劉 葉 李 越 苑名楊 衛(wèi)乃翠 關(guān)攀鋒 趙佳佳 武棒棒 鄭興衛(wèi) 郝宇瓊 喬 玲,* 鄭 軍,*

研究簡(jiǎn)報(bào)

小麥卷葉突變體的生理特性及遺傳研究

劉 葉1,2,**李 越1,**苑名楊1衛(wèi)乃翠1關(guān)攀鋒3趙佳佳1武棒棒1鄭興衛(wèi)1郝宇瓊1喬 玲1,*鄭 軍1,*

1山西農(nóng)業(yè)大學(xué)小麥研究所 / 農(nóng)業(yè)農(nóng)村部有機(jī)旱作農(nóng)業(yè)重點(diǎn)實(shí)驗(yàn)室(部省共建), 山西臨汾 041000;2山西大學(xué)生命科學(xué)學(xué)院, 山西太原 030006;3鄭州大學(xué)農(nóng)學(xué)院, 河南鄭州 450001

小麥葉片在逆境下會(huì)發(fā)生可逆的折疊或卷曲, 通過(guò)脫水回避的形態(tài)學(xué)變化降低非生物脅迫的損害。目前小麥葉片卷曲的生理和遺傳調(diào)控機(jī)制尚不清楚。本文利用EMS誘變晉麥47獲得了卷葉突變體(),在整個(gè)生育期葉片呈現(xiàn)卷曲, 初生葉片沿中軸脈向近軸面微卷, 隨著葉片生長(zhǎng)加速卷曲, 直至為筒狀。與野生型相比,株高降低、穗長(zhǎng)變短、旗葉變窄和千粒重降低。氯化三苯基四氮唑(TTC)染色結(jié)果表明種子活力低, 且種子發(fā)芽率降低了22%。抽穗10 d后,的葉綠素含量與野生型基本一致, 凈光合速率、蒸騰速率、氣孔導(dǎo)度、細(xì)胞間隙CO2濃度差異不顯著, 但水分利用率降低。低溫、高溫和干旱促進(jìn)的葉片卷曲; 石蠟切片觀察表明,的大葉脈和小葉脈偏少, 在中脈區(qū)域遠(yuǎn)軸面厚壁細(xì)胞和近軸面薄壁細(xì)胞數(shù)目減少, 且維管束間泡狀細(xì)胞的面積和數(shù)量均明顯低于野生型;葉片不同部位泡狀細(xì)胞縮小以及維管束減少導(dǎo)致整個(gè)葉片向近軸面極度卷曲。遺傳分析表明該性狀受1對(duì)不完全顯性的核基因控制, 位于1D染色體短臂上, 精細(xì)定位將目標(biāo)區(qū)間鎖定在9.42 Mb范圍內(nèi)。

小麥; 卷葉突變體; 細(xì)胞學(xué)分析; 生理特性; 遺傳分析

植物感受到外界不利環(huán)境后, 除生理和基因表達(dá)等內(nèi)在響應(yīng)外, 植株形態(tài)往往發(fā)生變化[1]。葉片卷曲是植物應(yīng)對(duì)強(qiáng)光、干旱、鹽和熱等非生物脅迫的自我保護(hù)機(jī)制, 葉片折疊或卷曲從而減少受光面積、削弱強(qiáng)光輻射、減輕葉片損傷, 進(jìn)而降低蒸騰作用、減少水分流失, 達(dá)到降低干旱影響的目的[2]。因此, 葉片卷曲是逆境條件下葉片免受光損傷的脫水回避形態(tài)學(xué)策略[3]。此外, 適當(dāng)?shù)娜~片卷曲也有利于保持葉片直立, 可改善群體整體的受光條件。因此, 研究葉片卷曲的生理特性和調(diào)控機(jī)制, 有利于解析作物對(duì)逆境響應(yīng)的適應(yīng)機(jī)制[4]。

