水稻低纖維突變體LCM527-1性狀鑒定、遺傳分析與基因定位

童杰鵬, 童川, 王艷, 任三娟, 沈圣泉*

(浙江大學原子核農業(yè)科學研究所,杭州310029)

摘要水稻莖稈機械強度是抗倒伏能力強弱的一項重要指標。為了深入研究水稻莖稈機械強度和抗倒伏特性的內在生理、生化、遺傳和分子等方面機制,經400 Gy60Co γ射線誘變和篩選,獲得了一個特異性低纖維突變體LCM527-1。經考查,該突變體與野生型品種527(WT)相比,除整個生長期間表現出莖稈、葉片、葉鞘、穗頸和枝梗均脆性及極易折斷外,還伴隨有生長勢偏弱、植株變矮、穗數減少、劍葉和穗變短等形態(tài)特性?？沟狗笖?、莖稈單位長度的鮮質量和干質量以及倒一節(jié)和倒二節(jié)抗折斷力均明顯降低,莖稈中Ca、Si、K等重要組成元素顯著減少,莖稈纖維素含量顯著下降,半纖維素含量增高。遺傳分析表明,該低纖維突變性狀受單隱性核基因控制(暫名為lcm527-1),利用SSR分子標記將lcm527-1基因定位于2號染色體的RM3774和RM1092之間,遺傳距離分別為10.6 cM和5.1 cM。這些結果將為今后研究低纖維突變及其精細定位提供依據,也為深入研究水稻抗倒伏機制提供分子生物學支持。

關鍵詞水稻; 低纖維; 突變體LCM527-1; 抗倒伏性; 遺傳分析; 基因定位

中圖分類號Q 343.17; S 511文獻標志碼A

Character identification, genetic analysis and gene mapping of a low cellulose mutant LCM527-1 in rice。 Journal of ZhejiangUniversity(Agric. & LifeSci.), 2015,41(3):261-268

Tong Jiepeng, Tong Chuan, Wang Yan, Ren Sanjuan, Shen Shengquan*(InstituteofNuclearAgriculturalSciences,ZhejiangUniversity,Hangzhou310029,China)

SummaryLow cellulose mutant in rice is a common type of crop culm mutants as reported in Arabidopsis, maize and barley previously. These mutants have obvious traits of fragility in stem and leaf, low mechanical strength and weaker breaking-resistance. A low cellulose mutant 527-1 (LCM527-1) obtained from the offspring of a rice variety 527 by 400 Gy60Co γ irradiation was applied to study it’s biological phenotype, genetic traits and mutant gene, attempting to find reasons for low cellulose mutation and rice lodging. It exhibited fragility with continuity and integrity during the whole period of rice growth. In the process of experiment, some main investigations were made to identify the variation in biological expression, genetic analysis and gene mapping, concerning phenotype observation, agronomic traits, rice quality, chemical mineral elements, inheritance pattern and location of mutation gene. Four crosses were made to inspect the inheritance pattern and genetic stability between the low cellulose mutant527-1 (female parent) and the wild-type 527, Zhe7954, 9311, Minghui63 (male parents). After then, 505 pairs of SSR molecular markers were chosen to map the mutant gene on chromosome, and three markers: RM3774, RM1092 and RM530 presented polymorphism difference. These mapping populations were derived from a cross between LCM527-1 and Zhe7954.

The mutant LCM527-1 showed dramatically decreased content of cellulose, high lodging index, big changes in some agronomic traits and mineral elements content. Genetic analysis indicated the character of mutant LCM527-1 was controlled by a single recessive gene. And genelcm527-1 was roughly located between SSR marker RM3774 and RM1092 on chromosome 2 with genetic distances of 10.6 cM and 5.1 cM, respectively.

Above all, it was concluded that mutant LCM527-1 was of great importance for research of mechanical strength, especially the ability of lodging resistance. The mutant traits appeared single locus mutation and inheritable stably, suggesting some specific traits of LCM527-1 might be controlled by this mutant genelcm527-1 and resulted in some related changes in agronomic traits and mineral elements content. These results are helpful to make a fine mapping and its gene cloning in the fragile mutant; meanwhile, they could also lay a theoretical basis on molecular biology mechanism of rice lodging resistance.

