韋荔全 羅延敏 王文強(qiáng) 池長(zhǎng)程 黃福燈 向 珣 程方民 潘 剛,*

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水稻斑點(diǎn)葉突變體spl的生理特性及其基因定位

韋荔全1,**羅延敏1,**王文強(qiáng)1池長(zhǎng)程1黃福燈2向 珣1程方民1潘 剛1,*

1浙江大學(xué)農(nóng)業(yè)與生物技術(shù)學(xué)院, 浙江杭州310058;2浙江省農(nóng)業(yè)科學(xué)院作物與核技術(shù)利用研究所, 浙江杭州310021

通過(guò)EMS誘變秈稻恢復(fù)系珍97獲得一個(gè)穩(wěn)定遺傳的褐色斑點(diǎn)葉突變體spl(ottedeaf, spl)。大田條件下, 突變體spl的斑點(diǎn)葉性狀始于分蘗期, 此后由葉緣向葉中下部迅速擴(kuò)散, 直至整個(gè)葉片, 嚴(yán)重時(shí)葉片部分或整體枯死, 從而致使突變體株高、每穗粒數(shù)及結(jié)實(shí)率極顯著低于野生型對(duì)照。生理分析表明, 與野生型珍97相比, 孕穗期突變體spl劍葉、倒二葉和倒三葉的葉綠素含量極顯著降低, 而POD (peroxidase, POD)活性、O2?含量及MDA (malondialdehyde, MDA)含量升高; 突變體spl倒二葉和倒三葉的CAT (catalase, CAT)活性和可溶性蛋白含量極顯著降低, 而SOD (superoxide dismutase, SOD)活性則極顯著增加。組織化學(xué)分析進(jìn)一步證實(shí), 突變體spl的葉片明顯累積O2?。此外, 突變體spl苗期經(jīng)鹽脅迫處理后, 其株高及根長(zhǎng)明顯受到抑制。遺傳分析表明, 突變體spl的斑點(diǎn)葉性狀受一對(duì)隱性核基因控制, 借助圖位克隆技術(shù)將該基因定位于第12染色體長(zhǎng)臂的RM28466與RM28485兩個(gè)SSR標(biāo)記之間, 物理距離為189 kb, 該結(jié)果為進(jìn)一步克隆SPL基因并研究其功能奠定了基礎(chǔ)。

水稻;spl; 斑點(diǎn)葉; 生理特性; 基因定位

植物斑點(diǎn)葉(spotted-leaf)、類病變(lesion mimic)或類病斑(lesion simulating disease)突變體是指突變體植物在沒(méi)有受到明顯逆境、機(jī)械和農(nóng)藥損傷或病原菌侵染的條件下, 植物葉片、葉鞘甚至莖稈等部位自發(fā)形成類病原菌侵染或類過(guò)敏反應(yīng)的程序性細(xì)胞死亡現(xiàn)象[1-2]。研究表明, 斑點(diǎn)葉突變體還與植物抗病防衛(wèi)反應(yīng)密切相關(guān)[3]。因此, 斑點(diǎn)葉突變體是研究植物程序性細(xì)胞死亡及病害響應(yīng)機(jī)理的理想材料[2-3]。

關(guān)于斑點(diǎn)葉的形成機(jī)理, 基于不同突變體的分子生理生化研究, 認(rèn)為斑點(diǎn)葉的形成不僅受外因, 如光[4]和溫度[5]的誘導(dǎo); 更受內(nèi)因, 如激素和ROS (reactive oxygen species, ROS)等信號(hào)因子[6-8], 以及基因的精細(xì)調(diào)控[7-8]。利用正向和反向遺傳學(xué)手段, 迄今已從水稻中克隆并經(jīng)遺傳轉(zhuǎn)化或T-DNA插入突變體驗(yàn)證的葉片類病變形成的相關(guān)基因有18個(gè), 根據(jù)基因功能可以將其分成6類, 包括轉(zhuǎn)錄因子, 如[9]和[10]; 蛋白酶或激酶基因, 如/[11][5]; 參與代謝加工降解的基因, 如[12]、/[13]、/[14]、[15]、/[16]、[17]、[18]、[19]等; 物質(zhì)轉(zhuǎn)運(yùn)相關(guān)基因, 如[20]、[21]、[22]等; 蛋白或RNA結(jié)合蛋白基因, 如[23]和[24]; 生物合成相關(guān)基因, 如[4]。

