小麥類黃酮的遺傳基礎與功能性小麥育種應用

陳杰，陳偉

小麥類黃酮的遺傳基礎與功能性小麥育種應用

陳杰，陳偉

華中農業大學植物科學技術學院/作物遺傳改良全國重點實驗室，武漢 430070

隨著人們生活水平的提高，對食物的要求逐漸由“吃飽”向“吃好”以及“吃入營養”“吃出健康”等方向轉變。小麥是我國以及世界最重要的糧食作物之一，育種家們認為小麥育種也要從“產量育種”向“品質育種”轉變，即產量基本不變的前提下使得小麥籽粒具有特定有益人體健康的“功能性”成分，這些成分一般是小分子代謝物。與之相對應，還進一步提出了“功能性小麥品種”的概念。黃酮類代謝物是目前受到廣泛關注的一類物質，由于它能夠影響植株表型以及人類健康，該類物質含量也是“功能性小麥”育種的范疇之一。為了更好地促進“功能性小麥”育種，需要使用多種手段解析影響特定“功能性”代謝物含量的分子機理和遺傳基礎。代謝組學手段與遺傳學設計相結合能夠高效鑒定影響代謝物含量的基因，然而由于小麥參考基因組信息公布較晚，小麥中這類研究進展相對滯后，導致對于代謝物的遺傳基礎揭示不足，從而限制其在“功能性小麥”育種中的應用。本文以黃酮類物質為例，概述了解析這類代謝物遺傳基礎的研究進展，相關研究結果將為以提高黃酮類物質含量為主要目標的“功能性小麥”育種提供分子資源和理論基礎，以及為研究其他“功能性”代謝物提供借鑒。與此同時，還初步構想了在相關基礎研究積累不足的前提下直接使用代謝組學手段輔助育種的方式，有望在獲得育種中間材料的同時“順便”解析關鍵遺傳因子或者候選基因，從而有效推動“功能性小麥”育種。

黃酮；功能性小麥；遺傳基礎；育種

0 引言

“功能性小麥品種”是最近提出的新名詞，廣義上來說，主要包含“加工功能性品種”和“營養功能性品種”兩類[1]。其中“加工功能性品種”主要指能改善食品結構及品質；“營養功能性品種”的基本概念為“含有對人體健康有益活性成分，可調節人體有益代謝，能給人體健康帶來某種益處或滿足特定人群的特殊需求，同時，可以作為日常食物的口感正常、無毒副作用的小麥品種類型”[1]。以上兩類功能性小麥品種中，營養功能性品種主要由于小麥籽粒中有益健康的活性成分能夠調節人體代謝或者滿足特定人群的需求，從而符合當下人群對于“吃得飽”向“吃得好”以及“吃得健康”的觀念轉變。與此同時，積極開展功能性小麥育種也符合“健康中國2030”規劃等國家戰略。

營養功能性小麥品種的主要育種目標包含高黃酮[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，黃酮類物質因其化學結構簡單、易于檢測鑒定、代謝通路清晰等原因，相關研究報道較多，針對這類物質代謝通路解析也是作物代謝組學中主要研究方向之一。本文將結合黃酮類物質的相關研究進展以及代謝組學手段在小麥研究中的應用，初步探討通過解析小麥黃酮代謝通路助力功能性小麥育種的理論基礎、分子資源和研究前景。

