水稻類病斑早衰突變體lmps1的表型鑒定與基因定位

夏賽賽 崔 玉 李鳳菲 譚 佳 謝園華 桑賢春 凌英華

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水稻類病斑早衰突變體的表型鑒定與基因定位

夏賽賽 崔 玉 李鳳菲 譚 佳 謝園華 桑賢春 凌英華*

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

經(jīng)甲基磺酸乙酯(EMS)誘變優(yōu)良秈型水稻恢復(fù)系縉恢10號(hào), 獲得一個(gè)穩(wěn)定遺傳的水稻類病斑早衰突變體()。該突變體苗期表型正常, 分蘗早期出現(xiàn)褐色類病斑, 且斑點(diǎn)數(shù)目隨植株生長(zhǎng)而增多, 孕穗期葉片開(kāi)始萎黃衰老。與野生型相比, 突變體的每穗總粒數(shù)下降8% (<0.05), 株高、穗長(zhǎng)、有效穗數(shù)、每穗實(shí)粒數(shù)、結(jié)實(shí)率以及千粒重分別下降14.3%、24.3%、27.2%、50%、45.7%與14.5%, 差異均達(dá)極顯著水平(<0.01)。遮光處理表明, 突變體的類病斑性狀受光照誘導(dǎo)。孕穗期葉片光合色素含量下降且光合效率降低, H2O2含量增加, 抗氧化酶SOD和CAT的活性顯著降低。透射電鏡觀察結(jié)果顯示, 突變體葉肉細(xì)胞中葉綠體數(shù)目減少, 葉綠體的類囊體片層結(jié)構(gòu)損傷降解。qRT-PCR結(jié)果顯示, 突變體中防衛(wèi)反應(yīng)相關(guān)基因除表達(dá)量降低外,、、、、、表達(dá)量均極顯著高于野生型。遺傳分析表明突變體的類病斑早衰性狀受1對(duì)隱性核基因控制, 利用西農(nóng)1A與突變體雜交所得F2群體中的突變株, 將目標(biāo)基因定位于第7染色體長(zhǎng)臂端粒附近約167.3 kb的物理區(qū)段內(nèi)。

水稻(L); 類病斑早衰突變體); 表型鑒定; 基因定位

植物類病斑突變體(lesion mimic mutant,)是指在沒(méi)有病原菌侵染或明顯逆境的情況下, 植物體自發(fā)形成類似于病原菌侵染產(chǎn)生壞死斑的一類突變體[1]。此類突變體首先在玉米中發(fā)現(xiàn)[1-2], 隨后在大麥[3]、擬南芥[4]、水稻[5-6]、花生[7]等植物中相繼被報(bào)道。植物類病斑突變體的壞死斑表型類似于過(guò)敏反應(yīng), 且大多數(shù)與過(guò)敏反應(yīng)以及細(xì)胞程序性死亡有關(guān), 一些類病斑突變體能提高植物抗病性并引發(fā)相關(guān)蛋白的表達(dá)。

國(guó)內(nèi)外研究工作者通過(guò)各種物理化學(xué)誘變方法已鑒定到大量水稻類病斑突變體, 其多數(shù)性狀受隱性基因調(diào)控, 只有少部分由顯性或半顯性基因控制[8]。在這些類病斑突變體中, 已被克隆的目標(biāo)基因相對(duì)較少[9]。從報(bào)道看, 水稻的類病斑主要是由抗病相關(guān)基因突變、某些代謝途徑中的相關(guān)基因突變、胞內(nèi)運(yùn)輸異常以及轉(zhuǎn)錄因子所致。其中一類是導(dǎo)致的抗病相關(guān)基因, 包括[6]、[10]、[11]、[12][13]等。另一類基因, 包括、等, 與葉綠素或其他細(xì)胞色素合成有關(guān)[14-16],參與脂氧物代謝過(guò)程[17],可能與糖類代謝有關(guān)[18]。此外, 細(xì)胞程序性死亡、熱擊蛋白、ROS相關(guān)基因的突變, 同樣會(huì)導(dǎo)致水稻植株出現(xiàn)表型。如[19]、[20]、[21]、[22]、[23]、[24]、[25]、[26]等。