Key wordsrice; low cellulose mutant; LCM527-1; lodging resistance; genetic analysis; gene mapping

水稻低纖維突變體是作物莖稈突變體中常見的一類,在玉米、擬南芥、大麥、小麥中均有報道[1-4]。這些突變體具有明顯的脆硬性狀,莖稈易于折斷,莖稈成分含量和結構發(fā)生變化,機械強度降低,從而影響其抗倒伏能力以及花穗孕育[5]。水稻低纖維相關性狀的產生涉及復雜的生理生化和遺傳調控過程。它的生長過程表現為連續(xù)性和整體性的低纖維脆硬性特點,從幼苗期到成熟期,從莖稈到葉片都有易斷易折的特點[6-7]。

關于水稻低纖維或脆性變異性狀及其控制基因的研究報道較多,迄今已在水稻中發(fā)現近十幾個控制該突變性狀的基因,如bc1-8、bc10-12、bc14-15等基因編碼產物，主要參與纖維素合成、細胞壁組裝、胞壁次級修飾等生物學過程[8]。Li等[9]通過γ誘變得到1個水稻突變體基因bc1,經定位和功能分析發(fā)現該基因位于3號染色體,編碼COBRA磷脂酰錨定蛋白,通過影響纖維素的裝配而影響細胞的生長,降低纖維素,增加木質素,從而調控細胞壁的化學組成和莖稈機械強度。Hirano等[10]研究發(fā)現,BC3突變體植株的纖維素含量降低28%～36%,厚壁細胞的細胞壁變薄,細胞壁結構異常,功能分析發(fā)現bc3編碼膜動力蛋白家族成員OsDRP2B,介導質膜與高爾基體反面管網區(qū)之間的囊泡轉運,可能影響纖維素的轉運組裝而導致胞壁變薄、莖稈變脆的原因。

由此可見,低纖維突變體的成因和產生途徑各有不同,基因突變位點多樣,編碼蛋白家族豐富,功能作用廣泛。為此,開展不同來源的水稻低纖維突變體分子遺傳的研究,對于深入理解復雜的水稻機械強度遺傳本質和抗倒伏機制意義重大。

本研究以60Co γ射線處理水稻品種527,經誘變后代篩選和鑒定,獲得了1份低纖維突變體LCM527-1。該突變體在生長發(fā)育過程中,莖、葉纖維素含量較低,有明顯脆硬性、機械強度較弱,與以往研究莖脆葉硬或葉脆莖硬的突變體具有明顯不同的表型。為此,筆者試圖通過對該突變體LCM527-1的生理、生化、遺傳等方面進行鑒定和分析,評價該突變體及其相關突變基因的理論意義和應用價值,以便為今后進一步開展分子水平的水稻纖維素合成、植株抗倒伏機制研究提供參考依據。

1材料與方法

1.1材料

于2009年利用400 Gy60Co γ射線輻照含水率為13.5%的水稻品種527干種子,在浙江大學華家池校區(qū)實驗農場種植M1。此后,在35 000株誘變M2群體中,發(fā)現了1株葉色稍淡,莖、鞘、葉、枝梗均表現脆性,成熟期易折斷的突變株。經后代M3和M4連續(xù)種植、鑒定和篩選,最終獲得了1份性狀遺傳穩(wěn)定,植株表現脆性,纖維素含量檢測較低的脆性、低纖維突變體,暫命名為LCM527-1(low cellulose mutant)。

1.2方法

1.2.1田間試驗低纖維突變體LCM527-1與527(WT)特征特性及各種抗倒伏相關性狀鑒定試驗于2011年夏在浙江大學紫金港校區(qū)實驗農場進行。5月25日播種,6月25日移栽,種植密度25 cm×25 cm,每小區(qū)種植100株,2次重復。田間管理同生產大田。

1.2.2農藝性狀考查在成熟期,每小區(qū)分別取5株樣品,室內進行水稻形態(tài)性狀和穗部特征的指標考查,包括株高、每株穗數、劍葉長、穗長、每穗總粒數、每穗實粒數、結實率、千粒重、著粒密度等。