1 黃酮類代謝物簡介

黃酮類代謝物（flavonoids）作為一類物質的總稱（為避免混淆下文使用“類黃酮”指代），是植物產生的一種多酚類化合物，基本骨架為C6-C3-C6，其中2個苯環（A環和B環）由一個三碳雜環吡喃環（C環）互連（圖1-A）[13]。根據C環3個碳原子的成環情況和氧化程度，以及B環的連接位置等，可將類黃酮大致分為黃酮（flavone）、黃酮醇（flavonol）、黃烷酮（flavanone）、黃烷酮醇（flavanonol）黃烷醇（flavanol）、花色素（anthocyanin）和異黃酮（isoflavone）等七大類（圖1-A，其中異黃酮僅存在于豆類植物等少數植物種類中[14]）。在每一種類黃酮中，其B環上羥基的數量差異也進一步增加了該類代謝物骨架的多樣性：例如在黃酮醇骨架下，當其B環上有1—3個羥基時，分別為山柰酚（kaempferol）、槲皮素（quercetin）和楊梅素（myricetin），而在黃烷酮醇中，3種對應的物質則分別為香橙素（aromadendrin）、花旗松素（taxifolin）和白蘞素（ampelopsin，圖1-B）。

類黃酮的生物合成被認為起始于苯丙烷途徑[15]，首先通過來自于苯丙氨酸的對香豆酰輔酶A（-Coumaroyl-CoA）與丙二酰輔酶A（malonyl-CoA）經由查兒酮合酶（chalcone synthase，CHS）的催化生成一種C環開環狀態的類黃酮前體物質：柚皮素查兒酮（naringenin chalcone）[16]，該物質可以被查兒酮異構酶（chalcone isomerase，CHI）轉化形成柚皮素（naringenin）[17]。自此之后，以柚皮素為代表的黃烷酮可以經由黃酮合酶（flavone synthase，FNS）生成黃酮骨架[18]并進一步經由類黃酮3-羥化酶（flavonoid 3-hydroxylase，F3H）生成對應的黃酮醇（圖1-B）[19]。與此同時，黃烷酮可以由F3H催化生成黃烷酮醇[20]，進一步通過二氫黃酮醇還原酶（dihydroflavonol reductase，DFR）和花青素合酶（anthocyanidin synthase，ANS）生成花色素[21]，最后通過花青素還原酶（anthocyanidin reductase，ANR）獲得黃烷醇（圖1-B）[22]。除了以上代謝路徑以外，黃酮醇還可以從黃烷酮醇經由黃酮醇合酶（flavonol synthase，FLS）催化生成[23]，黃烷醇則可以自黃烷酮醇經過DFR和無色花青素還原酶（leucoanthocyanidin reductase，LAR）催化生成[24]。在這些連續線性催化關系的同時，每一種骨架內B環上羥基數量的增加一般是由2種羥化酶（flavonoid 3′-hydroxylase，F3′H；flavonoid 3′, 5′-hydroxylase，F3′ 5′H）催化實現（圖1-B）[25]。由此可見，不同類黃酮骨架之間呈現清晰且復雜的網狀生物合成路徑。

2 類黃酮的多樣性與功能性小麥育種

代謝物的多樣性很大程度上是因為修飾的多樣性導致[26]。就類黃酮而言，最常見的修飾發生在羥基上。通過甲基轉移酶或者糖基轉移酶的催化，不同位置的羥基可能發生多種甲基化以及糖基化修飾組合，糖基所具有的羥基基團上還可以繼續發生糖基化或者酰基化等修飾，從而進一步豐富了類黃酮的物質多樣性以及生物活性范疇。如在玉米[27]、水稻[28]、大麥[29]和小麥[30]等作物中，病菌侵染與甲基化修飾類黃酮的含量變化相關；其中，7號位甲基化修飾的黃酮（芫花素：7--甲基芹菜素）[31]和黃烷酮（櫻花素：7--甲基柚皮素）[32]均表現出良好的抗植物真菌病害的活性。糖基化修飾的黃酮能夠幫助植株抵抗紫外線輻射逆境，其中5號位糖基化修飾的黃酮相較于7號位修飾的黃酮具有更好的紫外線抗性表型[13]。以山柰酚為代表的黃酮醇，其3號位發生糖基化修飾后，對于粉虱類昆蟲具有毒性，而粉虱在取食植物汁液后進一步在糖基上進行丙二酰化修飾，從而消除該物質對于自身的毒性[33]。