眾多水稻類病斑突變體中, 僅有少數(shù)伴隨早衰現(xiàn)象。水稻類病斑早衰突變體既能自發(fā)形成壞死病斑, 又伴有葉早衰現(xiàn)象, 故研究該類突變體對(duì)了解葉片衰老過(guò)程中的各種生理變化及其影響因素、解析植物抗病機(jī)制、研究細(xì)胞程序性死亡及植物防御反應(yīng)相關(guān)歷程具有十分重要的意義。本研究在秈型恢復(fù)系縉恢10號(hào)的EMS誘變體庫(kù)中, 鑒定到一個(gè)類病斑早衰突變體()。其前期表型正常, 從分蘗早期葉片開(kāi)始出現(xiàn)褐色小斑點(diǎn), 并隨植株生長(zhǎng)而數(shù)目增多直至布滿葉片。至孕穗期植株葉片開(kāi)始萎黃, 表現(xiàn)出早衰特征。本研究對(duì)該突變體進(jìn)行表型鑒定、光合特性、超微結(jié)構(gòu)、生理學(xué)特性、遺傳特性、基因定位等研究, 為目標(biāo)基因的克隆及后續(xù)的分子機(jī)制解析等奠定良好基礎(chǔ)。

1 材料與方法

1.1 研究材料及其表型鑒定

類病斑早衰突變體經(jīng)多代自交, 其表型穩(wěn)定遺傳。將縉恢10號(hào)作為該突變體的野生型(WT)參照, 自然條件下, 將WT與種植田間, 比較其全生育期植株表型。成熟期分別隨機(jī)選取WT和植株各10株, 分別考察其株高、穗長(zhǎng)、有效穗數(shù)、每穗總粒數(shù)、每穗實(shí)粒數(shù)、結(jié)實(shí)率、千粒重。

1.2 遮光處理及光合色素含量與光合效率參數(shù)分析

在分蘗期, 用錫箔紙包裹田間尚未發(fā)生病斑的突變體葉片, 連續(xù)遮光1周后恢復(fù)光照, 期間持續(xù)觀察葉片病斑的發(fā)生情況。在孕穗期用Beckman22S型分光光度計(jì)測(cè)定光合色素含量, 參照Wellburn[27]描述的方法, 于田間利用便攜式光合測(cè)定儀Li-6400測(cè)定凈光合速率(n, μmol m–2s–1)、氣孔導(dǎo)度(s, mmol m–2s–1)、胞間CO2濃度(i, μmol mol–1)與蒸騰速率(r, mmol m–2s–1)等光合參數(shù)。

1.3 透射電鏡觀察及生理指標(biāo)測(cè)定

參照Fang等[28]的方法, 用透射電子顯微鏡觀察WT與葉肉細(xì)胞和葉綠體的結(jié)構(gòu)。用相關(guān)試劑盒(南京建成科技有限公司)測(cè)定孕穗期倒一、倒二、倒三葉的過(guò)氧化氫酶(CAT)、過(guò)氧化物酶(POD)、超氧化物歧化酶(SOD)的活性和過(guò)氧化氫(H2O2)的含量, 每個(gè)樣本重復(fù)3次, 用DPS軟件進(jìn)行數(shù)據(jù)分析。

1.4 lmps1目標(biāo)基因的精細(xì)定位

分別將西農(nóng)1A、及西農(nóng)1A×獲得的F2群體種植于西南大學(xué)水稻研究所歇馬試驗(yàn)基地。在分蘗早期選取該群體中葉片上有褐色斑點(diǎn)的突變單株, 采用改良的CTAB法[29]提取親本和F2群體中突變表型植株與10個(gè)正常表型植株的DNA備用。參照劉寶玉等[9]的方法定位的基因, 所用引物由上海英駿生物技術(shù)公司合成。