1.2.3稻米品質性狀鑒定收取成熟期稻谷,按中華人民共和國農業(yè)部標準(NY147-88)進行稻米品質性狀的鑒定,包括米粒長、米粒寬、長寬比、堊白粒率、堊白度、透明度、堿消值(糊化溫度)、膠稠度、直鏈淀粉含量等。

1.2.4抗倒伏指數、莖稈單位長度的鮮質量和干質量以及抗折斷力測定參照Ookawa等[11]方法并稍加修改。具體做法:抗倒伏指數測定是水稻乳熟期在田間選取10株,用成串的回形針掛于穗頸節(jié)處,讓其自然垂至地面,計算出每單位長度莖稈自然彎曲下所承受的回形針質量(g/cm),該值即為抗倒伏指數。另取10株的主莖剝去葉鞘,測得莖高(根部與穗頸節(jié)之間距離),稱得莖稈的鮮質量和干質量(60 ℃烘干至恒質量),計算得到莖稈單位長度的鮮質量和干質量,g/cm。再取10株的主莖,剝去葉鞘,剪下倒一節(jié)(即穗頸下第一節(jié))和倒二節(jié),置于測定器,節(jié)間中點與測定器中點對應(支點間距5 cm),在節(jié)間中點處掛一盤子,加砝碼至莖稈斷裂,此時砝碼及盤子的質量即為節(jié)間的抗折力。

1.2.5莖稈礦質元素含量測定參照郝虎林等[12]的方法,取乳熟期生長正常植株的莖稈,先將待測樣品105 ℃殺青30 min,70 ℃烘干粉碎,200 ℃電熱板上完全碳化,馬弗爐中550 ℃左右灰化6 h,冷卻后用5 mL體積1∶1優(yōu)級純鹽酸溶解灰分,用純水定量到50 g左右,最后用ICP-MS(Agilent 7500a)測定Ca、Cu、Fe、K、Mg、Mn、Mo、Na、P、S、Si和Zn等12種礦質元素含量。

1.2.6莖稈纖維素、半纖維素、木質素和灰分含量測定按van Soest等[13]方法并稍加改動。取乳熟期植株的莖稈,85 ℃烘干,研磨成粉末;準確稱取1.000 g樣品,放入250-mL三角瓶中,加入100 mL酸性洗滌劑或中性洗滌劑(含2～3 g正辛醇),三角瓶套上冷凝裝置于電爐上加熱,在5～10 min內煮沸,從開始沸騰計時,回流60 min,煮沸完畢后,取下三角燒瓶,將內容物轉入已知質量的坩堝式抽濾器進行減壓抽濾,用90～100 ℃的沸水沖洗3～4次,直至酸性洗滌劑洗凈或者濾液呈中性為止。用40 mL丙酮沖洗濾器2～3次,抽濾。將坩堝置于100 ℃鼓風式干燥箱中干燥3 h,在干燥器中冷卻30 min,稱質量,直至恒質量。按van Soest方法測定和計算莖稈纖維素、半纖維素、木質素和灰分含量。

1.2.7遺傳分析以突變體LCM527-1為母本,水稻品種(系)527、9311、浙7954和明恢63為父本,配制LCM527-1×527、LCM527-1×9311、LCM527-1×浙7954、LCM527-1×明恢63等4個雜交組合。種植并觀察記錄F1的植株及其莖、葉部的特征;次年,種植F2并考查植株和莖、葉的性狀分離情況,分析突變基因的遺傳特性,計算分離比,進行2測驗。

1.2.8突變基因定位選取LCM527-1×浙7954組合,以F2的485個單株作為突變基因的定位群體,提取總DNA。同時,選擇F2分離群體中的正常型株和低纖維突變型株各10株提取DNA,分別混合,構成正常株和低纖維突變體的近等基因池。

