水稻矮化劍葉卷曲突變體dcfl1的鑒定與基因精細(xì)定位

張孝波，謝佳，張曉瓊，田維江，何沛龍，劉思岑，何光華，鐘秉強(qiáng)，桑賢春

（西南大學(xué)水稻研究所/轉(zhuǎn)基因植物與安全控制重慶市重點(diǎn)實(shí)驗(yàn)室，重慶 400715）

水稻矮化劍葉卷曲突變體dcfl1的鑒定與基因精細(xì)定位

張孝波，謝佳，張曉瓊，田維江，何沛龍，劉思岑，何光華，鐘秉強(qiáng)，桑賢春

（西南大學(xué)水稻研究所/轉(zhuǎn)基因植物與安全控制重慶市重點(diǎn)實(shí)驗(yàn)室，重慶 400715）

【目的】對(duì)一個(gè)水稻矮化劍葉卷曲突變體進(jìn)行鑒定與基因定位，為水稻等禾谷類作物劍葉形態(tài)發(fā)育及分子改良奠定基礎(chǔ)?！痉椒ā吭诙i型水稻恢復(fù)系縉恢10號(hào)的甲基磺酸乙酯（EMS）突變庫中篩選到一個(gè)隱性矮化劍葉卷曲突變體，命名為dcfl1（dwarf and curled flag leaf 1）。田間小區(qū)種植，全生育期內(nèi)觀察dcfl1和野生型的株型變化。苗期利用掃描電鏡觀察葉鞘內(nèi)表皮細(xì)胞大?。辉兴肫诤统樗肫诶檬炃衅^察劍葉基部形態(tài)；開花期測(cè)定劍葉、倒2葉和倒3葉的葉綠素含量；成熟期考查株高、有效穗數(shù)、穗實(shí)粒數(shù)、結(jié)實(shí)率和千粒重等主要農(nóng)藝性狀。配制西農(nóng)1A/dcfl1雜交組合，利用F1和F2群體進(jìn)行遺傳分析，并利用F2隱性群體進(jìn)行基因定位。【結(jié)果】生育期內(nèi)，突變體dcfl1都表現(xiàn)出矮化性狀。dcfl1葉鞘內(nèi)表皮細(xì)胞長(zhǎng)度明顯比野生型要短，達(dá)到了極顯著水平。與野生型相比，穗長(zhǎng)、倒1節(jié)間和倒2節(jié)間均顯著變短，倒3節(jié)間和倒4節(jié)間無顯著變化。抽穗期dcfl1劍葉的葉片和葉鞘連接處硬化，劍葉基部展開受阻，半邊葉片向內(nèi)卷曲，劍葉上部和中部正常，其他葉片也正常。農(nóng)藝性狀調(diào)查發(fā)現(xiàn)，dcfl1的有效穗數(shù)為14.24，極顯著高于野生型的11.62，穗粒數(shù)、實(shí)粒數(shù)、結(jié)實(shí)率和千粒重等則無顯著變化。此外，dcfl1的葉色略深，劍葉、倒2葉和倒3葉的葉綠素a含量均極顯著高于野生型，類胡蘿卜素含量也略有升高，但僅劍葉達(dá)到極顯著差異水平，葉綠素b的含量則無顯著變化。西農(nóng)1A/dcfl1的F1群體中，株高和劍葉表型與野生型一致。F2群體中分離出正常和突變兩種表型，突變表型與dcfl1類似，植株株高變矮，劍葉基部特異卷曲，說明矮化和劍葉基部特異卷曲是一對(duì)共分離性狀。且兩種表型分離比符合3﹕1，表明dcfl1突變型受1對(duì)隱性核基因控制。利用620株F2隱性單株，最終將DCFL1精細(xì)定位在第3染色體短臂InDel標(biāo)記Ind03-11和Ind03-6之間78 kb的物理范圍內(nèi)，包含15個(gè)注釋基因，為DCFL1的克隆和水稻劍葉形態(tài)發(fā)育機(jī)理研究奠定了基礎(chǔ)?！窘Y(jié)論】dcfl1是一個(gè)水稻矮化劍葉基部特異卷曲突變體，基因精細(xì)定位在第3染色體78 kb的物理范圍內(nèi)。