本課題組利用EMS誘變秈稻恢復(fù)系珍97, 獲得一個(gè)隱性斑點(diǎn)葉突變體, 暫命名為spl(spotted leaf Z97, spl)。該突變體斑點(diǎn)葉表型始于水稻分蘗期(約6~7葉期), 之后隨著葉齡的增加, 除上部1~2片展開(kāi)葉及心葉外, 其他葉片均不同程度出現(xiàn)斑點(diǎn)葉性狀; 抽穗開(kāi)花期除劍葉外所有葉片均出現(xiàn)斑點(diǎn)葉性狀。本文對(duì)突變體spl基本表型、生理變化及基因定位等方面的研究, 為進(jìn)一步克隆該基因并揭示其斑點(diǎn)葉分子生理機(jī)理奠定了基礎(chǔ)。

1 材料與方法

1.1 試驗(yàn)材料

EMS誘變秈稻恢復(fù)系珍97, 從誘變后代中獲得斑點(diǎn)葉突變體spl, 經(jīng)浙江杭州和海南連續(xù)6代回交和自交, 獲得斑點(diǎn)葉性狀穩(wěn)定株系。之后以spl為母本, 分別與原始野生型對(duì)照珍97、粳稻品種02428和秀水110雜交獲得F1, F1自交獲得的F2群體用于遺傳分析及基因定位。所有材料均種植于浙江大學(xué)紫金港農(nóng)業(yè)試驗(yàn)站。2014年和2015年在水稻成熟后, 分別取spl與珍97各20株, 調(diào)查其株高、穗長(zhǎng)、有效穗數(shù)、每穗粒數(shù)、結(jié)實(shí)率和千粒重等主要農(nóng)藝性狀。2015年在水稻生長(zhǎng)至孕穗期, 取突變體spl及其野生型珍97的劍葉、倒二葉和倒三葉, 測(cè)定有關(guān)生理指標(biāo); 同時(shí), 對(duì)F1及其F2群體分單株觀察其斑點(diǎn)葉表型并取樣, 提取DNA用于后續(xù)基因定位分析。

1.2 鹽處理

選取突變體spl及其野生型對(duì)照珍97的成熟種子并脫殼, 經(jīng)75%酒精消毒1 min及10%次氯酸鈉溶液消毒20 min, 之后滅菌水洗滌5次, 每次2 min。將消毒好的種子接種在含0、100和200 mmol L–1NaCl的1/2 MS[25]固體培養(yǎng)基上, 于水稻組織培養(yǎng)室培養(yǎng)6 d后統(tǒng)計(jì)根長(zhǎng)及株高并拍照。培養(yǎng)條件為光照16 h, 黑暗8 h, 溫度為28℃。