A：類黃酮的主要亞類；B：類黃酮B環不同羥基數量對應物質的信息

除了能夠幫助植物適應復雜環境中的生物脅迫和非生物脅迫外，類黃酮的不同修飾產物還有利于人體健康。Montbretin A（MbA）是一種強效的特異性人胰腺α-淀粉酶抑制劑，從而對于治療2型糖尿病具有良好的應用前景[34]。該物質在楊梅素黃酮醇骨架的4′和3號位羥基上具有包括葡萄糖、木糖和鼠李糖在內的多個糖基化修飾以及咖啡酸等酰基化修飾[35]，催化這些修飾的候選基因已被成功克隆[35-36]，從而使得MbA的異源生物合成成為可能。麥黃酮是五羥黃酮的3′和5′號位羥基分別有一個甲基化修飾的黃酮物質，該物質最早分離自二粒小麥葉片[37]，故而得名“麥黃酮”。麥黃酮具有潛在的食補和藥用價值[38]，包括降脂[39]、消炎[40]、抗病毒[41]、抑制腫瘤生長[42]和抗癌[43]等活性。與此同時，麥黃酮還被認為是小麥、水稻、玉米等單子葉植物以及苜蓿等少數雙子葉植物中木質素合成的起始位點[44–46]。除此以外，其他類黃酮物質也可能具有利于人體健康活性[47]以及參與木質素的合成[48-49]。多種類黃酮物質的存在有可能影響小麥的加工品質以及小麥制品的口感[50]。

在與類黃酮物質相關的功能性小麥育種范疇中，彩色小麥（即“彩麥”）是為人所熟知的領域之一。彩麥按照籽粒顏色區分主要有紅色、藍色和紫色等顏色類型，這些不同顏色主要是由于類黃酮途徑中的花色素物質積累導致的[51-52]。相應地，彩麥籽粒的下游制品也能夠具有相應的顏色[53]，從而極大地豐富了天然來源小麥制品的多樣性。在控制彩麥顏色的候選基因和分子機理方面，控制紅色的R基因被證實是一個MYB轉錄因子編碼基因[54]，且該基因在調控花色素合成通路的同時還能促進種子中脫落酸的合成來抑制種子發芽[55]，從而解釋了紅皮小麥一般更抗穗發芽[56]的現象。然而，關于調控藍色或者紫色的關鍵基因還在鑒定過程中[52]。因此，系統性解析小麥中的類黃酮代謝路徑，不僅符合當下消費者對營養品質日益增長的需求[57]，還能為與木質素相關的基礎研究提供新的視角[49, 58]，從而為功能性小麥育種提供分子資源和理論基礎。

3 類黃酮代謝通路的功能基因鑒定

由于類黃酮骨架生物合成的代謝路徑相對清晰（圖1-B），即使在小麥中鮮有針對具體基因如何參與代謝通路的報道，研究者們依然可以使用基于序列比對的反向遺傳學手段對同源基因進行相關研究。如通過與水稻中已經驗證的類黃酮通路基因進行序列比對，可以得到小麥中的同源基因（表1）。然而，這些基因如何參與類黃酮通路還需要進一步的試驗證實。如在水稻中麥黃酮合成路徑并不是預期的先由芹菜素2次羥基化生成五羥黃酮后再發生2次甲基化，而是羥基化與甲基化交替進行生成麥黃酮[59]。在這個過程中，序列相似的同源基因可能同時具有新的酶活功能，從而影響代謝通路的走向[60]。基于此，一方面可以比較容易地探究小麥中類黃酮通路同源基因響應脅迫或者參與生長過程中的表達規律[61-64]；另一方面也需要具體研究這些基因如何參與類黃酮通路[65]。

