本文關(guān)鍵詞：小麥TaRCR1和TaAGC1基因在防御紋枯病反應(yīng)中的功能與作用機(jī)制研究　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 小麥 紋枯病 NB-LRR 蛋白激酶 活性氧

【摘要】：近年來(lái),由于氣候變暖、耕作制度和種植密度的改變,由禾谷絲核菌(Rhizoctonia cerealis)引起的小麥紋枯病(wheat sharp eyespot),已發(fā)展為我國(guó)主要的病害,連續(xù)十多年發(fā)病面積超過(guò)一億畝,損失嚴(yán)重。生產(chǎn)上抗紋枯病小麥品種匱乏,因此分離克隆抗紋枯病相關(guān)基因,研究其功能和作用機(jī)制,為抗紋枯病小麥育種提供基因和理論支撐,具有重要意義。NB-LRR類(lèi)蛋白和蛋白激酶是植物抗病反應(yīng)中兩種重要的蛋白。本研究主要以紋枯病誘導(dǎo)的一個(gè)小麥NB-LRR類(lèi)蛋白編碼基因TaRCR1和一個(gè)蛋白激酶基因TaAGC1過(guò)表達(dá)的小麥和沉默表達(dá)的小麥為實(shí)驗(yàn)材料,研究了這兩個(gè)基因在小麥抗紋枯病反應(yīng)中功能和作用機(jī)制。TaRCR1編碼NB-LRR類(lèi)蛋白,參與小麥對(duì)紋枯病菌的防御反應(yīng)。本研究繼續(xù)對(duì)TaRCR1基因的分子生物學(xué)特性、抗病功能和作用機(jī)制進(jìn)行了研究,主要結(jié)果如下:(1)TaRCR1受紋枯菌誘導(dǎo)表達(dá),在接種紋枯菌7天后達(dá)到表達(dá)高峰,并且在根和莖中的表達(dá)量高于葉和穗中的表達(dá)量。(2)利用PEG介導(dǎo)GFP-TaRCR1融合蛋白在小麥原生質(zhì)體中瞬時(shí)表達(dá),進(jìn)行亞細(xì)胞定位分析,結(jié)果顯示TaRCR1分布于小麥的胞質(zhì)和細(xì)胞核中。(3)利用PCR、q RT-PCR和Westernblot技術(shù),對(duì)轉(zhuǎn)Ta RCR1基因小麥T2-T4代植株進(jìn)行轉(zhuǎn)基因分子特征分析,結(jié)果表明,在6個(gè)轉(zhuǎn)基因小麥株系中轉(zhuǎn)入的TaRCR1能夠遺傳、轉(zhuǎn)錄和過(guò)量表達(dá);對(duì)TaRCR1過(guò)表達(dá)轉(zhuǎn)基因小麥株系和未轉(zhuǎn)基因小麥揚(yáng)麥16(受體)接種紋枯菌進(jìn)行抗病性鑒定,結(jié)果表明TaRCR1基因的過(guò)表達(dá)提高了小麥對(duì)紋枯病的抗性。(4)應(yīng)用大麥條紋花葉病毒(barley stripe mosaic virus,BSMV)誘導(dǎo)的基因沉默(virus-induced gene silencing,VIGS)技術(shù),摩擦接種BSMV:TaRCR1病毒,以沉默抗紋枯病小麥CI12633中TaRCR1基因的表達(dá),再接種紋枯病菌進(jìn)行抗病程度分析,結(jié)果說(shuō)明TaRCR1基因表達(dá)量的降低,減弱了寄主小麥對(duì)紋枯病的抗性。(5)利用qRT-PCR分析14個(gè)防御相關(guān)基因在TaRCR1基因過(guò)表達(dá)小麥和受體揚(yáng)麥16以及Ta RCR1基因表達(dá)沉默和對(duì)照小麥植株中的轉(zhuǎn)錄水平,結(jié)果表明Ta RCR1正向調(diào)控PR1、PR2、chitinase2和Ta PIE1的表達(dá)水平,這四個(gè)防御相關(guān)基因的表達(dá)還受紋枯菌的誘導(dǎo)。(6)H_2O_2和茉莉酸(jasmonic,JA)處理誘導(dǎo)Ta RCR1基因的表達(dá)水平,且H_2O_2預(yù)處理可以提高紋枯菌侵染對(duì)Ta RCR1基因的誘導(dǎo)表達(dá)水平;Ta RCR1所調(diào)控的PR1、PR2、chitinase2和Ta PIE1基因也受H_2O_2的誘導(dǎo)表達(dá)。(7)接種紋枯菌12 h后,Ta RCR1正向調(diào)控CAT1、POX2等活性氧(reactive oxygen species,ROS)清除相關(guān)基因的轉(zhuǎn)錄表達(dá)以及過(guò)氧化物酶(peroxidase,POD)的活性,負(fù)向調(diào)控ROS合成關(guān)鍵基因NOX的轉(zhuǎn)錄表達(dá);DAB和NBT染色結(jié)果顯示,Ta RCR1基因過(guò)量表達(dá)減弱了紋枯菌侵染引起的ROS積累;對(duì)轉(zhuǎn)Ta RCR1基因小麥噴施POD合成的抑制劑Na N3降低了Ta RCR1過(guò)表達(dá)植株對(duì)紋枯病的抗性,說(shuō)明小麥體內(nèi)ROS的動(dòng)態(tài)平衡對(duì)于Ta RCR1介導(dǎo)的紋枯病抗性非常重要,Ta RCR1通過(guò)調(diào)控ROS相關(guān)基因的表達(dá)維持ROS的動(dòng)態(tài)平衡,進(jìn)而正向調(diào)控小麥對(duì)紋枯病的抗性反應(yīng)。(8)利用Anti-c-Myc Agarose純化Ta RCR1過(guò)表達(dá)小麥體內(nèi)總蛋白,用胰蛋白酶進(jìn)行酶解處理后進(jìn)行質(zhì)譜分析,篩選到一個(gè)Ta RCR1互作蛋白Ta PP2Ac,酵母雙雜交實(shí)驗(yàn)證明Ta RCR1與Ta PP2Ac相互作用;BSMV-VIGS實(shí)驗(yàn)證明,Ta PP2Ac的表達(dá)沉默提高了小麥對(duì)紋枯病的抗性;同時(shí),Ta PP2Ac的q RT-PCR分析結(jié)果顯示,Ta PP2Ac在Ta RCR1過(guò)表達(dá)小麥中的轉(zhuǎn)錄水平顯著低于受體小麥,而在Ta RCR1基因表達(dá)沉默小麥植株中的表達(dá)水平顯著高于對(duì)照植株中,推測(cè)Ta RCR1通過(guò)負(fù)調(diào)控Ta PP2Ac基因的表達(dá)水平提高小麥對(duì)紋枯病的抗性。