抽穗期按單株取葉片,DNA提取參考Porebski等[14]和韓美麗等[15]的方法并加以改正。根據Temnykh等[16-17]公布的相關SSR分子標記信息,選取水稻12條染色體中的505個SSR標記用于連鎖標記基因的初定位。連鎖標記得到后,在http://www.gramene.org搜索距離較近區(qū)段的SSR分子標記,尋找連鎖SSR分子標記。SSR引物由上海Sangon公司合成。PCR反應體系：10×PCR緩沖液2 μL,25 mmol/L dNTPs 1.5 μL,SSR引物2 μL,模板DNA 50～100 ng,5 U/μLTagDNA聚合酶0.2 μL,用ddH2O補足體積至20 μL。反應程序設計為94 ℃預變性5 min;94 ℃變性失活45 s,55 ℃退火復性45 s,72 ℃ 延伸1 min,共35個循環(huán),最后72 ℃延伸7 min。擴增產物用8%聚丙烯酰胺凝膠電泳,銀染檢測。

根據SSR分析結果,分別對低纖維突變體池和正常池的個體賦值,將表現為突變體帶型單株記為1,正常親本帶型單株記為2,同時具有2種帶型單株記為3,利用MAPMAKER 3.0[18]軟件對分離群體的表型和分子標記分離的數據進行連鎖分析,并用Kosambi函數將重組率轉化成遺傳圖距,構建目標基因區(qū)域的分子標記連鎖圖譜。

2結果與分析

2.1突變體LCM527-1的植株性狀和田間表現

LCM527-1生長較為正常,與527(WT)相比,生長勢稍弱、植株變矮、穗數減少、劍葉和稻穗變短,莖稈、葉片、葉鞘及至穗部枝梗均易折易斷,表現出明顯的脆性,不同于正常對照水稻折斷時的韌性和機械強度,憑手感即可分辨(圖1)。

圖1　突變體LCM527-1和527(WT)的表型特征 Fig.1　Phenotype characteristics of mutant LCM527-1 and 527 (WT)

對LCM527-1主要農藝性狀考查發(fā)現,株高、每株穗數、穗長、劍葉長、每穗總粒數、每穗實粒數、結實率等7個性狀均比527(WT)顯著降低,千粒重和著粒密度兩者則未見較大差異(表1),推測該基因突變可能對水稻的整體生長有著或大或小的影響。

2.2突變體LCM527-1稻米品質性狀表現

對LCM527-1和527(WT)進行主要稻米品質性狀鑒定,結果表明,LCM527-1與527(WT)相比,粒型相似,均屬細長粒,長寬比3.05。堊白粒率,堊白度、透明度、糊化溫度(堿消值)、膠稠度和直鏈淀粉含量均無顯著差異(表2),表明該突變基因對稻米品質影響不大。

2.3突變體LCM527-1莖稈抗倒伏指數和抗折斷力

取LCM527-1 和527(WT)乳熟期植株,田間測定抗倒伏指數,并將其帶回室內進行抗倒伏相關機械強度性狀檢測,包括莖稈高度、單位長度的鮮質量和干質量以及抗折斷力性狀。結果表明,LCM527-1與527(WT)相比,抗倒伏指數顯著較小,莖稈單位長度的鮮質量和干質量均明顯降低,倒一節(jié)和倒二節(jié)抗折斷力也顯著減弱(表3)?？梢?，低纖維突變基因對植株抗倒伏機械強度方面相關性狀有顯著影響。

表1　突變體LCM527-1和527(WT)主要農藝性狀比較

*表示在P<0.05水平差異有統(tǒng)計學意義。

* indicate statistically significant difference at the 0.05 probability level.

表2　突變體LCM527-1和527(WT)主要稻米品質性狀

表3　突變體LCM527-1和527(WT)莖稈機械強度性狀

*表示在P<0.05水平差異有統(tǒng)計學意義。

* indicate statistically significant difference at the 0.05 probability level.