除了使用序列比對這一反向遺傳學方式外，還可以結合多種組學手段來鑒定參與小麥類黃酮通路的候選功能基因。如對紫色小麥材料ZNM168籽粒不同發育時期進行轉錄組和代謝組聯合分析，成功檢測到4種以矢車菊素為主要骨架的糖基化修飾代謝物與籽粒顏色形成有關，并且推測包括BZ1、CHS和ANS等基因在顏色相關代謝物生物合成路徑中發揮關鍵作用[66]。另外，通過使用遺傳群體設計（如使用自然群體材料的GWAS分析或者人工構建分離群體的QTL定位）對代謝組學手段檢測到的多種類黃酮物質含量進行遺傳定位也有助于快速鑒定候選基因。然而，根據所定位到的遺傳位點推測候選基因需要借助參考基因組信息，因此，在小麥具備參考基因組之前的代謝組正向遺傳學研究都未能提供可能的候選基因[67-68]。隨著六倍體小麥高質量參考基因組的釋放[69]，使得隨后的小麥代謝物—GWAS（mGWAS）[70]或者代謝物—QTL（mQTL）[71]研究中批量鑒定候選基因成為可能，并以此解析代謝通路。如，通過鑒定并驗證麥黃酮及其修飾代謝物在自然群體材料中mGWAS位點的候選基因，Chen等[70]首次解析了小麥中的一條黃酮代謝通路。類似地，Shi等[71]也使用人工構建的分離群體進行mQTL定位，對影響多種代謝物含量的候選基因進行鑒定并驗證了其編碼產物能夠催化類黃酮物質酶活反應。以上研究表明，可以將代謝物相對含量作為“表型”數據，結合不同遺傳群體的多態性標記信息，發揮代謝組學檢測手段高通量的優勢，從而快速鑒定小麥中影響類黃酮等代謝物含量的候選基因（圖2），為以提高代謝物含量為目標的功能性小麥育種提供分子資源。

表1 水稻中已報道部分類黃酮代謝路徑基因在小麥中的同源基因

表中小麥同源基因按照與已報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 代謝組學手段在功能性小麥育種中的應用

傳統的育種流程一般使用農藝性狀有差異的材料作為親本進行雜交，通過該過程中的染色體重組來實現新的基因型組合，并在后代材料中對目標農藝性狀進行選擇。然而，由于小麥的連鎖不平衡程度較強[79]，導致雜交過程中染色體重組率較低，從而需要花費大量的時間和構建較大規模的群體以獲得所需基因型材料[80]。與此同時，可觀測農藝性狀數量較少以及變化幅度不大，并且從基因型到表型之間的調控關系較為復雜，進一步增加了育種的周期和難度。代謝物含量或者修飾狀態的變化能夠影響植株表型，因此，代謝組一直以來被認為是基因型和表型之間的橋梁[81]。其中，最廣為人知的例子莫過于植物激素能夠在植株生長發育以及逆境響應過程中發揮關鍵作用[82]，這些激素類代謝物的不同修飾形式往往對應激素分子的活性與否[83]。與此同時，不同類型的代謝物之間往往具有復雜的相關性，如糖基化修飾的類黃酮物質能夠抑制內源生長素在植物體內的極性運輸從而影響生長素含量及分布[84]，以及在小麥中一直以來觀察到的紅粒小麥更加抗穗發芽現象[56]是由于調控花色素的一個MYB轉錄因子也能影響小麥籽粒中的ABA含量，從而改變種子發芽性狀[55]。以上例子表明農藝性狀有可能受到復雜因素的影響，而通過對大量代謝物含量的數據進行建模預測等研究有可能解析代謝物含量與農藝性狀之間的內在聯系[85-86]，例如通過小麥葉片和小穗代謝組數據均能較好地預測小麥產量[87]。因此，通過對小麥不同組織及發育時期進行代謝組學研究，有助于更好地揭示功能性小麥育種過程中的相關分子機理以及育種目標的構成因素（如解析育種目標物質的代謝通路）。