1.3 生理指標(biāo)測(cè)定

選取突變體spl及其野生型對(duì)照珍97各10株處于孕穗期(劍葉葉枕與倒二葉葉枕平齊的分蘗)的劍葉、倒二葉和倒三葉, 分別測(cè)定葉綠素、可溶性蛋白、過(guò)氧化氫(H2O2)、超氧陰離子(O2?)、MDA含量以及CAT、SOD和POD活性。用80%丙酮于黑暗條件下浸泡葉片至發(fā)白, 將浸提液稀釋5倍, 以80%丙酮為空白, 在波長(zhǎng)663 nm和646 nm下測(cè)定其光密度, 計(jì)算葉綠素含量; 采用考馬斯亮藍(lán)G-250染色法測(cè)定可溶性蛋白; 采用碧云天生物技術(shù)有限公司的過(guò)氧化氫檢測(cè)試劑盒(s0038)測(cè)定H2O2含量; 參考《植物生理學(xué)實(shí)驗(yàn)技術(shù)》的相關(guān)方法測(cè)定其他生理指標(biāo)[26]。采用日本島津公司UV-2450紫外分光光度計(jì), 每個(gè)指標(biāo)測(cè)定5個(gè)生物學(xué)重復(fù)。

1.4 細(xì)胞組織化學(xué)染色

選取分蘗盛期突變體spl及其野生型對(duì)照珍97主莖倒二葉中上部3~5 cm長(zhǎng)葉片, 分別參考Kariola等[27]和Mahalingam等[28]的方法進(jìn)行NBT及DAB染色。每個(gè)試驗(yàn)重復(fù)3次。

1.5 基因遺傳分析與定位

大田栽培條件下, 首先分別觀察spl/珍97、spl/02428和spl/秀水110的雜種F1葉片表型, 確定控制突變體spl斑點(diǎn)葉性狀的顯隱性; 其次, 根據(jù)F2群體中具有正常葉色的水稻單株數(shù)與具有斑點(diǎn)葉表型的單株數(shù)的比例, 確定控制斑點(diǎn)葉性狀的基因數(shù)量。同時(shí), 分單株剪取spl/02428的F2定位群體中具有斑點(diǎn)葉性狀的單株、spl、02428以及F2群體中10株正常植株的葉片, 采用CTAB法提取基因組總DNA。利用均勻分布于水稻12條染色體的500對(duì)SSR標(biāo)記, 其引物序列來(lái)自Gramene數(shù)據(jù)庫(kù)(http://www.gramene.org/), 以及Shen等[29]所開(kāi)發(fā)的50對(duì)InDel標(biāo)記進(jìn)行基因定位。PCR總反應(yīng)體系為20 μL, 內(nèi)含0.8 UDNA聚合酶, 1 × PCR buffer (Mg2+Plus), 1 mmol L–1dNTP Mixture, 50 ng DNA, 上下游引物各0.25 μmol L–1。PCR反應(yīng)條件為95℃預(yù)變性5 min; 94℃, 30 s; 55℃, 30 s; 72℃, 30 s; 共35個(gè)循環(huán); 72℃, 10 min。PCR產(chǎn)物經(jīng)8%非變性聚丙烯酰胺凝膠電泳, 快速銀染后觀察[30]。

1.6 遺傳圖譜構(gòu)建

從spl/02428 F2群體中的853個(gè)單株中獲得207株具有斑點(diǎn)葉性狀的單株組成基因定位群體, 利用上述篩選到的基因連鎖分子標(biāo)記進(jìn)行定位群體的遺傳分析, 獲得每個(gè)標(biāo)記的重組交換單株數(shù)。同時(shí)在RAP-DB數(shù)據(jù)庫(kù)(http://rapdb.dna.affrc.go.jp/)查找基因連鎖分子標(biāo)記在染色體上的具體物理位置以確定其排列順序, 結(jié)合每個(gè)標(biāo)記的重組交換單株數(shù)確定SPL基因與連鎖標(biāo)記的順序, 構(gòu)建基因定位圖譜。