根據(jù)葉片近軸面和遠(yuǎn)軸面的極性發(fā)育可將葉片卷曲分為兩類, 即內(nèi)卷和外卷, 相關(guān)機(jī)制在擬南芥、水稻、玉米均有研究報(bào)道, 水稻中研究最為深入, 主要的卷曲機(jī)制主要有4種, 一是通過(guò)調(diào)節(jié)泡狀細(xì)胞形態(tài)、大小、數(shù)量以及分布來(lái)改變?nèi)~片卷曲程度, 如半卷葉抑制葉片近軸面泡狀細(xì)胞的生成, 導(dǎo)致葉片向近軸面卷曲[5];和也是通過(guò)影響泡狀細(xì)胞的發(fā)育使得葉片卷曲[6-7]。二是調(diào)節(jié)薄壁和厚壁細(xì)胞的大小和數(shù)量影響葉片卷曲, 如通過(guò)影響遠(yuǎn)軸面厚壁組織細(xì)胞的發(fā)育導(dǎo)致葉片極度內(nèi)卷[8]。三是改變維管束中細(xì)胞特性, 如由于葉脈維管束韌皮部中篩管細(xì)胞增多, 整個(gè)韌皮部的面積顯著增大, 使葉脈遠(yuǎn)軸面皺縮導(dǎo)致葉片外卷[9]。四是角質(zhì)層、表皮細(xì)胞和葉肉細(xì)胞的變化, 如通過(guò)與富亮氨酸拉鏈轉(zhuǎn)錄因子互作負(fù)向調(diào)節(jié)角質(zhì)層的發(fā)育, 過(guò)表達(dá)引起葉片內(nèi)卷[10]。在玉米中發(fā)現(xiàn)的葉片卷曲基因主要通過(guò)調(diào)節(jié)近軸面細(xì)胞大小,和改變近軸面細(xì)胞使得葉片卷曲[11-12]。

小麥?zhǔn)钱愒戳扼w, 基因組龐大(17G左右)且多為重復(fù)序列, 葉片卷曲基因克隆和調(diào)控機(jī)制的研究遠(yuǎn)落后于水稻和玉米, 目前只報(bào)道了少數(shù)QTL/基因。Zhu等[13]通過(guò)全基因組關(guān)聯(lián)分析在323份小麥材料中鑒定出調(diào)控葉片卷曲的候選基因。Aakriti等[14]通過(guò)構(gòu)建作圖群體及同源性比對(duì), 選定可能為卷葉候選基因。利用集群分離分析(BSA)結(jié)合660K芯片在7A染色體發(fā)現(xiàn)可能為葉片卷曲的候選基因[15]。最近, Bian等[16]利用卷葉突變體檢測(cè)到2個(gè)卷葉性狀的主效QTL (和), 并將精細(xì)定位到6 Mb的范圍內(nèi)?？梢?jiàn), 開(kāi)展小麥葉片卷曲相關(guān)基因的克隆及調(diào)控研究, 有助于深入了解小麥響應(yīng)逆境的調(diào)控機(jī)制。本文利用甲基磺酸乙酯(ethylmethane sulfonate, EMS)誘變著名旱地品種“晉麥47”, 獲得穩(wěn)定遺傳的卷葉突變體, 對(duì)農(nóng)藝性狀、葉片卷曲特征、細(xì)胞學(xué)及遺傳特性進(jìn)行了研究, 為后續(xù)基因克隆和基因功能研究奠定基礎(chǔ)。

1 材料與方法

1.1 試驗(yàn)材料

晉麥47號(hào), 旱地冬性品種, 1995年審定后, 一直是我國(guó)黃淮旱地生產(chǎn)上的主要品種, 也是國(guó)家黃淮旱地區(qū)試和山西省南部旱地區(qū)試的對(duì)照品種[17]。晉麥47和及相關(guān)材料種植于山西農(nóng)業(yè)大學(xué)(山西省農(nóng)業(yè)科學(xué)院)小麥研究所試驗(yàn)基地(山西省臨汾市, 36°2'N, 111°18'E), 每年10月上旬播種, 翌年6月中旬收獲。每個(gè)材料播種1行, 行長(zhǎng)2 m, 每行40粒。于越冬期和拔節(jié)期灌溉, 灌溉量均為700 m3hm–2。所有生育期內(nèi)未發(fā)生極端天氣和嚴(yán)重自然災(zāi)害, 小麥生長(zhǎng)情況良好。