水稻；矮化；劍葉卷曲；遺傳分析；精細(xì)定位

0 引言

【研究意義】葉片形態(tài)、大小和顏色直接決定了群體光能利用效率，進(jìn)而影響作物的產(chǎn)量和品質(zhì)，受到育種和分子生物學(xué)家的廣泛關(guān)注[1]。劍葉是水稻最重要的功能葉，葉片大小和形態(tài)與單株產(chǎn)量、單穗重、穗粒數(shù)均呈極顯著正相關(guān)，同時(shí)，劍葉葉型也是水稻理想株型的重要構(gòu)成部分[2-3]。因此，闡明劍葉發(fā)育的分子機(jī)理對(duì)水稻生產(chǎn)具有重要意義。【前人研究進(jìn)展】目前，利用突變體已在水稻中克隆了一系列調(diào)控葉片大小和形態(tài)發(fā)育的基因，如窄葉基因 NAL1[4]、NAL2/NAL3[5]、NAL7[6]，窄卷葉基因NRL1[7-8]，卷葉基因 CFL1[9]、SRL1[10]和 RL14[11]等，為闡釋水稻葉片發(fā)育的分子機(jī)理奠定了基礎(chǔ)。然而，這些突變體多表現(xiàn)為階段性或全生育期所有葉片發(fā)育缺陷，劍葉特異異常發(fā)育的突變體還鮮有報(bào)道。目前普遍認(rèn)為劍葉發(fā)育屬于復(fù)雜性狀，已利用 F2分離群體、雙單倍體（DH）、重組自交系（RILs）和單片段代換系（CSSLs）等材料，在水稻12條染色體上定位了系列控制劍葉大小的QTL。YAN等[12]利用IR64/Azucena衍生DH群體在第 4染色體上定位到一個(gè)控制劍葉大小的QTL。KOBAYASHI等[13]利用 Milyang23/Akihikari組合衍生的RIL群體（191個(gè)家系）定位到7個(gè)控制劍葉長(zhǎng)度和5個(gè)控制劍葉寬度的QTL。近來，BIAN等[14]利用CSSLs群體鑒定了4個(gè)控制水稻劍葉寬度、1個(gè)控制劍葉面積和 2個(gè)控制劍葉角度的 QTL；ZHANG等[3]則利用 RIL群體在海南和杭州兩地種植，檢測(cè)到9個(gè)控制劍葉長(zhǎng)度和14個(gè)控制劍葉寬度的QTL，其中僅7個(gè)為先前鑒定的QTL；CHEN等[15]利用 RIL群體也鑒定到 5個(gè)控制劍葉葉寬發(fā)育的QTL，并認(rèn)為主效QTL qFLW4是NAL1的同義突變，其通過選擇性剪切調(diào)控劍葉寬度的發(fā)育?！颈狙芯壳腥朦c(diǎn)】盡管目前已經(jīng)鑒定到大量控制劍葉發(fā)育的QTL，但這些研究主要集中在葉片大小上，對(duì)劍葉形態(tài)發(fā)育還鮮有報(bào)道。利用EMS誘變秈型水稻恢復(fù)系縉恢10號(hào)，從其后代鑒定到一個(gè)矮化，劍葉基部特異卷曲突變體，暫命名為dcfl1（dwarf and curled flag leaf 1）?！緮M解決的關(guān)鍵問題】本研究對(duì)突變體dcfl1進(jìn)行了形態(tài)鑒定、細(xì)胞學(xué)觀察和基因精細(xì)定位等研究，為DCFL1的克隆和功能研究奠定了基礎(chǔ)，有利于水稻劍葉形態(tài)發(fā)育的分子機(jī)理的闡釋。此外，劍葉基部卷曲導(dǎo)致葉片直立，有利于群體的通透性，dcfl1在水稻育種中也具有重要的應(yīng)用價(jià)值，是一類新型種質(zhì)資源。

1 材料與方法

1.1 試驗(yàn)材料

突變體 dcfl1來自西南大學(xué)水稻所培育的晚秈恢復(fù)系縉恢10號(hào)EMS誘變庫，經(jīng)過多世代連續(xù)種植，突變性狀已穩(wěn)定遺傳。配置西農(nóng)1A/ dcfl1雜交組合，調(diào)查西農(nóng)1A、dcfl1及其雜交組合F1和F2的株高及劍葉形態(tài)，進(jìn)行遺傳分析，利用F2群體隱性單株進(jìn)行基因定位。西農(nóng)1A是西南大學(xué)水稻所選育的不育系，整個(gè)生育期株高及葉片均正常。

1.2 主要農(nóng)藝性狀鑒定

田間小區(qū)種植dcfl1和野生型縉恢10號(hào)，成熟期分別測(cè)量10株材料的株高和節(jié)間長(zhǎng)并調(diào)查穗長(zhǎng)、有效穗數(shù)、穗粒數(shù)、穗實(shí)粒數(shù)、結(jié)實(shí)率和千粒重等主要農(nóng)藝性狀。

1.3 光合色素含量測(cè)定

開花期，參照文獻(xiàn)[16]描述的方法測(cè)定dcfl1和野生型的光合色素含量，測(cè)定部位為劍葉、倒2葉和倒3葉的葉片中部。

1.4 石蠟切片分析

孕穗期和抽穗期分別選取未全展的劍葉基部和全展劍葉基部，根據(jù)文獻(xiàn)[17]略有改動(dòng)。用FAA固定液固定后，依次進(jìn)行乙醇脫水、二甲苯透明、石蠟包埋、切片、番紅固綠染色等步驟。并在體視鏡下觀察葉片形態(tài)。

1.5 掃描電鏡觀察

田間種植三葉期秧苗，移至掃描電鏡室，利用日立 SU350型掃描電鏡在-20℃冷凍條件下觀察第二片葉的葉鞘內(nèi)表皮細(xì)胞。

1.6 DNA的提取

在西農(nóng)1A/ dcfl1的F2群體中分別選取正常和突變單株各10株，構(gòu)成正?；虺睾屯蛔兓虺?。采取改良的CTAB法[18]提取基因池DNA，采用堿煮法[19]提取定位群體單株基因組DNA。