除了使用以代謝組學為主的手段解析代謝通路從而挖掘分子資源用于小麥育種以外，還可以充分發揮其具有高靈敏度和高通量的特點，對以具體代謝物（如麥黃酮）含量為目標的功能性小麥育種過程中相關物質含量進行高通量檢測，有可能達到即使不解析代謝通路也能獲得育種材料的目的，并且在獲得目標材料的同時“順便”鑒定關鍵調控基因[88]。如圖3所示，通過對小麥種質資源進行代謝譜檢測，能夠快速獲得目標代謝物在不同（籽粒）材料中的相對含量。從中選取具有優良性狀的當地主推品種（目標代謝物含量相對較低）為受體，與具有高目標代謝物含量的品系為供體進行雜交。雜交后代不斷與受體回交以維持優良農藝性狀，在此過程中也可以使用育種芯片或者分子標記來檢驗回交后代基因型與受體的一致性[89]。為了更好地監測雜交后代代謝通路變化情況，可以在獲得每一代種子時將其一分為二，通過代謝組學手段檢測不包含胚的半粒種子中目標代謝物含量，具有高含量對應的半粒含胚種子則繼續種植并進入下一輪回交流程。該育種過程可以在快速育種溫室中進行，每一代材料生長周期最短（如春小麥）可在3個月內完成[90]，從而縮短育種周期。最后，由于所獲得品系遺傳背景與受體基本一致，保持了對應的優良農藝性狀，而控制改良后性狀的候選基因則由供體提供，因此，通過進一步分析基因組中供體區段所包含的基因編碼序列，結合代謝物化學特性及其所在通路等信息，可以鑒定候選基因以及開發分子標記。

5 結論和展望

本文以類黃酮為例，概述了解析代謝物遺傳基礎對于功能性小麥育種的意義，以及代謝組學手段在育種過程中的應用方式。在與類黃酮通路代謝物含量相關的小麥育種中，“彩麥”因其表型可以僅憑肉眼容易分辨，目前已經育成了包括綠色（如“靈綠麥”和“秦綠”等系列）、藍色（如農大5321藍和山農藍麥1號）、紫色（如紫麥4179和紫麥19）和黑色（如“靈黑麥”和“秦黑”等系列）在內的多個“彩麥”品種。其余類黃酮物質由于不具有明顯的顯色性，其在育種過程中的含量變化一般需要通過高效液相色譜或者質譜等儀器進行測定。因此，盡管目前與類黃酮通路相關的功能性小麥育種已經有相當的研究積累與嘗試[52, 91-94]，除了“彩麥”外已經審定的高類黃酮小麥僅有山農101（魯審麥20206035，總黃酮含量1.013 mg·g-1）。造成這種差異性現象除了檢測成本較高以外，相關標準缺位也是重要原因之一。最近，由中國國際科技促進會發布了《高麥黃酮小麥籽粒中游離態麥黃酮及總麥黃酮含量指標和測定方法》行業標準（T/CI 004-2022），其中，約定了高麥黃酮小麥品種定義、麥黃酮含量指標，即非彩色小麥籽粒中自然產生和積累的游離態麥黃酮含量大于等于0.5 mg·kg-1或者總麥黃酮含量大于等于1 mg·kg-1以及檢測方法。因此，通過加深對關鍵代謝物遺傳基礎的認識以及隨著影響代謝物含量分子資源的豐富，能夠更好地實現不同目標功能性的小麥育種，以便滿足消費者的差異化需求以及符合“健康中國2030”規劃等國家戰略。在推動“功能性小麥”育種過程中將獲得一系列小麥種質資源，從而掌握品種芯片并夯實功能農業基礎。

圖2 快速鑒定影響代謝物含量的候選基因并構建代謝物與表型之間的網絡

以高麥黃酮育種目標為例，綜合利用代謝組學檢測手段、多代雜交回交流程以及快速育種溫室體系，可以在關鍵基因未知的情況下快速獲得育種中間材料，并且幫助篩選控制麥黃酮含量的候選基因

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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

國家自然科學基金（32001541）、中國博士后科學基金（2021T140246）

通信作者陳杰，E-mail：lqlcj@126.com

（責任編輯 李莉）