本文選題：水稻　切入點(diǎn)：葉片衰老　出處：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：葉片衰老是葉片生長(zhǎng)發(fā)育的最后一個(gè)階段,研究葉片衰老對(duì)整株植物而言具有重要意義。蛋白磷酸酶催化的蛋白質(zhì)去磷酸化在植物多種生命過(guò)程中有重要地位,其中2C型蛋白磷酸酶(PP2C)是植物中數(shù)量較多的一類(lèi),但關(guān)于PP2C參與水稻葉片衰老進(jìn)程調(diào)控的報(bào)道還比較少。因此,關(guān)于參與水稻葉片衰老過(guò)程中蛋白磷酸酶基因的鑒定、克隆及功能分析具有重要意義。1、水稻參與葉片衰老調(diào)控的PP2C候選基因的篩選在前期實(shí)驗(yàn)中,通過(guò)公共Microarray芯片數(shù)據(jù)分析、生物信息學(xué)計(jì)算結(jié)合序列同源性比對(duì),篩選到7個(gè)可能參與水稻葉片衰老進(jìn)程調(diào)控的PP2C基因,命名OsS4PP1-7.在此基礎(chǔ)上利用熒光定量RT-PCR檢測(cè)OsSAPP1-7在人工黑暗誘導(dǎo)水稻衰老過(guò)程中的表達(dá)情況,發(fā)現(xiàn)OsSAPPl、OsSAPP2、OsAPP3、OsSAPP5和OsSAPP7變化較顯著,隨后在自然衰老中發(fā)現(xiàn)OsS4PP2、OsS4PP3表達(dá)變化最強(qiáng)烈,因此將OsSAPP2、OsSAPP3作為后續(xù)研究的目標(biāo)基因。2、OsS4PP2基因的克隆與植物雙元表達(dá)載體構(gòu)建構(gòu)建了過(guò)表達(dá)植物雙元表達(dá)載體(35S-OsSAPP2、35S-OsSAPP3)、啟動(dòng)子-GUS報(bào)告基因植物雙元表達(dá)載體(ProossAPP2-GUS、ProossAP3-GUS),利用農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化法對(duì)擬南芥和水稻進(jìn)行了遺傳轉(zhuǎn)化,通過(guò)卡那霉素或潮霉素抗性篩選、GUS組織化學(xué)染色鑒定、PCR檢測(cè)、熒光定量RT-PCR檢測(cè),獲得相應(yīng)的轉(zhuǎn)基因株系。3、35S-OsSAPP3轉(zhuǎn)基因植物的獲得實(shí)驗(yàn)中無(wú)法獲得35S-OsSAPP3轉(zhuǎn)基因水稻陽(yáng)性植株,同樣組成型啟動(dòng)子過(guò)表達(dá)OsS4PP3基因?qū)е罗D(zhuǎn)基因擬南芥早期衰亡無(wú)法完成生活史,推測(cè)OsSAPP3在組成型啟動(dòng)子驅(qū)動(dòng)下過(guò)表達(dá)會(huì)導(dǎo)致植物死亡。因此將組成型啟動(dòng)子替換成可誘導(dǎo)型啟動(dòng)子,重新構(gòu)建植物雙元表達(dá)載體、獲得轉(zhuǎn)基因植物后再進(jìn)行分析。4、35S-OsSAPP2轉(zhuǎn)基因植物的表型觀察35S-OsSAPP2轉(zhuǎn)基因水稻和擬南芥葉片褪色較快,萎蔫衰老情況加劇,葉綠素含量下降速率高于對(duì)照。35S-OsSAPP2轉(zhuǎn)基因擬南芥抽苔和開(kāi)花時(shí)間較早,蓮座葉小且少,株高較高,種子千粒重較小和籽粒長(zhǎng)寬比較大。另外,在黑暗誘導(dǎo)衰老下,35S-OsSAPP2轉(zhuǎn)基因擬南芥葉片黃化嚴(yán)重、葉綠素含量下降幅度大。以上結(jié)果表明OsSAPP2過(guò)表達(dá)會(huì)引起轉(zhuǎn)基因植物早衰。5、35S-OsSAPP2轉(zhuǎn)基因植物的基因表達(dá)分析水稻衰老標(biāo)志基因OsSM-1和OsSM-2在35S-OsSAPP2轉(zhuǎn)基因水稻葉片中的表達(dá)量均上升,而OsTZF1的表達(dá)量下降;35S-OsSAPP2轉(zhuǎn)基因擬南芥葉片衰老相關(guān)基因(S4G2)、衰老關(guān)鍵轉(zhuǎn)錄因子(NAC1NAC2、AN4CP、WRKY6)和葉綠素降解關(guān)鍵酶編碼基因(ACD1)的表達(dá)量較野生型高且上升快;光合作用相關(guān)基因(RbcL、RbcS)的表達(dá)量大幅度下降,在人工黑暗誘導(dǎo)衰老下也觀察到了類(lèi)似結(jié)果。6、35S-OsSAPP2轉(zhuǎn)基因植物對(duì)植物激素的響應(yīng)植物衰老促進(jìn)激素乙烯和脫落酸會(huì)加速35S-OsSAPP2轉(zhuǎn)基因水稻的離體葉片早衰,而植物衰老抑制激素生長(zhǎng)素可以有效緩解由OsSAPP2基因過(guò)表達(dá)造成的水稻葉片早衰。綜上所述,本研究發(fā)現(xiàn)過(guò)表達(dá)OsS4PP2轉(zhuǎn)基因植物具有早衰表型,因此認(rèn)為OsSAPP2基因是水稻葉片衰老的正向調(diào)節(jié)因子。
[Abstract]:Leaf senescence is the last stage of leaf growth, leaf senescence research has important significance for the whole plant. Protein phosphatase catalytic protein phosphorylation plays an important role in many biological processes in plants, including type 2C protein phosphatase (PP2C) is a kind of large number of plants, but the involvement of PP2C in rice regulation of leaf senescence was rarely