本課題組前期研究表明,一個(gè)小麥蛋白激酶基因TaAGC1的過(guò)量表達(dá)可以顯著增強(qiáng)轉(zhuǎn)基因小麥對(duì)紋枯病的抗性反應(yīng),而該基因的表達(dá)沉默削弱了小麥對(duì)紋枯病的抗性。本文對(duì)TaAGC1抗紋枯病作用的機(jī)制進(jìn)行了研究,取得以下結(jié)果:(1)TaAGC1響應(yīng)H_2O_2的脅迫,H_2O_2處理后TaAGC1基因轉(zhuǎn)錄水平顯著升高且在處理后3h達(dá)到表達(dá)高峰,這暗示TaAGC1基因可能參與H_2O_2信號(hào)轉(zhuǎn)導(dǎo)路徑。(2)DAB和NBT染色結(jié)果顯示,紋枯菌的侵染誘導(dǎo)小麥植株產(chǎn)生大量的ROS(H_2O_2和O2-),TaAGC1基因過(guò)表達(dá)可以降低紋枯菌侵染引起的過(guò)量ROS的積累水平。qRT-PCR分析結(jié)果表明ROS消除相關(guān)基因POX2、CAT1和SOD3在Ta AGC1過(guò)表達(dá)株系中的表達(dá)量顯著上調(diào),而其在TaAGC1沉默表達(dá)植株中的表達(dá)量明顯降低;ROS合成關(guān)鍵基因NOX的表達(dá)模式與以上3個(gè)基因的相反,即在TaAGC1過(guò)表達(dá)中表達(dá)量最低,在TaAGC1沉默表達(dá)植株中的表達(dá)量最高。(3)ns LTP1、defensin、chitinase2和PR10等防御標(biāo)記基因的表達(dá)受紋枯菌誘導(dǎo);TaAGC1正向調(diào)控nsLTP1和defensin的表達(dá),而負(fù)向調(diào)控chitinase2和PR10的表達(dá)。由此可知,TaAGC1作為一個(gè)正向調(diào)節(jié)因子,通過(guò)調(diào)控ROS相關(guān)基因和防御基因的表達(dá)介導(dǎo)小麥對(duì)紋枯病的抗性反應(yīng)。
[Abstract]:In recent years , due to the changes of climate warming , cropping system and planting density , wheat sheath blight caused by Fusarium cerealis has been developed as the main disease in China . The results showed that the expression of TaRCR1 gene was higher than that in leaves and ears . The results showed that the expression of TaRCR1 gene was higher than that in leaf and ear . ( 4 ) The expression of TaRCR1 gene in wheat CI12633 induced by barley stripe mosaic virus was studied . The results showed that the expression level of TaRCR1 gene was decreased and the expression level of TaRCR1 gene was reduced . The results showed that the expression of TaRCR1 gene was decreased and the expression level of Ta RCR1 gene was decreased . ( 7 ) After 12 hours of inoculation , the expression of reactive oxygen species ( ROS ) , such as CAT1 , POX2 and other reactive oxygen species ( ROS ) , was positively regulated by Ta RCR1 . The expression of ROS was negatively regulated by reactive oxygen species ( ROS ) . ( 1 ) The expression level of TaAGC1 gene TaAGC1 was significantly higher than that in control plants . ( 3 ) The expression of defensive marker genes , such as ns LTP1 , deferent , chitinase2 and PR10 , was induced by stridicum ; TaAGC1 regulates the expression of nsLTP1 and deferent and negatively regulates the expression of chitinase2 and PR10 . Thus , TaAGC1 serves as a positive regulation factor to mediate the resistance of wheat to sheath blight by regulating the expression of ROS - related genes and defense genes .

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S435.121.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 蔡士賓;任麗娟;顏偉;吳紀(jì)中;陳懷谷;吳小有;張仙義;;小麥抗紋枯病種質(zhì)創(chuàng)新及QTL定位的初步研究[J];中國(guó)農(nóng)業(yè)科學(xué);2006年05期

2 任麗娟,蔡士賓,湯，

本文編號(hào)：1405365