1.7 SSR分析

RM系列SSR引物來源于http://www.gramene.org/網(wǎng)站。根據(jù)縉恢10號(hào)和西農(nóng)1A的DNA序列差異，利用Vector 10軟件設(shè)計(jì)InDel標(biāo)記。SSR引物及InDel標(biāo)記均由上海英俊生物公司合成。PCR反應(yīng)總體積12.5 μL，包括1.25 μL的10×PCR緩沖液、0.65 μL的25 mmol·L-1MgC12、0.5 μL 2.5 mmol·L-1dNTPs、8.0 μL的ddH2O、1.0 μL的10 μmol·L-1引物、1.0 μL的模板DNA和0.1 μL的5 U·μL-1Taq酶。PCR反應(yīng)程序?yàn)?4℃ 5 min；94℃ 20 s，55℃ 20 s，72℃ 20 s，35個(gè)循環(huán)； 72℃ 10 min。PCR產(chǎn)物用10%的非變性聚丙烯酰胺凝膠電泳后，0.1%AgNO3染色10 min，去離子水漂洗2次，1%質(zhì)量濃度的氫氧化鈉和0.1%甲醛混合液顯色，觀察照相。

1.8 遺傳圖譜構(gòu)建

西農(nóng) 1A/dcfl1雜交組合的 F2群體中，具有西農(nóng)1A帶型的單株記為A，具有dcfl1帶型的單株記為B，具有 F1雜合體帶型的單株記為 H。利用公式[(H+2A)/2n]×100%計(jì)算遺傳重組率，其中，H代表定位群體中雜合體帶型單株的數(shù)量，A代表正常株帶型單株的數(shù)量，n表示F2群體隱性單株總株數(shù)。

2 結(jié)果

2.1 dcfl1的形態(tài)鑒定

突變體dcfl1整個(gè)生育期都表現(xiàn)出矮化性狀。播種后5 d的dcfl1籽苗地上部長(zhǎng)度只有野生型的一半，二葉期也明顯矮于野生型。dcfl1的倒1節(jié)間、倒2節(jié)間和穗長(zhǎng)顯著短于野生型，進(jìn)而導(dǎo)致株高半矮化（圖1）。掃描電鏡觀察野生型和 dcfl1葉鞘內(nèi)表皮細(xì)胞，發(fā)現(xiàn)dcfl1的細(xì)胞長(zhǎng)度明顯比野生型短，達(dá)到了極顯著水平，而細(xì)胞寬度并無顯著差異（圖 2），暗示突變體的矮化性狀可能是由于細(xì)胞長(zhǎng)度變短而導(dǎo)致的。農(nóng)藝性狀分析（表1）發(fā)現(xiàn)，dcfl1的有效穗數(shù)為14.24，極顯著高于野生型的11.62，穗粒數(shù)、實(shí)粒數(shù)、結(jié)實(shí)率和千粒重等則無顯著變化。此外，dcfl1的葉片呈深綠色（圖1-D），光合色素測(cè)定表明劍葉、倒2葉和倒3葉的葉綠素a含量均極顯著增加，葉綠素b含量無顯著變化，進(jìn)而導(dǎo)致dcfl1的葉綠素a/b值極顯著增加；dcfl1的類胡蘿卜素含量雖略有升高，但僅劍葉中的含量極顯著高于野生型。

抽穗期dcfl1劍葉的葉片和葉鞘連接處硬化，葉基部展開受阻，半邊葉片向內(nèi)卷曲（圖3-A），而劍葉上部和中部無卷曲現(xiàn)象（圖3-B）。為進(jìn)一步觀察劍葉基部形態(tài)，將未展開的劍葉和全展開時(shí)期的劍葉基部進(jìn)行石蠟切片。發(fā)現(xiàn)在劍葉發(fā)育前期，野生型的劍葉有規(guī)律地向內(nèi)卷曲排列（圖3-C），而dcfl1的內(nèi)卷葉排列不規(guī)則（圖3-D）。劍葉全展開后，野生型的劍葉基部呈向外展開狀（圖3-E），而dcfl1的劍葉基部展開受阻，一半葉片呈向外展開狀，但內(nèi)卷葉始終無法向外展開（圖3-F），從而形成了劍葉基部半邊葉片向內(nèi)卷曲這一突變表型。盡管目前已經(jīng)報(bào)道了許多矮化突變體，但劍葉基部特異卷曲的水稻矮化突變體還沒有報(bào)道。

圖1 突變體dcfl1和野生型(WT)縉恢10號(hào)形態(tài)鑒定Fig. 1 Morphology identification of the dcfl1 and the wild type

表1 突變體dcfl1與野生型的農(nóng)藝性狀Table 1 Agronomic traits of dcfl1 mutant and the wild type