2.4突變體LCM527-1莖稈礦質元素含量

乳熟期,取植株莖稈進行主要礦質元素檢測。結果發(fā)現,LCM527-1和527(WT)相比,Ca、K、Mn、Na、Si、Zn等6種元素含量均有明顯降低,Fe含量有所增高,Cu、Mo、P、S含量無顯著差異(表4)。由于Ca元素是細胞壁重要組成,Si元素對加強植株硬度有顯著作用,說明低纖維突變基因表達使植株對Ca和Si的吸收和積累減少,可能是導致莖稈脆性和易倒的主要原因之一。

2.5突變體LCM527-1纖維素、半纖維素和木質素含量

取LCM527-1和527(WT)乳熟期植株,測定莖稈中纖維素、半纖維素和木質素含量。結果表明,LCM527-1莖稈中纖維素含量顯著低于527(WT),表現出明顯低纖維、脆硬性狀;半纖維素含量增高,其差異達顯著水平;木質素含量有升高趨勢,但差異不顯著(表5)。因而,推測該突變體可以通過植物自身“補救”途徑維持莖稈生長。

2.6突變體LCM527-1遺傳分析

表4　突變體LCM527-1和527(WT)莖稈礦質元素含量

*表示在P<0.05水平差異有統(tǒng)計學意義。

* indicate statistically significant difference at the 0.05 probability level.

表5　突變體LCM527-1和527(WT)莖稈纖維素、半纖維素和木質素含量

*表示在P<0.05水平差異有統(tǒng)計學意義。

* indicate statistically significant difference at the 0.05 probability level.

表6　突變體LCM527-1制配的4個雜交組合遺傳性狀分離比

2.7突變體LCM527-1基因定位

選擇覆蓋整個水稻基因組,均勻分布于12條染色體的505對SSR標記,逐條進行2個池之間SSR標記多態(tài)性分析,篩選出80對具有多態(tài)性差異的SSR標記。利用這80對分子標記對F2的群體進行驗證和分析,以明確這種低纖維變異性狀是否與這些SSR之間存在連鎖關系。結果顯示,在第2號染色體上存在有3對微衛(wèi)星引物RM3774,RM1092和RM530,在低纖維突變池與正常株混合池間表現出多態(tài)性,可能與低纖維突變性狀連鎖。利用這些多態(tài)性的微衛(wèi)星標記進一步檢測,確認低纖維突變基因與RM3774、RM1092和RM530這3個分子標記存在不同程度的連鎖(圖2、圖3)。

1: RM3774; 2: RM1092; 3: RM530. 圖2　3個標記在親本間的多態(tài)性 Fig.2　Polymorphisms of three markers between parents

M:DNA標志物; P1:野生親本;P2:突變親本;*:基因型交換植株. M: 100 bp DNA ladder; P1: Wild parent; P2: Mutant parent; *: Genotypes of the plant exchanged. 圖3　SSR標記RM3774,RM1092和RM530在F 2突變株中的差異分離 Fig.3　Separation of F 2 mutants by RM3774, RM1092 and RM530

根據 F2群體的表型和SSR分子標記分離的數據進行連鎖分析,構建了局部連鎖圖,低纖維突變基因lcm527-1位于第2染色體微衛(wèi)星標記RM3774和RM1092之間,與RM3774和RM1092的遺傳距離分別為10.6 cM和5.1 cM(圖4)。

圖4　水稻低纖維突變基因lcm527-1在2號染色體上的部分連鎖圖譜 Fig.4　Linkage map of a low cellulose mutant lcm527-1 on chr2

3討論

水稻低纖維、脆性突變體的產生途徑多樣,如化學誘變、電離輻射、基因插入等,涉及的生理、生化、遺傳和分子機制也不盡相同[19]。目前有關水稻低纖維脆稈相關基因主要有bc1、bc2、bc3、bc4、bc7、bc8、bc12、bc15等,分別位于水稻第3、5、2、6、1、7、9、4等染色體上[20-26]。其中bc1編碼1種COBRA樣蛋白,通過作用于纖維素的組裝而影響細胞壁的厚度和機械強度;bc3通過影響纖維素次生細胞壁合成相關基因CesA4的表達豐度而導致纖維素合成減少,脆性增加;bc12主要參與組織細胞分裂和次生細胞壁加厚過程,其編碼蛋白結合赤霉素合成關鍵基因,導致細胞生長和分裂受到影響;諸如上述不同調控水稻細胞壁成分的基因還有很多,它們以不同的方式作用,最終都導致水稻莖稈易折變脆。