1.5 RNA提取及qRT-PCR

在分蘗期, 用RNA提取試劑盒(北京天根生化科技有限公司)提取野生型和突變體植株葉片總RNA, 以之為模板通過(guò)反轉(zhuǎn)錄得到相應(yīng)cDNA (TaKaRa公司生產(chǎn)的PrimeScript 1st Strand cDNA Synthesis Kit)。用作為內(nèi)參基因, 進(jìn)行相關(guān)基因cDNA擴(kuò)增, 以2–ΔΔCT計(jì)算各基因的相對(duì)表達(dá)量, 對(duì)每個(gè)樣品至少做3個(gè)重復(fù)。qRT-PCR主要用于定位區(qū)間內(nèi)部分基因和一些防衛(wèi)反應(yīng)相關(guān)標(biāo)記基因的表達(dá)分析。

2 結(jié)果與分析

2.1 突變體lmps1的表型特性

與WT相比, 突變體苗期表型正常, 分蘗早期葉片開(kāi)始出現(xiàn)褐色小斑點(diǎn), 隨著植株生長(zhǎng), 斑點(diǎn)數(shù)目持續(xù)增加(圖1-A, B)。孕穗期開(kāi)始出現(xiàn)早衰表型; 至成熟期, 斑點(diǎn)布滿所有葉片, 衰老加速, 葉片嚴(yán)重黃化, 整個(gè)植株呈黃褐色(圖1-C, D)。突變體的類病斑與早衰表型, 最早出現(xiàn)的時(shí)期不同, 但均由葉尖發(fā)展到葉片中部和基部, 并隨葉齡增長(zhǎng)而程度加深(圖1-A, B, C, D)。成熟期突變體的株高、穗長(zhǎng)、有效穗數(shù)、每穗總粒數(shù)、每穗實(shí)粒數(shù)、結(jié)實(shí)率和千粒重較野生型均顯著或極顯著降低(表1)。

2.2 突變體lmps1對(duì)光的響應(yīng)

分蘗期對(duì)突變體即將出現(xiàn)但尚未有類病斑表型的葉片進(jìn)行遮光處理表明, 未遮光部位逐漸產(chǎn)生大量褐色小斑點(diǎn), 而錫箔紙遮光部位沒(méi)有斑點(diǎn)或僅產(chǎn)生少量斑點(diǎn), 且葉片呈綠色, 與野生型基本一致(圖2-A, C)。移去錫箔紙恢復(fù)光照后, 該部位又開(kāi)始逐漸變黃, 并出現(xiàn)褐色小斑點(diǎn)(圖2-D)。說(shuō)明突變體表型受光誘導(dǎo)。

圖1 野生型(WT)與突變體lmps1的表型

A: 分蘗期WT與植株; B: 分蘗期WT與的葉片; C: 成熟期WT與植株; D: 成熟期WT與的葉片; 1: 倒一葉; 2: 倒二葉; 3: 倒三葉; 標(biāo)尺=5 cm。

A: plants of the wild type and themutant at tillering stage; B: leaves of the wild type and themutant at tillering stage; C: plants of the wild type and themutant at mature period; D: leaves of the wild type and themutant at mature period; 1: the flag leaves; 2: the second leaves; 3: the third leaves; bar = 5 cm.

表1 野生型與突變體lmps1之間的表型差異

表中各性狀的表現(xiàn)以“平均值±標(biāo)準(zhǔn)差” 格式呈現(xiàn)。**表示在0.01水平上差異顯著;*表示在0.05水平上差異顯著。

Phenotypic performance of each trait was expressed as means ± standard deviation.**means significant difference at< 0.01 by-test;*means significant difference at< 0.05 by-test.