2.2 遺傳分析

西農(nóng) 1A/dcfl1雜交組合的 F1代植株劍葉基部均無卷曲現(xiàn)象，且株高正常。F2群體中分離出正常和突變2種類型，正常型1 940株，突變型620株，突變型植株也表現(xiàn)為劍葉基部卷曲和矮化現(xiàn)象，與dcfl1類似，表明劍葉基部卷曲和矮化是1對(duì)共分離性狀。χ2測(cè)驗(yàn)顯示正常植株與突變植株的分離比符合 3﹕1（χ2=0.79＜χ20.05=3.84），暗示dcfl1的突變性狀受1對(duì)隱性核基因控制。

圖2 突變體dcfl1和野生型(WT)縉恢10號(hào)細(xì)胞大小比較Fig. 2 Comparison of cell length and width between the dcfl1 and the wild type

2.3 基因定位

利用平均分布在水稻12條染色體上、在西農(nóng)1A和野生型縉恢10號(hào)間呈多態(tài)性的96對(duì)SSR標(biāo)記篩選正常基因池和突變基因池，發(fā)現(xiàn)第3染色體上的SSR標(biāo)記RM6297、RM14347、RM5474和RM5955在基因池間呈現(xiàn)多態(tài)性，暗示可能與 DCFL1連鎖。利用140個(gè) F2隱性單株進(jìn)行驗(yàn)證，確定了連鎖關(guān)系并將DCFL1初步限定在RM14347和RM5474之間，遺傳距離分別為4.32和3.81 cM。

為進(jìn)一步確定DCFL1的物理位置，在DCFL1初步定位區(qū)間內(nèi)設(shè)計(jì)開發(fā)了20對(duì)InDel標(biāo)記，多態(tài)性篩選發(fā)現(xiàn) 4對(duì)在親本之間具有多態(tài)性，分別命名為Ind03-8、Ind03-11、Ind03-6和Ind03-4（表2）。利用這些多態(tài)性標(biāo)記對(duì)620株西農(nóng)1A/dcfl1雜交組合的F2隱性單株進(jìn)行分析，結(jié)果表明，Ind03-8、Ind03-11、Ind03-6和Ind03-4的交換株分別為9、2、3和7個(gè)，且前 2個(gè)標(biāo)記的交換株不同于后者，從而將 DCFL1精細(xì)定位在InDel標(biāo)記Ind03-11和Ind03-6之間，物理距離約為78 kb（圖4）。

圖3 突變體dcfl1和野生型（WT）縉恢10號(hào)的劍葉形態(tài)Fig. 3 Flag leaf phenotype of the dcfl1 and the wild type

根據(jù)Gramene（http://ensembl.gramene.org/ Oryza_ sativa/Info/Index）和 Rice genome annotation project（http://rice.plantbiology.msu.edu/）提供的信息，發(fā)現(xiàn)在DCFL1精細(xì)定位區(qū)間內(nèi)，包含15個(gè)注釋基因（表3），5個(gè)編碼逆轉(zhuǎn)錄轉(zhuǎn)座子蛋白，6個(gè)編碼表達(dá)蛋白，4個(gè)為功能基因，分別編碼細(xì)胞色素P450蛋白、60S核糖體蛋白L21-2、β-淀粉酶和RNA基序識(shí)別蛋白。

表2 基因定位InDel引物Table 2 Primers of InDel markers for gene mapping

3 討論

圖4 DCFL1在水稻第3染色體上的分子定位Fig. 4 Molecular mapping of DCFL1 gene on rice chromosome 3

在水稻生產(chǎn)中，葉片適度卷曲有利于葉的挺直，從而改善了群體結(jié)構(gòu)、提高了光能利用率，在水稻高產(chǎn)育種中具有重要的應(yīng)用價(jià)值。因此，揭示水稻葉片卷曲的遺傳機(jī)制不僅有利于葉片發(fā)育的分子機(jī)理闡釋，也為水稻株型育種提供了基礎(chǔ)材料和理論支撐。目前，在水稻中至少報(bào)道了20多份卷葉突變體，這些突變體的形成多受植物葉片極性建成、泡狀細(xì)胞大小和數(shù)量變化以及環(huán)境因素的影響。蔥狀卷曲突變體sll1[20]是由于葉片遠(yuǎn)軸面厚壁組織細(xì)胞發(fā)育異常而表現(xiàn)出卷曲。位于第3染色體上的突變體srl2[21]表現(xiàn)出葉片半卷，葉片變窄，株高降低。類似于 sll1，srl2也是由于遠(yuǎn)軸面厚壁組織細(xì)胞異常導(dǎo)致葉片卷曲。突變體adl1[22]表現(xiàn)為下表皮泡狀細(xì)胞異位發(fā)育，從而導(dǎo)致葉片向遠(yuǎn)軸面卷曲。另一個(gè)外卷突變體oul1[23]來源于水稻最外層細(xì)胞特異基因 Roc5的敲除，表現(xiàn)出近軸側(cè)泡狀細(xì)胞體積變大而引起葉片外卷。SRL1編碼糖基磷脂酰肌醇固定蛋白，調(diào)控葉片上表皮泡狀細(xì)胞數(shù)量從而控制葉片卷曲[10]。突變體rl14[11]卷葉表型是由于近軸面泡狀細(xì)胞萎縮引起，RL14通過調(diào)控次生細(xì)胞壁組分合成從而影響葉片水分運(yùn)輸，并進(jìn)一步影響泡狀細(xì)胞形態(tài)。環(huán)境誘導(dǎo)型卷葉突變體rl15(t)葉片卷曲行為受環(huán)境誘導(dǎo)，濕度是誘導(dǎo)突變體卷曲的主要因素[25]。值得注意的是突變體cfl1[9]，由于劍葉卷曲，所以該突變體被命名為 curly flag leaf1，但其他葉片也受到影響，因此，cfl1也屬于所有葉片全卷突變體。此外，rl12(t)葉片卷曲特性隨著發(fā)育進(jìn)程而發(fā)生變化，卷曲表型主要發(fā)生在葉片中上部1/3處，中下部正常，劍葉亦是如此[24]。本文報(bào)道的 dcfl1突變體，僅劍葉基部卷曲，明顯不同于已報(bào)道的水稻卷葉突變體，也不同于劍葉發(fā)育缺陷相關(guān)突變體，因此，dcfl1是一類新型劍葉基部特異卷曲突變體。