本文的低纖維突變體LCM527-1生物學性狀不僅表現出莖、葉、鞘、穗部枝梗均脆,抗折斷力變弱,抗倒伏指數減小,Ca、Si等主要礦質成分下降,纖維素含量明顯降低,還伴隨著株高變矮、穗數減少、結實率下降等農藝性狀的改變。農藝性狀和纖維素等理化測定結果與劉斌美等[27]和王川麗等[28]的研究結果相近,而抗折斷力和礦質元素等測定更加全面考察了突變體脆性相關因素。從實驗結果可以推測突變基因lcm527-1的表達具有多效性,除了影響植株機械強度外,對植株營養(yǎng)生長和生殖生長也存在一定的負效應。本試驗遺傳分析和基因定位發(fā)現,lcm527-1位于第2染色體SSR標記RM3774和RM1092之間。鑒于與目前國內外已發(fā)表的相關或類似突變基因的比對,尤其是水稻2號染色體上有關控制低纖維突變體的基因至今發(fā)現較少的事實,作者認為該突變基因lcm527-1很可能是新的。推測本試驗低纖維變異性狀的產生可能受lcm527-1控制,通過表達產物影響水稻莖葉細胞壁化學成分的含量改變,或影響細胞壁成分的組裝,從而導致細胞壁組裝材料不足,壁變薄,結構形態(tài)有別于正常生長型水稻,最終表現出機械強度減小,抗倒伏能力減弱的低纖維特性。

由于本試驗采用秈/秈交構建遺傳群體的多態(tài)性較少,難以對該突變基因lcm527-1進行精細定位。因此,作者正構建相關新的秈/粳交遺傳群體,并借助于上述基因初定位和多個性狀的鑒定結果,旨在對該基因進行精細定位、測序和克隆等,進而深入了解突變基因的遺傳表達機制,為闡明水稻植株纖維素合成、代謝及抗倒性分子機制提供理論依據。

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Foundation item： Project supported by National Natural Science Foundation of China (No. 31260179).

*Corresponding author： Yang Bin, E-mail: yangbin48053@163.com

Biography： Wang Dawei, E-mail: wdwchem@163.com

Received： 2014-11-11; Accepted: 2014-12-12; Published online： 2015-05-19

URL:http://www.cnki.net/kcms/detail/33.1247.s.20150519.1252.004.html

Interruption effects of green leaf volatiles to a forest pest pine shoot beetle,Tomicusyunnanensis(Coleoptera, Scolytidae)

Wang Dawei1, Zhao Ning1, Ze Sangzi2, Zhu Jiaying3, Yang Bin1*(1.KeyLaboratoryofForestDisasterWarningandControlinYunnanProvince,SouthwestForestryUniversity,Kunming650224,China; 2.YunnanForestryTechnologicalCollege,Kunming650224,China; 3.CollegeofForestry,SouthwestForestryUniversity,Kunming650224,China)

SummaryThe pine shoot beetle,Tomicusyunnanensis, is one of the most damaging pests of Yunnan pine (Pinusyunnanensis) in southwestern China. In this study, the laboratory behavioral bioassays and field investigations were employed to examine the effects of three most abundant green leaf volatiles (GLVs) [(E)-2-hexenal, (E)-2-hexen-1-ol, (Z)-3-hexen-1-ol] from non-host trees on host location behavior of this forest pest. Y-tube olfactometer bioassays showed that adultT.yunnanensiswere significantly repelled by single GLVs compound or their blends. Field experiments showed that, the increase rates of infested twigs (fed byT.yunnanensis) in all the Yunnan pine groups treated with GLVs compound were lower than control group, and had significant differences from control group. The GLVs and their blends played a negative role in the feeding behavior ofT.yunnanensisin field, and decreased the damage to Yunnan pine. The above results show that, as the interrupting factors, GLVs from non-host plants have the potential to be used in field integrated managements ofT.yunnanensisin the future.

Key wordspine shoot beetle;Tomicusyunnanensis; green leaf volatiles; non-host plant; olfactory bioassay

CLC numberQ 968Document codeA