春化后種植在溫室中, 在光照14 h、22℃的條件下生長(zhǎng), 拔節(jié)期設(shè)置不同光照(400、800和1000 μmol m–2s–1)、不同平均溫度(10℃、22℃和30℃)及不同土壤水分含量(27%～ 30%和干旱處理14%～17%), 研究環(huán)境因素對(duì)葉片卷曲的影響; 每個(gè)處理設(shè)置3次重復(fù), 調(diào)查葉片卷曲指數(shù)。

1.2 農(nóng)藝性狀和葉片卷曲指數(shù)的測(cè)定

選擇長(zhǎng)勢(shì)均勻的材料各10株, 抽穗20 d后測(cè)量株高、穗長(zhǎng)、各節(jié)間長(zhǎng)度、穗粒數(shù)、小穗數(shù)等主要農(nóng)藝性狀, 同時(shí)測(cè)量旗葉長(zhǎng)和寬, 通過(guò)葉長(zhǎng)×葉寬×0.77計(jì)算葉面積[18]。籽粒收獲后, 測(cè)量千粒重。參考Shi等[19]的卷曲指數(shù)(LRI)測(cè)定方法進(jìn)行計(jì)算, 公式為L(zhǎng)RI (%)=(w–n)/w×100%, 其中w為測(cè)量葉片最寬處展開(kāi)后的葉緣間距,n為葉片最寬處卷曲狀態(tài)下的葉緣間距, 平展葉當(dāng)LRI為0; LRI大于0, 葉片向近軸面對(duì)折為微卷, 葉片呈筒狀為高度卷曲。

1.3 種子活力及苗期性狀檢測(cè)

在適宜的條件下, 分別取100粒野生型和結(jié)構(gòu)完整的種子播種于育苗盤, 15 d后統(tǒng)計(jì)發(fā)芽數(shù), 計(jì)算發(fā)芽率[20]; 采集10株長(zhǎng)勢(shì)一致、根系完整的幼苗統(tǒng)計(jì)根系性狀。選取成熟的野生型和籽粒, 用溫水浸泡4 h, 使種子充分吸脹, 沿種胚中央準(zhǔn)確切開(kāi)將切好的種子放在培養(yǎng)皿中, 加入現(xiàn)配的質(zhì)量體積濃度為0.1%氯化三苯基四氮唑(TTC), 25℃染色40～60 min, 觀察種胚著色情況。

1.4 葉綠素含量和光合參數(shù)的測(cè)定

抽穗10 d后利用葉綠素測(cè)定儀SPAD-502 (Konica- Minolta, 日本)測(cè)定旗葉葉綠素含量, 取旗葉底部、中部和頂部的均值作為測(cè)定結(jié)果, 每個(gè)材料測(cè)5株計(jì)算平均值。于晴天上午09:30—10:30, 利用便攜式光合測(cè)定儀(TARGAS-1, PP Systems, Amesbury, 美國(guó))測(cè)定旗葉中部的凈光合速率(n)、蒸騰速率(r)、氣孔導(dǎo)度(s)、細(xì)胞間隙CO2濃度(i)等氣體交換參數(shù), 每個(gè)材料測(cè)5株, 通過(guò)n/r計(jì)算葉片水分利用效率(WUE)[21]。

1.5 突變體RL1的組織學(xué)分析

選取抽穗10 d后的旗葉進(jìn)行組織學(xué)分析。葉片在FAA固定液中浸泡24 h以上, 經(jīng)脫水浸蠟后包埋于石蠟中, 蠟塊冷卻后使用組織攤片機(jī)(KD-P)切片, 厚4 μm左右。將切片依次放入環(huán)保型脫蠟透明液、無(wú)水乙醇和水中進(jìn)行漂洗; 置于番紅染色液2 h, 固綠染色6～20 s, 在正置光學(xué)顯微鏡(NIKON ECLIPSE E100, 日本)下觀察。