表3 定位區(qū)間內(nèi)注釋基因Table 3 Annotated genes of the gene mapping range

目前報(bào)道的葉片發(fā)育調(diào)控基因，多具有“一因多效”性。如，Ghd7調(diào)控抽穗期和產(chǎn)量性狀，同時(shí)也影響劍葉的葉面積[26-27]；dtl1是一個(gè)矮化突變體，同時(shí)也表現(xiàn)葉片卷曲、分蘗減少和不育等性狀[28]。dcf1并不是傳統(tǒng)的葉卷曲突變體，只是劍葉基部特異卷曲，劍葉中上部和其他葉片均正常，因此它的育性并沒有受影響。另外，盡管dcfl1的穗長(zhǎng)變短了，但dcfl1的穗型更緊湊，使dcfl1穗粒數(shù)無明顯變化，而且有效穗增加了，從而暗示該突變體的產(chǎn)量有所提高。突變體dcfl1除劍葉特異卷曲外，還表現(xiàn)植株的矮化和葉片顏色深綠。這可能是由于 DCFL1的多效性造成的，引起這些突變表型的原因還有待進(jìn)一步研究。

利用SSR等分子標(biāo)記最終將DCFL1定位在第3染色體InDel標(biāo)記Ind03-11和Ind03-6之間78 kb物理距離內(nèi)，包含15個(gè)注釋基因，其中4個(gè)為功能基因。細(xì)胞色素 P450編碼基因已克隆，可能通過脂類代謝途徑調(diào)控細(xì)胞伸長(zhǎng)，其突變體oscyp96b4主要表現(xiàn)為植株半矮化和育性降低。它的矮化表型并不受激素的調(diào)控，而是通過轉(zhuǎn)錄劑量的方式來降低水稻株高[29]。ZHANG等[30]鑒定到一個(gè) oscyp96b4的等位突變體sd37，除全生育期植株矮化外，還表現(xiàn)稻穗和花軸變短，籽粒變小。另一個(gè)等位突變體dss1萌發(fā)以及早期生長(zhǎng)均延遲，它的矮化表型也不受外源激素影響。但內(nèi)源ABA的積累和GA的缺陷可能是dss1矮化的原因，而且其耐旱性增強(qiáng)[31]。此外，WANG等[32]鑒定了oscyp96b4第三個(gè)等位突變體bsh1，其表現(xiàn)出株高、千粒重以及每株產(chǎn)量均顯著降低，葉鞘角質(zhì)層蠟質(zhì)含量降低，暗示BSH1可能參與蠟質(zhì)生物合成。其他3個(gè)基因則沒有克?。害?淀粉酶是一個(gè)非生物脅迫蛋白，響應(yīng)磷和鉀的缺失[33-34]；60S核糖體蛋白L21-2編碼基因在不育系和保持系之間具有表達(dá)差異[35]；RNA基序識(shí)別蛋白則尚沒有描述。從表型和定位結(jié)果推測(cè)，DCFL1可能是一個(gè)調(diào)控劍葉發(fā)育的新基因。

4 結(jié)論

EMS誘變獲得一個(gè)矮化和劍葉基部卷曲的新型水稻突變體dcfl1，其表現(xiàn)為分蘗數(shù)增多、葉色深綠。產(chǎn)量性狀除有效穗極顯著升高外，其他無明顯變化。dcfl1的葉綠素a含量極顯著高于野生型，導(dǎo)致葉綠素a/b比值極顯著增加。劍葉基部卷曲和植株矮化受同1對(duì)隱性核基因調(diào)控，利用西農(nóng)1A和dcfl1雜交組合的F2分離群體，最終將DCFL1定位在水稻第3染色體InDel標(biāo)記Ind03-11和Ind03-6之間78 kb的物理距離內(nèi)，包含15個(gè)注釋基因。

[1] 徐靜, 王莉, 錢前, 張光恒. 水稻葉片形態(tài)建成分子調(diào)控機(jī)制研究進(jìn)展. 作物學(xué)報(bào), 2013, 39(5): 767-774.