圖2 遮光對(duì)野生型(WT)和突變體lmps1葉片的影響

A: WT葉片; B:葉片; C:遮光1周后; D:遮光處理后復(fù)光1周后; 標(biāo)尺=6 cm。

A: leaf of the wild type; B: leaf ofthemutant; C: leaf of themutantafter one week shading; D: leaf of themutant illuminated for one week after shading; bar = 6 cm.

2.3 光合色素含量分析

孕穗期突變體光合色素含量均低于WT, 差異達(dá)到極顯著水平(<0.01)(圖3-A, B, C, D), 并且突變體的倒三、倒四葉光合色素含量明顯低于倒一葉、倒二葉, 其中倒四葉最低(圖3-E, F)。表明在孕穗期突變體葉片已在生理水平上進(jìn)入衰老階段, 且衰老是從倒四葉向倒一葉發(fā)展。

2.4 光合效率變化

孕穗期, 突變體除倒一葉的氣孔導(dǎo)度(s)外, 其他光合效率參數(shù)與WT差異均極顯著:的凈光合速率(n)較WT極顯著降低(<0.01); 胞間CO2濃度(i)均極顯著升高; 氣孔導(dǎo)度(s)倒二葉、倒三葉極顯著升高;的蒸騰速率(r)倒一葉、倒二葉均極顯著降低, 而倒三葉則極顯著升高(圖4-A, B, C, D)。光合效率顯著降低表明突變體光合能力受到嚴(yán)重影響。

圖3 野生型(WT)與突變體lmps1孕穗期光合色素含量

A~D: 孕穗期WT與的倒一葉(A)、倒二葉(B)、倒三葉(C)和倒四葉(D)光合色素含量; E: WT光合色素含量; F:光合色素含量。條柱表示平均值, 誤差線表示標(biāo)準(zhǔn)差; **表示在0.01水平上差異顯著; E, F圖中標(biāo)的不同字母的柱值在0.05水平上差異顯著。

A-D: photosynthetic pigments contents of the flag leaves, the second leaves, the third leaves and the fourth leaves respectively in the wild type and themutant at booting stage; E: photosynthetic pigments contents of the wild type; F: photosynthetic pigments contents of themutant. The column shows the mean value and the error line shows the standard deviation; ** means significant difference at< 0.01 by-test; in Figs. E and F, bars superscripted by different letter are significantly different at< 0.05.

2.5 細(xì)胞超微結(jié)構(gòu)分析

利用透射電鏡觀察孕穗期WT和突變體倒四葉葉尖和葉中部的細(xì)胞超微結(jié)構(gòu)表明, 突變體的葉綠體結(jié)構(gòu)較WT出現(xiàn)明顯變化: 細(xì)胞內(nèi)葉綠體數(shù)目明顯減少(圖5-D, J), 嗜鋨小體的數(shù)量和體積增加(圖5-E, F, K, L)。葉尖部分存在結(jié)構(gòu)完好的葉綠體, 但部分葉綠體嚴(yán)重受損, 類囊體片層結(jié)構(gòu)損傷解體(圖5-E, F); 葉片中部葉綠體基本完好, 基質(zhì)片層結(jié)構(gòu)相對(duì)葉尖部分而言較為完整, 但結(jié)構(gòu)松散(圖5-K, L)。表明突變體葉綠體結(jié)構(gòu)損傷, 葉綠體開(kāi)始降解, 致使其葉綠素含量和凈光合速率n顯著降低; 其葉綠體的完整程度與衰老進(jìn)程一致。

圖4 孕穗期野生型(WT)與突變體lmps1功能葉的光合特性

A: WT和的凈光合速率; B: WT和的氣孔導(dǎo)度; C: WT和的細(xì)胞間CO2濃度; D: WT和的蒸騰速率。條柱表示平均值, 誤差線表示標(biāo)準(zhǔn)差; **表示在0.01水平上差異顯著, *表示在0.05水平上差異顯著。

A: net photosynthetic rate of the wild type and themutant; B: stomatal conductance of the wild type and themutant; C: intercellular CO2concentration of the wild type and themutant; D: transpiration rate of the wild type and themutant. The column shows the mean value and the error line shows the standard deviation; ** means significant difference at< 0.01 by-test, * means significant difference at< 0.05 by-test.