1.6 突變體RL1的遺傳分析和基因定位

分別與晉麥47和臨汾5064進(jìn)行正反交, 以/晉麥47和/臨汾5064構(gòu)建F2定位群體, 大田觀察F2表型并通過(guò)F3進(jìn)行驗(yàn)證, 卡方檢測(cè)分離比例。DNA提取采用改良的CTAB法, 從F2中選取平展葉與高度卷葉各20株構(gòu)建極端混池用于基因初定位。混池及親本由北京中玉金標(biāo)記公司進(jìn)行660K SNP芯片檢測(cè)。篩選親本間及后代間純合有差異的SNP位點(diǎn), 在小麥多組學(xué)數(shù)據(jù)庫(kù)(http://wheatomics.sdau.edu.cn/)的中國(guó)春參考基因組序列中查找SNP位點(diǎn)上下游100 bp堿基序列, 設(shè)計(jì)KASP (kompetitive allele specific PCR)特異性引物進(jìn)行基因精細(xì)定位(表1)。引物由北京博邁德生物技術(shù)有限公司合成。

2 結(jié)果與分析

2.1 突變體RL1的獲得及表型觀察

卷葉突變體由晉麥47經(jīng)EMS誘變處理后, M2篩選到葉片卷曲的突變株, 連續(xù)3代套袋自交后, 與晉麥47回交3代, 自交獲得到的一個(gè)穩(wěn)定遺傳的卷葉突變體株系。選取不同染色體上的共42對(duì)SSR引物(附表1), 對(duì)突變體和野生型進(jìn)行遺傳背景測(cè)定, 未發(fā)現(xiàn)多樣性差異, 說(shuō)明該卷葉植株是晉麥47的誘變株, 命名為。

葉片卷曲特性隨生育進(jìn)程而發(fā)生變化(圖1-c), 新抽出的葉片基本上沿中軸脈縱向微卷, 伴隨著生長(zhǎng)葉片加速卷曲, 直到卷曲為筒狀。與野生型相比,生長(zhǎng)勢(shì)較弱, 從苗期開(kāi)始出現(xiàn)輕微卷曲現(xiàn)象, LRIs為0.11～ 0.25 (圖1-b)。越冬前LRIs為0.31～0.60。返青期后, 新抽出的葉片仍然微卷, 隨著葉片生長(zhǎng)沿中軸逐漸向內(nèi)卷曲, 直至葉片內(nèi)卷成為近似圓筒狀, LRIs為0.43～0.62, 且葉片的中下部約2/3卷曲、中上部正常。拔節(jié)期, LRIs為0.57～0.70。抽穗后, 旗葉、倒二葉卷曲程度高, 倒三、倒四葉依然為輕微卷曲, 葉鞘高度卷曲, 此時(shí)的旗葉LRIs為0.91～0.98 (圖1-a)。而野生型植株的葉片始終接近為平展葉。

2.2 突變體RL1的農(nóng)藝性狀分析

對(duì)比和野生型株高相關(guān)性狀, 發(fā)現(xiàn)穗長(zhǎng)及節(jié)間縮短從而導(dǎo)致植株的矮化(圖2)。通過(guò)比較和野生型的其余9個(gè)主要農(nóng)藝性狀, 發(fā)現(xiàn)的株高、旗葉寬、穗粒數(shù)和千粒重均極顯著低于野生型, 而抽穗期、成熟期、旗葉面積、旗葉長(zhǎng)和小穗數(shù)差異不顯著(表2)。

2.3 種子活力及苗期性狀檢測(cè)

種子發(fā)芽率進(jìn)行統(tǒng)計(jì)發(fā)現(xiàn), 野生型的發(fā)芽率為98%, 而發(fā)芽率僅為76%。苗期地上部長(zhǎng)勢(shì)較弱, 地下部長(zhǎng)勢(shì)無(wú)顯著差異, 其中根數(shù)目、主根長(zhǎng)也無(wú)明顯差異(圖1-b和表2)。TTC染色結(jié)果表明, 野生型籽粒的胚著色較深, 而胚染色較淺, 說(shuō)明種子活力較弱(圖3)。

圖1 野生型與突變體RL1的表型鑒定

a、b: 分別代表WT與的抽穗期、苗期表型; a中, 標(biāo)尺為20 cm; b中, 標(biāo)尺為10 cm。c: 野生型與不同時(shí)期卷曲程度的比較, 左為野生型, 右為, 標(biāo)尺為1 cm。

a, b: plant phenotypes of WT andat heading stage and the seeding stage, respectively; Bar: 20 cm, in a; Bar: 10 cm, in b. c: the comparison of curl degree between wild type andat different stages, wild type on the left andon the right, Bar: 1 cm.