XU J, WANG L, QIAN Q, ZHANG G H. Research advance in molecule regulation mechanism of leaf morphogenesis in rice (Oryza sativa L.). Acta Agronomica Sinica, 2013, 39(5): 767-774. (in Chinese)

[2] YUE B, XUE W Y, LUO L J, XING Y Z. QTL analysis for flag leaf characteristics and their relationships with yield and yield traits in rice. Acta Genetica Sinica, 2006, 33(9): 824-832.

[3] ZHANG B, YE W J, REN D Y, TIAN P, PENG Y L, GAO Y, RUAN B P, WANG L, ZHANG G H, GUO L B, QIAN Q, GAO Z Y. Genetic analysis of flag leaf size and candidate genes determination of a major QTL for flag leaf width in rice. Rice, 2015, 8: 2.

[4] QI J, QIAN Q, BU Q Y, LI S Y, CHEN Q, SUN J Q, LIANG W X, ZHOU Y H, CHU C C, LI X G, REN F G, PALME K, ZHAO B R, CHEN J F, CHEN M S, LI C Y. Mutation of rice Narrow leaf1 gene, which encodes a novel protein, affects vein patterning and polar auxin transport. Plant Physiology, 2008, 147(4): 1947-1959.

[5] CHO S H, YOO S C, ZHANG H, PANDEYA D, KOH H J, HWANG J Y, KIM G T, PAEK N C. The rice narrow leaf2 and narrow leaf3 loci encode WUSCHEL related homeobox 3A (OsWOX3A) and function in leaf, spikelet, tiller and lateral root development. New Phytologist, 2013, 198(4): 1071-1084.

[6] FUJINO K, MATSUDA Y, OZAWA K, NISHIMURA T, KOSHIBA T, W. FRAAIJE M, SEKIGUCHI H. NARROW LEAF 7 controls leaf shape mediated by auxin in rice. Molecular Genetic Genome, 2008, 279(5): 499-507.

[7] HU J, ZHU L, ZENG D, GAO Z, GUO L, FANG Y, ZHANG G, DONG G, YAN M, LIU J, QIAN Q. Identification and characterization of NARROW AND ROLLED LEAF 1, a novel gene regulating leaf morphology and plant architecture in rice. Plant Molecular Biology, 2010, 73(3): 283-292.

[8] WU C, FU Y P, HU G C, SI H M, CHENG S H, LIU W Z. Isolation and characterization of a rice mutant with narrow and rolled leaves. Planta, 2010, 232(2): 313-324.

[9] WU R H, LI S B, HE S, WA?MANN F, YU C H, QIN G J, SCHREIBER L, QU L J, GU H Y. CFL1, a WW domain protein, regulates cuticle development by modulating the function of HDG1, a class IV homeodomain transcription factor, in rice and Arabidopsis. The Plant Cell, 2011, 23(9): 3392-3411.

[10] XIANG J J, ZHANG G H, QIAN Q, XUE H W. Semi-rolled leaf1 encodes a putative glycosylphosphatidylinositol-anchored protein and modulates rice leaf rolling by regulating the formation of bulliform cells. Plant Physiology, 2012, 159(4): 1488-1500.

[11] FANG L K, ZHAO F M, CONG Y F, SANG X C, DU Q, WANG D Z, LI Y F, LING Y H, YANG Z L, HE G H. Rolling-Leaf14 is a 2OG-Fe (II) oxygenase family protein of unknown function that modulates rice leaf rolling by affecting secondary cell wall formation in leaves. Plant Biotechnology Journal, 2012, 10(5): 524-532.

[12] YAN J P, ZHU J, HE C X, BENMOUSSA M, WU P. Molecular marker-assisted dissection of genotype× environment interaction for plant type traits in rice (Oryza sativa L.). Crop Science, 1999, 39(2): 538-544.

[13] KOBAYASHI S, FUKUTA Y, MORITA S, SATO T, OSAKI M, KHUSH G S. Quantitative trait loci affecting flag leaf development in rice (Oryza sativa L.). Breed Science, 2003, 53(3): 255-262.

[14] BIAN J M, HE H H, SHI H, ZHU G Q, LI C J, ZHU C L, PENG X S, YU Q Y, FU J R, HE X P, CHEN X R, HU L F, OUYANG L J. Quantitative trait loci mapping for flag leaf traits in rice using a chromosome segment substitution line population. Plant Breeding, 2014, 133(2): 203-209.

[15] CHEN M L, LUO J, SHAO G N, WEI X J, TANG S Q, SHENG Z H, SONG J, HU P S. Fine mapping of a major QTL for flag leaf width in rice, qFLW4, which might be caused by alternative splicing of NAL1. Plant Cell Reports, 2012, 31(5): 863-872.

[16] LICHTENTHALER H K. Hlorophylls and carotenoids pigments of photosynthetic biomembranes. Methods in Enzymology, 1987, 48: 350-382.

[17] SANG X C, LI Y F, LUO Z K, WANG N, LING Y H, ZHAO H M, YANG Z L, LUO H F, LIU Y S, HE G H. CHIMERIC FLORAL ORGANS 1, encoding a monocot-specific MADS box protein, regulates floral organ identity in rice. Plant Physiology, 2012, 160(2): 788-807.