圖5 孕穗期野生型(WT)和突變體lmps1的葉肉細(xì)胞超微結(jié)構(gòu)

A: WT倒四葉尖; B、C、D: WT葉尖細(xì)胞超微結(jié)構(gòu); E:倒四葉尖; F、G、H:葉尖細(xì)胞超微結(jié)構(gòu); I: WT倒四葉中部; J、K、L: WT葉中部細(xì)胞超微結(jié)構(gòu); M:倒四葉中部; N、O、P:葉中部細(xì)胞超微結(jié)構(gòu); Chl: 葉綠體; Os: 嗜鋨小體; TM: 類囊體膜; N: 細(xì)胞核。標(biāo)尺: 2 μm (A, D), 1 μm (B, E), 200 nm (C, F)。

A: tip of the fourth leaves of the wild type; B, C, D: ultrastructure of the blade tip of the wild type; E: tip of the fourth leaves of themutant; F, G, H: ultrastructure of the blade tip ofthemutant; I: middle part of the fourth leaves of the wild type; J, K, L: ultrastructure of the middle part of blade of the wild type; M: middle part of the fourth leaves of themutant; N, O, P: ultrastructure of the middle part of blade ofthemutant. Chl: chloroplast; Os: osmiophilic granule; TM: thylakoid membranes; N: ribosome. Bars: 2 μm (A, D), 1 μm (B, E), 200 nm (C, F).

2.6 抗氧化酶活性及H2O2含量分析

在孕穗期, 除倒一葉外,倒二、倒三葉的CAT、SOD活性較WT均顯著或極顯著降低(圖6-A, C)。的POD活性除倒三葉較WT顯著降低外, 其倒一、倒二葉差異不顯著(圖6-B)。而的H2O2含量較WT均顯著或極顯著增加(圖6-D)。抗氧化酶活性降低和活性氧H2O2的增加表明葉片發(fā)生衰老。

圖6 孕穗期野生型(WT)和突變體lmps1的抗氧化酶活性及H2O2含量

A~C: WT和的過(guò)氧化氫酶(CAT)、過(guò)氧化物酶(POD)、超氧化物歧化酶(SOD)活性; D: WT和各功能葉過(guò)氧化氫(H2O2)含量。條柱表示平均值, 誤差線表示標(biāo)準(zhǔn)差; **表示在0.01水平上差異顯著, *表示在0.05水平上差異顯著。

A-C: the enzyme activity of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in functional leaves of the wild type and themutant; D: H2O2content of different functional leaves of the wild type and themutant. The column shows the mean value and the error line shows the standard deviation; ** means significant difference at< 0.01 by-test, * means significant difference at< 0.05 by-test.

2.7 遺傳分析與目標(biāo)基因的定位

用秈稻西農(nóng)1A與突變體構(gòu)建的F1單株表型均與WT一致, F2代發(fā)生性狀分離。共收獲正常單株1214株、類病斑早衰突變單株420株, 其比例為2.89∶1。經(jīng)卡平方測(cè)驗(yàn), 符合3∶1的理論分離比例(χ2= 0.3949,= 0.5211), 表明突變體的類病斑早衰性狀受1對(duì)隱性核基因控制。