2 結(jié)果與分析

2.1 突變體spl的表型及主要農(nóng)藝性狀

突變體spl葉片在分蘗期(6~7葉期)就表現(xiàn)斑點(diǎn)葉性狀(圖1-A), 而后除上部1~2片展開(kāi)葉及心葉外, 其他葉片的褐色斑點(diǎn)始于葉尖及邊緣, 并向葉中部及下部迅速蔓延, 嚴(yán)重時(shí)葉中上部枯死。而且自分蘗開(kāi)始至抽穗期, 相對(duì)于野生型珍97, 突變體spl葉片的葉色明顯更黃(圖1-B, C)。突變體spl生長(zhǎng)至抽穗開(kāi)花前期, 除劍葉外所有葉片均不同程度出現(xiàn)斑點(diǎn)葉癥狀(圖1-D, E, F)。由于葉片出現(xiàn)類病變癥狀, 導(dǎo)致突變體spl的主要農(nóng)藝性狀, 如株高、穗長(zhǎng)、每穗粒數(shù)和結(jié)實(shí)率分別比野生型對(duì)照珍97下降25.16%、12.78%、47.29%和42.60% (表1)。

2.2 突變體spl對(duì)鹽脅迫的響應(yīng)特性

正常條件下, 突變體spl與野生型珍97之間的株高和根長(zhǎng)均無(wú)明顯差異; 而在鹽脅迫條件下, 兩者的均受到抑制, 但與野生型相比, 突變體spl的根長(zhǎng)和株高抑制更明顯(圖2-A)。與突變體spl對(duì)照相比, 經(jīng)100 mmol L–1NaCl處理后的突變體株高和根長(zhǎng)分別降低19.70%和21.76%; 而經(jīng)200 mmol L–1NaCl處理的則分別下降26.71%和56.68% (圖2-B, C)。

A: 分蘗期; B: 抽穗期; C: 抽穗期葉片; D: 開(kāi)花期; E: 開(kāi)花期葉片; F: 開(kāi)花期倒二葉; 1~4代表劍葉至倒四葉。Bar=20 cm。

A: tillering stage; B:heading stage; C:leaves at heading stage; D:flowering stage; E:leaves at flowering stage; F: the 2nd leaf from top at the flowering stage; 1–4 mean the 1st leaf to 4th leaf from top. Bar=20 cm.

表1 突變體及其野生型的農(nóng)藝性狀

*在0.05水平上差異顯著;**在0.01水平上差異顯著。

*Significantly different at< 0.05;**significantly different at< 0.01 (-test).

A: 突變體spl及其野生型的表型, Bar=5 cm; B: 芽長(zhǎng); C: 根長(zhǎng)。*在0.05水平上差異顯著;**在0.01水平上差異顯著。

A: phenotype ofspland its WT plants, Bar=5 cm; B: Shoot lengths; C: primary root lengths of seedlings.*Significantly different at<0.05;**Significantly different at<0.01 (-test).

2.3 突變體spl的生理分析

2.3.1 葉綠素含量 孕穗期突變體spl的劍葉、倒二葉和倒三葉的葉綠素(圖3-A)、葉綠素(圖3-B)、葉綠素總含量(圖3-C)及葉綠素/比值(圖3-D)依次下降且顯著低于野生型, 其中突變體spl的倒二葉和倒三葉的總?cè)~綠素含量分別比其劍葉下降7.47%和39.46% (圖3-C)。與野生型相比, 突變體spl的劍葉、倒二葉和倒三葉的總?cè)~綠素含量分別下降43.18%、47.62%和58.59% (圖3-C)。

2.3.2 POD、CAT和SOD的活性 圖4-A顯示, 相對(duì)于野生型對(duì)照珍97, 突變體spl的劍葉、倒二葉與倒三葉的POD活性均極顯著增加, 分別為113.49%、982.69%和929.22%。圖4-B顯示, 野生型對(duì)照珍97的劍葉與倒二葉間的CAT活性無(wú)顯著性差異, 但倒三葉則分別比劍葉與倒二葉下降11.72%和8.58%; 而突變體spl的劍葉、倒二葉與倒三葉間的CAT活性則依次極顯著下降, 且其倒二葉與倒三葉的CAT活性極顯著低于野生型珍97, 分別下降21.16%和37.00%。圖4-C顯示, 突變體spl的劍葉、倒二葉與倒三葉間的SOD活性無(wú)顯著性差異, 而野生型珍97則依次下降。與野生型珍97相比, 突變體spl的倒二葉與倒三葉的SOD活性分別增加14.75%和23.13%。

1: 劍葉; 2: 倒二葉; 3: 倒三葉。*在0.05水平上差異顯著;**在0.01水平上差異顯著。

1: flag leaves; 2: 2nd leaves from top; 3: 3rd leaves from top.*Significantly different at<0.05;**significantly different at<0.01 (-test).