[18] MURRAY M G, THOMPSON W F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 1980, 8(19): 4321-4325.

[19] 桑賢春, 何光華, 張毅, 楊正林, 裴炎. 水稻PCR擴(kuò)增模板的快速制備. 遺傳, 2003, 25(6): 705-707.

SANG X C, HE G H, ZHANG Y, YANG Z L, PEI Y. The simple gain of templates of rice genomes DNA for PCR. Hereditas, 2003, 25(6): 705-707. (in Chinese)

[20] ZHANG G H, XU Q, ZHU X D, QIAN Q, XUE H W. SHALLOT-LIKE1 is a KANADI transcription factor that modulates rice leaf rolling by regulating leaf abaxial cell development. The Plant Cell, 2009, 21(3): 719-735.

[21] LIU X F, LI M, LIU K, TANG D, SUN M F, LI Y F, SHEN Y, DU G J, CHENG Z K. Semi-Rolled Leaf 2 modulates rice leaf rolling by regulating abaxial side cell differentiation. Journal of Experimental Botany, 2016, 67(8): 2139-2150.

[22] HIBARA K, OBARA M, HAYASHIDA E, ABE M, ISHIMARU T, SATOH H, ITOH J, NAGATO Y. The ADAXIALIZED LEAF 1 gene functions in leaf and embryonic pattern formation in rice. Developmental Biology, 2009, 334(2): 345-354.

[23] ZOU L P, SUN X H, ZHANG Z G, LIU P, WU J X, TIAN C J, QIU J L, LU T G. Leaf rolling controlled by the homeodomain leucine zipper class IV gene Roc5 in rice. Plant Physiology, 2011, 156(3): 1589-1602.

[24] 羅遠(yuǎn)章, 趙芳明, 桑賢春, 凌英華, 楊正林, 何光華. 水稻新型卷葉突變體 rl12(t)的遺傳分析和基因定位. 作物學(xué)報(bào), 2009, 35(11): 1967-1972.

LUO Y Z, ZHAO F M, SANG X C, LING Y H, YANG Z L, HE G H. Genetic analysis and gene mapping of a novel rolled leaf mutant rl12(t) in rice. Acta Agronomica Sinica, 2009, 35(11): 1967-1972. (in Chinese)

[25] 張禮霞, 劉合芹, 于新, 王林友, 范宏環(huán), 金慶生, 王建軍. 水稻卷葉突變體 rl15(t)的生理學(xué)分析及基因定位. 中國農(nóng)業(yè)科學(xué), 2014, 47(14): 2881-2888.

ZHANG L X, LIU H Q, YU X, WANG L Y, FAN H H, JIN Q S, WANG J J. Molecular mapping and physiological characterization of a novel mutant rl15(t) in rice. Scientia Agricultura Sinica, 2014, 47(14): 2881-2888. (in Chinese)

[26] XUE W Y, XING Y Z, WENG X Y, ZHAO Y, TANG W J, WANG L, ZHOU H J, YU S B, XU C G, LI X H, ZHANG Q F. Natural variation in Ghd7 is an important regulator of heading date and yield potential in rice. Nature Genetics, 2008, 40(6): 761-767.

[27] 談聰, 翁小煜, 鄢文豪, 白旭峰, 邢永忠. 多效性基因Ghd7調(diào)控水稻劍葉面積. 遺傳, 2012, 34(7): 901-906.

TAN C, WENG X Y, YAN W H, BAI X F, XING Y Z. Ghd7, a pleiotropic gene controlling flag leaf area in rice. Hereditas, 2012, 34(7): 901-906. (in Chinese)

[28] 張帆濤, 方軍, 孫昌輝, 李潤寶, 羅向東, 謝建坤, 鄧曉建, 儲(chǔ)成才. 水稻矮稈突變體dtl1的分離鑒定及其突變基因的精細(xì)定位. 遺傳, 2012, 34(1): 79-86.

ZHANG F T, FANG J, SUN C H, LI R B, LUO X D, XIE J K, DENG X J, CHU C C. Characterization of a rice dwarf and twist leaf 1 (dtl1) mutant and fine mapping of DTL1 gene. Hereditas, 2012, 34(1): 79-86. (in Chinese)

[29] RAMAMOORTHY R, JIANG S Y, RAMACHANDRAN S. Oryza sativa cytochrome P450 family member OsCYP96B4 reduces plant height in a transcript dosage dependent manner. PLoS ONE, 2011, 6(11): e28069.

[30] ZHANG J, LIU X Q, LI S Y, CHENG Z K, LI C Y. The rice semi-dwarf mutant sd37, caused by a mutation in CYP96B4, plays an important role in the fine-tuning of plant growth. PLoS ONE, 2014, 9(2): e88068.

[31] TAMIRU M, UNDAN J R, TAKAGI H, ABE A, YOSHIDA K, UNDAN J Q, NATSUME S, UEMURA A, SAITOH H, MATSUMURA H, URASAKI N, YOKOTA T, TERAUCHI R. A cytochrome P450, OsDSS1, is involved in growth and drought sress responses in rice (Oryza sativa L.). Plant Moecular Biology, 2015, 88(1): 85-99.