圖2 野生型與突變體RL1的株型比較

Fig. 2 Comparing of plant type between RL1 mutant and its wild type counterpart

a: WT與的莖稈, 標(biāo)尺為 20 cm; b、c: 成熟期WT與穗長(zhǎng)及各節(jié)間長(zhǎng)(I、II、III、IV), 標(biāo)尺為10 cm;*: 在< 0.05區(qū)間差異顯著,**: 在< 0.01差異極顯著。

a: stem of WT and, Bar: 20 cm; b and c: the length of spike and internodes of WT andat mature stage, Bar: 10 cm; *: significant difference at< 0.05 by-test; **: significant difference at< 0.01 by-test.

2.4 旗葉葉綠素和光合參數(shù)的測(cè)定分析

抽穗10 d后, 對(duì)的葉綠素含量和光合參數(shù)進(jìn)行測(cè)定。與野生型相比,的凈光合速率、蒸騰速率、氣孔導(dǎo)度、細(xì)胞間隙CO2濃度無(wú)顯著差異, 葉綠素含量基本一致。單位面積內(nèi)野生型和的葉綠素含量和光合能力差異不大, 但在自然生長(zhǎng)過(guò)程中過(guò)度卷曲, 葉片整體受光面積明顯小于野生型。此外, 葉片水分利用率數(shù)值表明,的葉片水分利用率極顯著降低(圖4)。

2.5 光照、溫度和干旱處理對(duì)突變體葉片卷曲的影響

正常生長(zhǎng)條件下的LRIs為0.10～0.31, 降低光照強(qiáng)度(400 μmol m–2s–1)和增加光照強(qiáng)度(1000 μmol m–2s–1)時(shí),的LRIs分別為0.10～0.28和0.12～0.32, 表明光照強(qiáng)度基本不影響葉片卷曲。與22℃培養(yǎng)條件下的葉片卷曲度(LRIs為0.10～0.31)相比, 在平均溫度10℃生長(zhǎng)條件下,葉片卷曲度增加(LRIs為0.24～0.36); 平均溫度30℃的培養(yǎng)條件也增加了葉片卷曲度(LRIs為0.32～ 0.38)。與正常澆水的葉片卷曲度(LRIs為0.10～0.31)相比, 干旱明顯增加了葉片的卷曲程度(LRIs為0.46～0.61)?？梢?jiàn), 低溫、高溫和干旱促進(jìn)的葉片卷曲。

圖3 野生型與突變體RL1成熟籽粒的TTC染色

a: 種胚著色圖, 標(biāo)尺為2 mm; b、c: 種胚放大圖, 標(biāo)尺為200 μm。

a: seed embryo staining, Bar: 2 mm; b, c: magnified diagrams of seed embryos, Bar: 200 μm.

圖4 野生型與突變體RL1的光合參數(shù)與SPAD的分析

a: 蒸騰速率; b: 氣孔導(dǎo)度; c: 凈光合速率; d: 細(xì)胞間隙CO2濃度; e: 葉綠素含量; f: 水分利用率。**:< 0.01差異極顯著。

a: transpiration rate; b: stomata conductance; c: net photosynthetic rate; d: the intercellular CO2concentration; e: chlorophyll content; f: water use efficiency. **: significant difference at< 0.01 by-test.

2.6 突變體RL1的組織學(xué)分析

為了進(jìn)一步了解葉片卷曲的機(jī)制, 對(duì)的葉片組織切片觀察發(fā)現(xiàn), 葉片泡狀細(xì)胞、近遠(yuǎn)軸面的細(xì)胞層均發(fā)生了變化。與野生型相比,具有較少的大葉脈和小葉脈。野生型中脈一側(cè)含有5～6個(gè)大葉脈和18～22個(gè)小葉脈, 而中僅有3～4個(gè)大葉脈和13～15個(gè)小葉脈(圖5-a)。另外,的中脈區(qū)域, 遠(yuǎn)軸面厚壁細(xì)胞和近軸面薄壁細(xì)胞數(shù)目減少(圖5-c, e)。維管束間泡狀細(xì)胞的面積和數(shù)量都顯著低于野生型, 發(fā)生卷曲部位的泡狀細(xì)胞, 以及葉片邊緣的泡狀細(xì)胞都顯著皺縮減少(圖5-b, d, f)。葉片各部位泡狀細(xì)胞縮小、維管束減少, 且葉肉細(xì)胞層數(shù)變薄, 導(dǎo)致整個(gè)葉片向近軸面極度卷曲。