選取西南大學(xué)水稻研究所現(xiàn)存均勻分布于水稻12條染色體上的400個(gè)SSR和InDel標(biāo)記, 在西農(nóng)1A和突變體之間進(jìn)行多態(tài)性篩選。用篩選到的106對(duì)多態(tài)性引物擴(kuò)增正常葉片和類病斑早衰葉片基因池,發(fā)現(xiàn)位于水稻第7染色體的InDel標(biāo)記ZTQ66在兩組基因池之間表現(xiàn)出差異, 利用F2群體中正常單株10株和類病斑早衰單株21 株進(jìn)行單株驗(yàn)證, 初步確定ZTQ66與目標(biāo)基因連鎖。進(jìn)一步在該標(biāo)記上下游開(kāi)發(fā)新的多態(tài)性標(biāo)記, 初步將目標(biāo)基因定位于第7染色體CY7-6和CY7-5之間(圖7-A)。利用CY7-6和CY7-5之間的多態(tài)性標(biāo)記, 最終將基因定位在標(biāo)記CY7-8和CY7-60之間, 遺傳距離分別為0.83 cM和0.71 cM, 物理距離約為167.3 kb (圖7-B)。

2.8 定位區(qū)間內(nèi)基因及防衛(wèi)反應(yīng)相關(guān)基因表達(dá)量分析

根據(jù)Rice Genome Annotation Project網(wǎng)站上的信息預(yù)測(cè), 定位區(qū)間內(nèi)共25個(gè)注釋基因。根據(jù)遺傳距離挑選部分基因并利用網(wǎng)站提供序列信息設(shè)計(jì)qRT-PCR引物, 對(duì)部分基因進(jìn)行表達(dá)分析發(fā)現(xiàn), 與野生型相比,、、表達(dá)量沒(méi)有明顯變化,上調(diào)接近1.5倍,上調(diào)2倍, 其他、、分別下調(diào)37.3%、76.6%和44.4%,下調(diào)近20倍(圖8-A)。

圖7 LMPS1基因在第7染色體的分子定位

A:基于F2群體的初定位; B:的精細(xì)定位。

A: QTL mapping of; B: fine mapping of.

圖8 野生型(WT)和突變體lmps1相關(guān)基因的表達(dá)量

A:精細(xì)定位物理區(qū)間內(nèi)部分基因的表達(dá)量; B: 抗病相關(guān)基因相對(duì)表達(dá)量。條柱表示平均值, 誤差線表示標(biāo)準(zhǔn)差; *表示0.05水平上差異顯著; **表示在0.01水平上差異顯著。

A: relative expression level of annotated genes within the physical fine mapping region of; B: relative expression level of genes related to disease resistance. The column shows the mean value and the error line shows the standard deviation; * refers to the significant difference at< 0.05; ** denotes significant difference at< 0.01.

類病斑突變體中常伴隨防衛(wèi)反應(yīng)相關(guān)基因表達(dá)的變化, 這可能是類病斑突變體對(duì)病原菌抗性增強(qiáng)的原因之一。圖8-B表明,除相對(duì)WT下調(diào)9倍外,、、、、、均為上調(diào)。和分別上調(diào)28.2%和9.8%,上調(diào)約1.4倍、上調(diào)15.8倍、上調(diào)27倍、上調(diào)接近1倍。說(shuō)明的突變可能激活部分病程相關(guān)因子, 引起水稻防衛(wèi)反應(yīng)。