[32] WANG X L, CHENG Z J, ZHAO Z C, GAN L, QIN R Z, ZHOU K N, MA W W, ZHANG B C, WANG J L, ZHAI H Q, WAN J M. BRITTLE SHEATH1 encoding OsCYP96B4 is involed in secondary cell wall formation in rice. Plant Cell Reports, 2016, 35: 745-755.

[33] PARK M R, BAEK S H, DE LOS REYES B G, YUN S J, HASENSTEIN K H. Transcriptome profiling characterizes phosphate deficiency effects on carbohydrate metabolism in rice leaves. Journal of Plant Physiology, 2012, 169(2): 193-205.

[34] SHANKAR A, SINGH A, KANWAR P, SRIVASTAVA A K, PANDEY A, SUPRASANNA P, KAPOOR S, PANDEY G K. Gene expression analysis of rice seedling under potassium deprivation reveals major changes in metabolism and signaling components. PLoS ONE, 2013, 8(7): e70321.

[35] YANA J J, TIAN H, WANG S Z, SHAO J Z, ZHENG Y Z, ZHANG H Y, GUO L, DING Y. Pollen developmental defects in ZD-CMS rice line explored by cytological, molecular and proteomic approaches. Journal of Proteomics, 2014, 108: 110-123.

（責(zé)任編輯 李莉）

Identification and Gene Mapping of a Dwarf and Curled Flag Leaf Mutant dcfl1 in Rice (Oryza sativa L.)

ZHANG XiaoBo, XIE Jia, ZHANG XiaoQiong, TIAN WeiJiang, HE PeiLong, LIU SiCen, HE GuangHua, ZHONG BingQiang, SANG XianChun
(Rice Research Institute of Southwest University/Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crops, Chongqing 400715)

【Objective】Leaf blade is an important factor of plant type, which is directly related to leaf photosynthetic area and light energy utilization. Flag leaf is most prominently in the formation of rice production. Study of the genes which regulate flag leaf development in rice is of very significance in rice functional genomics research and molecular breeding. A novel flag leaf mutant has been identified and the results of study will provide a foundation for the research of leaf morphological formation and plant type breeding in Oryza sativa L.【Method】A dwarf and curled flag leaf mutant (dcfl1) was discovered from the progeny of indica restorerline Jinhui10 with seeds treated by ethyl methane sulfonate (EMS) and the traits of dwarf and curled flag leaf base inherited steadily after multi-generations’ self-fertility. The second leaf sheath was observed by scanning electron microscopy (SEM) at the three-leaf stage. The flag leaf base was used for paraffin section at the booting and heading stages. At the blooming stage, the characteristics of chloroplast pigment of the flag, second and third leaf blades were measured. At the maturity stage, agronomic traits such as plant height, panicle length, efficient panicle per plant, seed number per panicle, filled grain number per panicle, seed setting ratio, and 1000-seed weight were measured. The dcfl1 was crossed with indica sterile line Xinong 1A, and the F1and F2generations were used for genetic analysis. Additionally, gene mapping was performed based on the recessive individuals of the F2generation of Xinong 1A/ dcfl1.【Result】The dcfl1 was dwarf in all phases of plant development. The cell length of the 2nd leaf sheath surface of the dcfl1 was significantly shorter than the wild type. The panicle length, the first and the second internode of the dcfl1 were all significantly shorter than those of the wild type. The dcfl1 displayed a severe curl at the base of flag leaf blade after the heading stage, while the upper of flag leaf blade was nearly normal in the flag leaf. Meanwhile, the other leaf blades appeared as normal as the wild type. No significant differences were detected in grain number per panicle, filled grain number per panicle, seed setting rate and 1000-seed weight between the dcfl1 and the wild type. However, the number of the tiller in the dcfl1 was more than the wild type and the efficient panicle per plant was increased significantly than the wild type. Having the dark green leaves, the contents of chlorophyll a and total chlorophyll in the dcfl1 increased significantly compared with those of the wild type for the flag leaves, the second and the third leaves. Genetic analysis indicated that the dwarf and curled flag leaf traits of dcfl1 were controlled by a recessive nuclear gene. Based on the F2population derived from a cross between the dcfl1 and an indica sterile line, Xinong 1A, the gene was fine mapped on chromosome 3 between InDel marker Ind03-11 and Ind03-6 with the physical distance 78 kb, containing fifteen annotated genes.【Conclusion】The dcfl1 is a novel recessive dwarf and curled flag leaf mutant coming from EMS-inducement. The DCFL1 was mapped on chromosome 3 with 78 kb physical distance. These results will provide a foundation for map-based cloning of DCFL1 gene and understanding of the molecular mechanism of the rice flag leaf.

rice (Oryza sativ L.); dwarf; curled flag leaf; genetic analysis; gene mapping

2016-11-09；接受日期：2017-01-04

中央高?；究蒲袠I(yè)務(wù)費(fèi)項(xiàng)目（XDJK2013A023）、重慶市研究生科研創(chuàng)新項(xiàng)目（CYS14043）

聯(lián)系方式：張孝波，E-mail：zhangxiaobo@163.com。通信作者桑賢春，E-mail：sangxianchun@163.com