1: 劍葉; 2: 倒二葉; 3: 倒三葉。**在0.01水平上差異顯著。

1: flag leaves; 2: 2nd leaves from top; 3: 3rd leaves from top.**Significantly different at<0.01 (-test).

2.3.3 超氧陰離子(O2?)和過(guò)氧化氫(H2O2)的累積

圖5-A顯示, 野生型對(duì)照珍97和突變體spl倒二葉和倒三葉間的O2?含量差異不顯著, 但顯著高于劍葉。與野生型相比, 突變體spl劍葉、倒二葉和倒三葉的O2?含量分別增加15.71%、17.31%和13.66%。圖5-B結(jié)果顯示, 野生型及突變體的劍葉、倒二葉和倒三葉間的H2O2含量無(wú)顯著差異。同時(shí), 抽穗期突變體spl劍葉的部分組織細(xì)胞明顯被NBT染成更多更深的藍(lán)色(圖5-C), 進(jìn)一步證實(shí)其體內(nèi)累積O2?; 而突變體spl及其野生型對(duì)照葉片組織均沒(méi)有被DAB染成明顯的棕紅色(圖5-D), 證實(shí)兩者之間的H2O2含量無(wú)明顯差異。

2.3.4 MDA和可溶性蛋白含量 圖6-A顯示, 與野生型相比, 突變體劍葉、倒二葉和倒三葉的MDA含量分別增加29.63%、34.48%和56.25%, 達(dá)極顯著水平; 且突變體spl倒二葉和倒三葉的MDA含量分別比劍葉增加11.42%和42.86%。圖6-B顯示, 野生型和突變體劍葉、倒二葉和倒三葉間的可溶性蛋白含量依次降低, 尤其是兩者倒三葉的含量極顯著低于其劍葉和倒二葉(圖6-B), 與野生型相比, 除劍葉外, 突變體倒二葉和倒三葉的可溶性蛋白含量分別下降12.26%和16.74%。

2.4 遺傳分析及基因定位

spl/珍97、spl/02428和spl/秀水110三個(gè)雜種F1單株的葉片均表現(xiàn)正常, 說(shuō)明突變體spl的斑點(diǎn)葉性狀由隱性位點(diǎn)控制。而這3個(gè)雜種F1的自交F2后代的田間結(jié)果表明, 具正常葉片表型的單株與具斑點(diǎn)葉表型的單株數(shù)之比均符合孟德?tīng)?∶1的分離比(表2), 證實(shí)突變體spl的斑點(diǎn)葉性狀受一對(duì)隱性核基因控制。

1: 劍葉; 2: 倒二葉; 3: 倒三葉。**在0.01水平上差異顯著。 C: NBT染色。D: DAB染色。

1: flag leaves; 2: 2nd leaves from top; 3: 3rd leaves from top.**Significantly different at<0.01 (-test). C: NBT staining. D: DAB staining.