2.7 突變體RL1的遺傳分析

分別與晉麥47和臨汾5064正反交得到的F1表型一致(附表2), 灌漿期的旗葉微卷, 介于野生型和突變體之間, 說(shuō)明為細(xì)胞核遺傳。在/晉麥47和/臨汾5064的F2群體中出現(xiàn)明顯的分離, 旗葉分別表現(xiàn)平展、微卷和高度卷曲, 經(jīng)卡方檢驗(yàn), 平展葉單株∶微卷葉單株(中間型)∶高度卷葉單株符合1∶2∶1分離比(表3), 表明卷葉性狀由1對(duì)不完全顯性的單基因控制。

2.8 突變體RL1基因定位

小麥660K SNP芯片結(jié)果表明, 親本及混池中有2170個(gè)多態(tài)性SNPs, 其中1115個(gè)分布在1DS上(51.38%), 表明決定卷葉形成有關(guān)基因可能在1D染色體上。通過(guò)計(jì)算每0.1 Mb的SNP數(shù)量, 初步將定位在62.82 Mb。利用8個(gè)高質(zhì)量的KASP標(biāo)記掃描320個(gè)F3分離家系, 根據(jù)目標(biāo)區(qū)段內(nèi)的重組家系, 將進(jìn)一步定位在標(biāo)記RLI和RLK間, 物理距離為9.42 Mb (圖6), 其中包含了66個(gè)高可信度注釋基因。

a: WT和的旗葉橫切圖, 標(biāo)尺為1000 μm; b、c: 為a圖的放大圖, 標(biāo)尺為 500 μm; d、e: 分別為b、c圖的放大圖, 標(biāo)尺為200 μm; f: 兩維管束之間的泡狀細(xì)胞的數(shù)量和面積。SC: 厚壁細(xì)胞; PC: 薄壁細(xì)胞; BC: 泡狀細(xì)胞; LV: 大葉脈; SV: 小葉脈; ad: 近軸面; ab:遠(yuǎn)軸面。**: 在0.01概率水平上差異顯著。

a: cross cutting diagrams of flag leaves in WT and, Bar: 1000 μm; b, c: magnified diagrams of picture a, Bar: 500 μm; d, e: magnification of figure b and c, respectively, Bar: 200 μm; f: the number and area of bulliform cells between two vein. SC: sclerenchymatous cell; PC: parenchyma cell; BC: bulliform cell; LV: large vein; SV: small vein; ad: adaxial; ab: abaxial. **: significant difference at< 0.01 by-test.

表3 F2代群體材料表型分離統(tǒng)計(jì)

χ2(0.05)(1)=3.84.

圖6 RL1的精細(xì)定位

3 討論

葉片卷曲作為一種植物抵抗非生物脅迫脫水回避的形態(tài)學(xué)策略, 可以減輕逆境帶來(lái)的損傷, 因此研究葉片卷曲有利于了解作物對(duì)響應(yīng)和抵御逆境脅迫的調(diào)控機(jī)制。根據(jù)植物葉片的近軸面和遠(yuǎn)軸面的極性發(fā)育, 可將卷葉性狀分為正向和反向卷曲; 依照植株葉位表現(xiàn), 可分為全株葉片卷曲和部分葉片卷曲; 從卷曲程度看, 有高度卷曲成筒狀的, 也有中度卷曲和微卷[22]。本文獲得的為全株正向卷曲, 新抽出的葉片微卷, 隨著葉片生長(zhǎng)沿著中軸向內(nèi)卷曲, 直至葉片內(nèi)卷成為近似圓筒狀, 苗期卷曲程度輕,抽穗后的旗葉、倒二葉卷曲程度增高, 而倒三、倒四葉依然為輕微卷曲, 且旗葉葉鞘也出現(xiàn)卷曲。已發(fā)現(xiàn)的水稻卷葉突變體大多長(zhǎng)勢(shì)較弱, 育性差, 如的莖稈變短, 種子干癟, 根細(xì)長(zhǎng), 根數(shù)量減少, 葉和穗變短小[23];株高變矮, 每穗粒數(shù)與結(jié)實(shí)率均下降, 且葉綠素含量和光合速率也顯著降低[24]。的株高、旗葉寬、千粒重分別降低了13.25%、27.81%和35.50%, 這與、等類似[25-26]; 但苗期根系與野生型相比無(wú)明顯差異, 單位面積內(nèi)葉綠素含量和光合特性影響較小, 因?yàn)楣夂献饔眯孰m不受影響, 但其葉片卷曲造成受光面積減小, 因而造成長(zhǎng)勢(shì)較弱。雖長(zhǎng)勢(shì)和育性較差, 但其光合作用并未受到太大影響, 與水稻等[24]不同, 在研究作物應(yīng)對(duì)外界脅迫響應(yīng)和平衡基礎(chǔ)代謝, 保證正常光合作用方面具有一定的研究利用價(jià)值。

