of the most widely reported categories of metabolites, the contents of which have been included within the “functional wheat variety” breeding program for its connection with plant phenotypes and its contribution to human health. The combination of metabolomics approach and genetics design has been proved to be efficient in identifying the candidates that responsible for metabolite contents, that said its application in wheat was lagged behind due to the lately released wheat reference genome. Further, the deficient knowledge upon the genetic basis of metabolites has in turn constrained the application of breeding “functional wheat variety”. In the current manuscript, the research progresses on genetic basis of flavonoids are briefly summarized, and its application for wheat breeding is highlighted. Meanwhile, the metabolomics-assisted breeding frame is concepted. Ultimately, the “functional wheat variety” breeding program will be achieved through the combination of the fundamental researches and breeding applications.

flavonoid; functional wheat; genetic basis; breeding

10.3864/j.issn.0578-1752.2023.13.001

2023-03-29；

2023-05-10

国家自然科学基金（32001541）、中国博士后科学基金（2021T140246）

通信作者陈杰，E-mail：lqlcj@126.com

（责任编辑 李莉）