利用spl/02428的F2群體中的207株具斑點(diǎn)葉性狀的單株作為定位群體。選取均勻分布于水稻12條染色體上的500對(duì)SSR標(biāo)記及50對(duì)InDel標(biāo)記逐條對(duì)02428和突變體spl進(jìn)行親本多態(tài)性分析,利用篩選到的多態(tài)性標(biāo)記分析正常基因池和突變基因池。發(fā)現(xiàn)在突變體基因池和正?；虺厮镜?2染色體上的SSR標(biāo)記RM511、RM1246和RM1300 (引物序列見(jiàn)附表1)存在明顯偏分離, 利用207個(gè)突變體單株驗(yàn)證以上標(biāo)記, 初步把SPL定位在RM511和RM1300之間(圖7)。為進(jìn)一步定位SPL基因, 利用RM511和RM1300之間的5對(duì)在兩親本之間具有多態(tài)性的SSR標(biāo)記(包括RM28204、RM1246、RM28466、RM28485和RM28502)(引物序列見(jiàn)附表1)將該基因定位在RM28466與RM28485之間, 物理距離約189 kb, 橫跨AL935072、AL928753、BX072546和AL713908等4個(gè)BAC (圖7), 其間有EST支持的22個(gè)ORF (http://rapdb.dna. affrc.go.jp/viewer/gbrowse/irgsp1/)(表3)。

3 討論

斑點(diǎn)葉突變體最明顯的外在表現(xiàn)是葉片上出現(xiàn)壞死斑點(diǎn)或斑塊, 而內(nèi)在表現(xiàn)則為程序性細(xì)胞死亡(programmed cell death, PCD), 并由此帶來(lái)葉綠體降解、蛋白質(zhì)降解和ROS (reactive oxygen species, ROS)累積等眾多生理生化變化[7-8]。研究表明, 葉綠體降解不僅引起ROS累積, 也介導(dǎo)PCD形成[7], 因此葉綠素含量及葉綠素的比值是衡量細(xì)胞死亡的重要生理指標(biāo)。利用EMS誘變獲得斑點(diǎn)葉突變體spl, 其斑點(diǎn)葉癥狀始于分蘗期(圖1-A), 孕穗期后除劍葉外所有葉片均不同程度出現(xiàn)褐色斑點(diǎn)(圖1-B, C, D, E, F)。葉綠素含量測(cè)定表明, 突變體劍葉、倒二葉和倒三葉的葉綠素/比值均低于其野生型珍97且依次顯著下降, 說(shuō)明突變體葉肉細(xì)胞已發(fā)生PCD, 而且其葉綠素比葉綠素下降更快, 究其原因可能是倒二葉和倒三葉中的ROS累積, 特別是O2?的累積顯著高于劍葉(圖5-A), 導(dǎo)致葉綠素對(duì)活性氧離子的敏感性高于葉綠素[31]。至于孕穗期突變體光合色素含量明顯低于野生型的原因, 可能是突變體基因的突變影響葉綠素的合成[32], 從而造成突變體葉色偏黃(圖1-B, C)。

1: 劍葉; 2: 倒二葉; 3: 倒三葉。**在0.01水平上差異顯著。

1: flag leaves; 2: 2nd leaves from top; 3: 3rd leaves from top. ** Significantly different at<0.01 (-test).