3 討論

目前, 通過(guò)圖位克隆或其他手段, 已有多個(gè)控制水稻類病斑形成的基因被定位或克隆。已報(bào)道的類病斑突變體中, 僅有少數(shù)伴隨葉片早衰現(xiàn)象, 包括已克隆的[19]、[18]、[30]基因?qū)?yīng)的突變體, 它們分別被定位在第1、第8和第2染色體。未克隆的[31]、[32]、[33]等被定位在第7、第10和第11染色體。編碼一個(gè)網(wǎng)格蛋白相關(guān)銜接蛋白復(fù)合物1, 其突變體苗期表型正常, 分蘗初期開(kāi)始出現(xiàn)紅褐色小斑點(diǎn), 抽穗期斑點(diǎn)面積達(dá)到最大, 開(kāi)花早期葉片開(kāi)始黃化, 開(kāi)花后期葉片枯萎, 成熟期植株死亡, 該突變體的籽粒干癟, 產(chǎn)量嚴(yán)重下降[19];編碼UDP-N-乙酰葡萄糖胺焦磷酸化酶, 在種子萌發(fā)后23 d左右開(kāi)始出現(xiàn)黑褐色類病斑, 30 d左右開(kāi)始出現(xiàn)葉片早衰現(xiàn)象, 主要農(nóng)藝性狀指標(biāo)均嚴(yán)重低于野生型[18];編碼一個(gè)羧基末端磷酸酶結(jié)構(gòu)域及2個(gè)雙鏈的RNA結(jié)合區(qū), 相應(yīng)突變體在播種30~40 d左右出現(xiàn)紅棕色類壞死斑點(diǎn), 開(kāi)花后葉片早衰, 籽粒形態(tài)與野生型類似[30]。和尚未被克隆, 前者分蘗早期出現(xiàn)褐色類病斑, 后者五葉期自發(fā)出現(xiàn)壞死斑, 都在孕穗期出現(xiàn)葉片黃化衰老[31-32]。本研究中的突變體, 從分蘗早期出現(xiàn)褐色類病斑, 之后類病斑數(shù)量逐漸增加, 直至整個(gè)植株葉片被斑點(diǎn)覆蓋。至孕穗期開(kāi)始衰老直至整個(gè)植株呈黃褐色, 此時(shí)野生型植株呈綠色。對(duì)進(jìn)行相應(yīng)的生理指標(biāo)測(cè)定, 與野生型相比,的光合色素含量極顯著降低, 光合效率顯著降低, 抗氧化酶活性降低, 活性氧H2O2顯著增加。此外, 突變體較野生型, 成熟期每穗總粒數(shù)顯著降低而株高、穗長(zhǎng)、有效穗數(shù)、每穗實(shí)粒數(shù)、結(jié)實(shí)率與千粒重極顯著降低。

類病斑突變體中常伴隨防御反應(yīng)相關(guān)基因表達(dá)的變化, 這可能是類病斑突變體對(duì)病原菌抗性增強(qiáng)原因之一。本研究中, 除下調(diào)9倍外, 其余、、、、、表達(dá)量均高于野生型。為PR9家族基因編碼過(guò)氧化物酶, 與H2O2代謝相關(guān), 突變體的表達(dá)量的下降符合葉片H2O2含量的變化[26];編碼過(guò)氧化物酶[36];編碼的蛋白屬類甜蛋白質(zhì), 類甜蛋白質(zhì)具有抗真菌活性[26];編碼的苯丙氨酸解氨酶, 是植物防衛(wèi)反應(yīng)的一個(gè)關(guān)鍵蛋白, 涉及SA信號(hào)途徑[36];為PR10家族基因, 編碼一種噻菌靈誘導(dǎo)蛋白;編碼的蛋白屬于類絲氨酸羧肽酶, 常被看作水稻抗病反應(yīng)的標(biāo)記基因[37];為系統(tǒng)獲得抗性中SA信號(hào)途徑的標(biāo)記[10]。結(jié)果表明,的突變可能激活部分病程相關(guān)因子, 引起水稻防衛(wèi)反應(yīng)。

目前在第7染色體已報(bào)道的類病斑突變體有、?；蛞驯豢寺? 定位在第7染色體的短臂, 與定位區(qū)域相距較遠(yuǎn), 且表型不同, 即基因與不是同一基因[20];目標(biāo)基因定位區(qū)間包含了已報(bào)道類病斑早衰突變體的定位區(qū)間, 兩者表型極為相似, 細(xì)微區(qū)別之處在于單株有效穗數(shù)以及每穗總粒數(shù)沒(méi)有差異或差異不明顯, 而表現(xiàn)出極顯著的差異[31], 鑒于與的目的基因均尚不確定, 兩者是否由同一基因突變有待進(jìn)一步研究。