禾本科葉片泡狀細(xì)胞分布在兩維管束之間, 位于葉片的近軸面, 一般只有4～5個(gè), 中間大兩邊小, 類似扇形, 是調(diào)節(jié)水分的薄壁細(xì)胞。已有研究報(bào)道, 卷葉基因可通過(guò)調(diào)控泡狀細(xì)胞的發(fā)育來(lái)控制葉片的卷曲, 借助大液泡內(nèi)水分的得失來(lái)調(diào)控葉片的平展和卷曲度, 從而改變?nèi)~片的光合作用和蒸騰作用[27]。泡狀細(xì)胞數(shù)量或體積的改變是調(diào)控葉片形態(tài)的關(guān)鍵因素, 增加泡狀細(xì)胞的數(shù)量或大小會(huì)導(dǎo)致葉片外卷, 減少泡狀細(xì)胞的數(shù)量或大小通常會(huì)導(dǎo)致葉片內(nèi)卷[28]。的過(guò)表達(dá)導(dǎo)致泡狀細(xì)胞增加,進(jìn)而引起葉片反向卷曲[29]。過(guò)表達(dá)植株葉片表現(xiàn)為正向卷曲[30], 而過(guò)表達(dá)葉片則為反向卷曲[6]。此外, 增加的表達(dá)量會(huì)引起泡狀細(xì)胞的縮小使得植株呈現(xiàn)出葉片正向卷曲的表型[31]。泡狀細(xì)胞數(shù)量和面積均減小, 維管束及大小葉脈數(shù)量也減少, 兩者共同作用引起葉片卷曲。

禾本科作物中卷葉基因克隆已有大量的研究報(bào)道。水稻葉片卷曲絕大多數(shù)為隱性性狀, 受1對(duì)或多基因控制, 如、、、、、、、、、等都是隱性基因[32-33]。普通小麥含有3個(gè)亞基因組, 性狀調(diào)控受基因冗余性影響表型分離不明顯, 增加了QTL和圖位克隆的難度。Zhu等[13]利用323份小麥材料通過(guò)全基因組關(guān)聯(lián)分析鑒定了一個(gè)調(diào)控卷葉的候選基因。Aakriti等[14]通過(guò)QTL在5D染色體上定位到1個(gè)卷葉候選基因, 該基因是水稻外卷葉基因的同源基因。Yang等[15]通過(guò)BSA結(jié)合660K芯片在7A染色體717.82～720.18 Mb間定位到一個(gè)卷葉候選基因, 與擬南芥中的一個(gè)氣孔發(fā)育調(diào)控因子同源。最近, EMS誘變卷葉突變體的1A和5A染色體上分別存在2個(gè)主效QTL, 其中被最終定位到6 Mb的范圍內(nèi)[16]。是連續(xù)自交得到的穩(wěn)定遺傳的卷葉突變體, 受環(huán)境影響小, 控制卷葉的主效基因定位在1DS染色體。水稻中克隆的卷葉基因有[6]、[28]、[10]、[31]、[34]、[19]、[35]、[36]、[24]、[37]、[29]、[38]、[39]、[40]、[41]、[7]、[5]、[8]、[25]、[42]、[43]、[26]、[30]、[44]、[45]、[46], 它們?cè)谛←溨械耐椿虿⑽闯霈F(xiàn)在功能區(qū)段中(表4), 因此可能是一個(gè)新的控制葉片卷曲的基因, 對(duì)其進(jìn)一步精細(xì)定位和功能研究有助于深入了解小麥卷葉“脫水回避”發(fā)育機(jī)制, 解析小麥對(duì)于抵御逆境脅迫的應(yīng)答調(diào)控。