表2 突變體splZ97的遺傳分析

表3 定位區(qū)間內(nèi)的基因及功能注釋

引起葉片斑點(diǎn)葉形成的原因極其復(fù)雜, 它是由植物自身的生理生化原因及外界環(huán)境因素共同作用決定的。其中, 作為植物體內(nèi)重要信號(hào)分子ROS, 在抵御各種逆境脅迫、調(diào)控植物生長(zhǎng)發(fā)育中起重要作用, 其重要來(lái)源是葉綠體, 伴隨葉片葉綠體的降解或解體, 葉綠體中的電子傳遞鏈?zhǔn)艿揭种? 致使1O2、H2O2和O2?大量形成[33]。研究顯示, O2?是引起擬南芥突變體類病斑形成的重要因素, 外源SOD能有效抑制該突變體葉片出現(xiàn)類病斑癥狀[34]。本研究中野生型珍97的劍葉、倒二葉和倒三葉間的ROS(主要包括H2O2和O2?)無(wú)顯著性差異, 但突變體的O2?含量則極顯著高于野生型且依次增加(圖5-A), 葉片NBT組織化學(xué)染色進(jìn)一步證實(shí)突變體的O2?明顯累積(圖5-C)。因此推測(cè)O2?是引起spl斑點(diǎn)葉形成的重要因素。同時(shí), 由于葉片中大量累積ROS, 導(dǎo)致細(xì)胞膜脂質(zhì)過(guò)氧化而產(chǎn)生大量MDA (圖5-A), 加劇膜損傷并致使細(xì)胞壞死[35]。當(dāng)然, 在正常植株體內(nèi), 存在重要的抗氧化酶系統(tǒng), 如SOD、POD和CAT, 調(diào)控ROS的產(chǎn)生和降解, 維持ROS含量處于一個(gè)動(dòng)態(tài)平衡而使細(xì)胞免于氧化脅迫傷害[36]。其中, CAT專一作用于H2O2, 將其分解成無(wú)毒的H2O和O2。本研究中突變體倒二葉和倒三葉的CAT活極顯著低于劍葉, 致使倒三葉細(xì)胞中存在H2O2累積(圖4-B)。至于突變體葉片, 尤其是倒二葉和倒三葉的POD活性(圖4-A)和SOD活性(圖4-C)極顯著高于野生型且依次顯著升高的原因, 可能是斑點(diǎn)葉形成初期細(xì)胞自我保護(hù), 誘導(dǎo)SOD和POD合成相關(guān)基因的表達(dá)而使SOD和POD活性增加, 該結(jié)果進(jìn)一步證實(shí)了汪媛[37]和趙晨晨等[38]的研究。

此外, 任何與葉片生理生化代謝相關(guān)基因的突變, 均有可能破壞細(xì)胞內(nèi)部的生理生化平衡, 導(dǎo)致ROS累積及內(nèi)源激素的失衡, 引起細(xì)胞程序性死亡并最終造成斑點(diǎn)葉的形成。研究表明,、和是植物中最重要的三大轉(zhuǎn)錄因子, 調(diào)控各種分子生理生化過(guò)程, 進(jìn)而影響植物生長(zhǎng)發(fā)育[39-42]。過(guò)量表達(dá)基因, 將導(dǎo)致轉(zhuǎn)基因擬南芥出現(xiàn)類病斑癥狀, 究其原因, 可能是該基因的過(guò)量表達(dá)促進(jìn)了SA合成途徑中基因的上調(diào)表達(dá), 進(jìn)而導(dǎo)致SA的累積而造成斑點(diǎn)葉的形成[43-44]。而編碼水稻熱激轉(zhuǎn)錄因子的基因的突變將導(dǎo)致突變體從水稻分蘗期到抽穗期, 整個(gè)葉片表面出現(xiàn)紅棕色小斑點(diǎn)[9]。此外, 在高粱中表達(dá)水稻蛋白激酶基因, 將導(dǎo)致轉(zhuǎn)基因后代表現(xiàn)類病斑癥狀且對(duì)鹽敏感[45]。迄今為止, 在第12染色體長(zhǎng)臂上已定位或克隆[15]和[46]等斑點(diǎn)葉相關(guān)基因, 而這些基因均不在本研究的定位區(qū)間, 因此SPL是一個(gè)新的斑點(diǎn)葉相關(guān)基因。與報(bào)道相關(guān)的導(dǎo)致植物類病斑形成的基因則僅有蛋白激酶基因(LOC_ Os12g37570)和MYB轉(zhuǎn)錄因子(LOC_Os12g37690)?；谕蛔凅w的MDA含量、CAT活性、O2–含量及H2O2含量等結(jié)果初步認(rèn)為spl基因與ROS密切相關(guān), 而依據(jù)突變體對(duì)鹽敏感的結(jié)果則進(jìn)一步將候選基因指向蛋白激酶基因(LOC_Os12g37570)。當(dāng)然,SPL基因的最終確定要依賴于候選基因的測(cè)序分析及遺傳互補(bǔ)驗(yàn)證。