4 結(jié)論

是EMS誘變得到的類病斑早衰突變體, 早期表型正常, 分蘗早期出現(xiàn)病斑, 孕穗期開(kāi)始衰老, 斑點(diǎn)數(shù)目隨植株生長(zhǎng)而增多。植株變矮, 結(jié)實(shí)率降低, 千粒重下降。葉片中光合色素含量降低, 細(xì)胞內(nèi)葉綠體數(shù)目減少, 葉綠體的類囊體片層結(jié)構(gòu)損傷降解。光合色素的減少和葉綠體的降解引起光合效率降低, 進(jìn)而引起突變體植株表型的相應(yīng)變化。突變體類病斑的產(chǎn)生受光照誘導(dǎo), 并受ROS脅迫, 其突變能激活部分病程相關(guān)因子。該性狀受1對(duì)隱性核基因控制, 被定位于第7染色體長(zhǎng)臂端粒附近的CY7-8和CY7-60兩個(gè)分子標(biāo)記之間, 物理距離約為167.3 kb。

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Phenotypic characterizing and gene mapping of aand() mutant in rice (L.)

XIA Sai-Sai, CUI Yu, LI Feng-Fei, TAN Jia, XIE Yuan-Hua, SANG Xian-Chun, and LING Ying-Hua*

Rice Research Institute of Southwest University / Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crops, Chongqing 400715, China

An elite ricerestorer line, Jinhui 10 was chemically mutated with ethyl methane sulfonate (EMS). A novel and stable mutant,() was identified from the descendant library. The growth and development ofplant was normal at seedling stage, but its leaves began to exhibit brown spots at the beginning of tillering stage, and the number of the spots increased as the plant growing up. When mutant plant entered booting stage, leaves started to wilt and became yellowish, then exhibited premature senescence. Compared with wild type, grains per panicle ofdecreased by 8%, significantly lower than that of wild type (< 0.05). Besides, plant height, panicle length, effective panicle number, filled grain number per panicle, seed setting rate, and 1000-grain weight ofcorrespondingly decreasedly 14.3%, 24.3%, 27.2%, 50.0%, 45.7%, and 14.5%, statistically lower than those of wild type (< 0.01). The shading treatment suggested that the mutant performance ofwas light induced. Within the cells of, both of photosynthetic pigment content and photosynthetic efficiency decreased. At the same time, H2O2content increased, and activities of protective enzymes, SOD and CAT, reduced in. The observation by transmission electronic microscope (TEM) demonstrated that chloroplast number reduced in, and the chloroplast thylakoid lamellar structure seriously damaged and degraded. As a result of qRT-PCR, the expression levels of,,,,, andwere higher inthan in wild type, while that ofwas lower than that of wild type. The genetic analysis indicated that the lesion mimic and premature senescence ofwas under the control of a recessive nuclear gene. Mapping result via F2population derived from the cross between Xinong 1A andshowed that, the target gene was located in a 167.3 kb physical interval near the telomere of the long arm of chromosome 7 in rice (L.).

rice (L.);(); phenotyping; genemapping

通信作者(Corresponding author): 凌英華, E-mail: lingyh003@126.com

2018-04-25;

2018-08-20;

2018-09-18.

10.3724/SP.J.1006.2019.82022

本研究由重慶市社會(huì)事業(yè)與民生保障科技創(chuàng)新專項(xiàng)項(xiàng)目(cstc2016shms-ztcx80012)和中央高?；究蒲袠I(yè)務(wù)費(fèi)專項(xiàng)資金(XDJK2016A013)資助。

This study was supported by the project of Chongqing Science and Technology Commission Grants (cstc2016shms-ztcx80012) and Fundamental Research Funds for the Central Universities (XDJK2016A013).

URL:http://kns.cnki.net/kcms/detail/11.1809.S.20180915.1120.006.html