表4 水稻葉片卷曲相關(guān)基因在小麥中的同源基因

(續(xù)表4)

附表1 42對(duì)SSR引物信息(國(guó)標(biāo): NY/T 2859–2015)

(續(xù)附表1)

附表2 RL1與晉麥47/臨汾5064正反交F1

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Physiological characteristics and genetic research of() in wheat (L.)

LIU Ye1,2,**, LI Yue1,**, YUAN Ming-Yang1, WEI Nai-Cui1, GUAN Pan-Feng3, ZHAO Jia-Jia1, WU Bang-Bang1, ZHENG Xing-Wei1, HAO Yu-Qiong1, QIAO Ling1,*, and ZHENG Jun1,*

1Institute of Wheat Research, Shanxi Agriculture University / Key Laboratory of Sustainable Dryland Agriculture (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Linfen 041000, Shanxi, China;2School of Life Science, Shanxi University, Taiyuan 030006, Shanxi, China;3School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, Henan, China

Wheat leaves tend to fold or curl when exposure to stresses, the dehydration avoidance in morphology can reduce the damage of abiotic stress. At present, the physiological and genetic regulation mechanism associated with leaf curling is not clear in wheat. This study reported a novel() from the ethyl methyl sulphonate (EMS) mutagenesis cultivar Jinmai 47. The leaves of the mutantwere curled during the growth period, and the primary leaves were slightly curled along axial vein to paraxial plane. Leaf curling was accelerated with the growth until the leaf was tubular. Compared to wild type (WT), plant height, ear length, flag leaf narrowing, and 1000-grain weight were decreased in mutant. Triphenyltetrazolium Chloride (TTC) staining showed that seed vigor ofwas low, together with the decreased germination rate by 22%. Additionally, there was no significant differences in chlorophyll content, net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO2concentration betweenand WT, while water utilization rate was decreased inafter heading for 10 days. Low temperature, high temperature, and drought led to the leaf rolling in.showed fewer leaf/lobular veins via paraffin section assay, and the number of abaxial sclerenchyma and adaxial parenchyma cells were reduced in midrib region of. Moreover, the area and counts of vesicular cells between the vascular bundles were significantly reduced in, together with the vesicular cells at the midvein region of leaves compared with WT. Vesicular cells and vascular bundles shrunk and decreased, respectively, resulting in the situation that the entire blade was extremely crimped to the adaxial plane. Genetic analysis demonstrated that the mutant trait was localized on the short arm of chromosome 1D, regulated by a pair of nuclear genes with incomplete dominance and fine mapping analysis further locked the target interval at 9.42 Mb.

wheat; rolled leaf mutant; cytological analysis; physiological characteristics; genetic analysis

2023-06-29;

2023-07-18

10.3724/SP.J.1006.2023.31004

通信作者(Corresponding author): 喬玲, E-mail: qiaolingsmile@163.com;鄭軍, E-mail: sxnkyzj@126.com

**同等貢獻(xiàn)(Contributed equally to this work)

劉葉, E-mail: liuye12345202@126.com

2023-01-09;

本研究由山西農(nóng)業(yè)大學(xué)省部共建有機(jī)旱作農(nóng)業(yè)國(guó)家重點(diǎn)實(shí)驗(yàn)室自主研發(fā)項(xiàng)目(202002-1)和山西省科技重大專項(xiàng)計(jì)劃“揭榜掛帥”項(xiàng)目(202201140601025-2-01)資助。

This study was supported by the Research Program Sponsored by State Key Laboratory of Integrative Sustainable Dryland Agriculture, Shanxi Agricultural University (202002-1) and the Science and Technology Major Special Plan Project “Reveal the Title” of Shanxi Province (202201140601025-2-01).

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This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).