4 結(jié)論

spl是一個(gè)新的斑點(diǎn)葉且對(duì)鹽敏感的突變體, 其斑點(diǎn)葉癥狀始于分蘗期, 此后由葉邊緣向葉片內(nèi)部擴(kuò)散, 直至遍布整個(gè)葉片。與野生型對(duì)照相比, 突變體劍葉、倒二葉和倒三葉的葉綠素含量極顯著降低, ROS明顯累積, MDA含量、SOD和POD活性極顯著升高; 而CAT活性顯著下降, 清除H2O2的能力也顯著下降。spl斑點(diǎn)葉性狀受1對(duì)隱性核基因控制, 位于第12染色體長(zhǎng)臂的RM28466和RM28485兩個(gè)標(biāo)記的189 kb區(qū)間, 本研究結(jié)果為進(jìn)一步克隆SPL基因并揭示斑點(diǎn)葉形成的分子生理機(jī)制奠定了基礎(chǔ)。

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附表1 用于SPL基因定位的SSR標(biāo)記

Supplementary table 1 SSR markers used forSPLgene mapping

Physiological Characters and Gene Mapping of a Spotted-leaf Mutantsplin Rice

WEI Li-Quan1,**, LUO Yan-Min1,**, WANG Wen-Qiang1, CHI Chang-Cheng1, HUANG Fu-Deng2, XIANG Xun1, CHENG Fang-Min1, and PAN Gang1,*

1College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China;2Institute of Crop and Nuclear Technology Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

A spotted-leaf mutantsplwas isolated from a mutant bank generated by EMS mutagenesis ofrestore line Zhen 97. Under field conditions, the brown lesion-mimics mutant splfirstly displayed at the tip and edge of leaf blade at tillering stage, and then gradually spread to whole leaf, resulting in the death of the whole blade when the symptom was severe. At the same time, the major agronomic traits including plant height, grain number per panicle and seed-setting rate were markedly affected. Compared with the wild-type the flag leaf, the second leaf from top and the third leaf from top at heading stage, chlorophyll contents in the mutantsplsignificantly decreased, while POD (peroxidase, POD) activity, O2?level and MDA (malondialdehyde, MDA) content increased. In addition, CAT (catalase, CAT) activity and soluble protein content of the second leaf from top and the third leaf from top of the mutant decreased as compared with the wild type; on the contrary, the SOD (superoxide dismutase, SOD) activity significantly increased. The histochemical analysis further indicated thatO2?accumulated in the leaf blade of the mutantspl. In addition, under salt stress at seedling stage, the shoot length and root length of the mutant splwere significantly shorter than these of the wild type. Genetic analysis and gene mapping showed thatsplwas controlled by a single recessive nuclear gene, which was mapped to a region of 189 kb flanked by two SSR markers RM28466 and RM28485 on the long arm of chromosome 12. These results achieved in the present study would further facilitate the cloning and functional analysis of the geneSPL.

Rice;spl; Spotted-leaf; Physiological characters; Gene mapping

10.3724/SP.J.1006.2017.00648

本研究由國(guó)家自然科學(xué)基金項(xiàng)目(31271691)和國(guó)家轉(zhuǎn)基因生物新品種培育重大專項(xiàng)(2016ZX08001-002)資助。

This study was supported by the Natural Science Foundation of China (31271691) and the National Major Project for Developing New GM Crops (2016ZX08001-002)

(Corresponding author): 潘剛, E-mail: pangang12@126.com**同等貢獻(xiàn)(Contributed equally to this work)

(收稿日期): 2016-08-13; Accepted(接受日期): 2017-01-21; Published online(網(wǎng)絡(luò)出版日期):2017-02-17.

URL: http://www.cnki.net/kcms/detail/11.1809.S.20170217.1001.012.html