reported. Therefore, the identification of protein phosphatase involved during leaf senescence in rice gene, cloning and functional analysis of.1 has an important significance, screening the candidate gene of PP2C in rice leaf senescence regulation in the previous experiment, through the public Microarray microarray data analysis, bioinformatics computing with sequence homology comparison on screening to 7 may be involved in rice leaf senescence regulation of PP2C gene, named OsS4PP1-7. on the basis of the use of fluorescent quantitative RT-P CR OsSAPP1-7 was detected in dark induced expression of artificial aging process in rice, OsSAPPl, OsSAPP2, OsAPP3, OsSAPP5 and OsSAPP7 change significantly, then found OsS4PP2 in natural senescence, the expression of OsS4PP3 is the strongest, so OsSAPP2, OsSAPP3 as the following target genes of.2, OsS4PP2 gene cloning and plant expression vector construction of over expression of plant binary expression vector (35S-OsSAPP2,35S-OsSAPP3), binary expression vector of -GUS promoter reporter gene plants (ProossAPP2-GUS, ProossAP3-GUS), using Agrobacterium mediated genetic transformation method for genetic transformation of Arabidopsis and rice by kanamycin or hygromycin resistance screening, GUS histochemical staining, PCR assay, fluorescence quantitative RT-PCR detection, to obtain the corresponding transgenic lines of.3,35S-OsSAPP3 transgenic plants was not obtained in the experiment 35S-O SSAPP3 transgenic rice plants were the same, the constitutive promoter of OsS4PP3 gene in transgenic Arabidopsis early decline to complete life history expression, suggesting that OsSAPP3 in the drive constitutive promoter over expression may lead to the death of plants. So the constitutive promoter replaced inducible promoter, construction of plant binary expression vector observation 35S-OsSAPP2 transgenic rice and Arabidopsis leaves fade faster phenotype of.4,35S-OsSAPP2 transgenic plants were obtained after analysis of transgenic plants, increased the chlorophyll content decreased wilting and aging, the rate is higher than that of the control.35S-OsSAPP2 transgenic Arabidopsis bolting and flowering time earlier, rosette Ye Xiaoqie, higher plant height, smaller seed weight and grain length width is relatively large. In addition, in the dark induced senescence, 35S-OsSAPP2 transgenic Arabidopsis leaves chlorophyll content decreased seriously. Large amplitude. The above results indicated that overexpression of OsSAPP2 can cause analysis of rice senescence marker gene expression of OsSM-1 and OsSM-2 in 35S-OsSAPP2 transgenic rice leaves were significantly increased the expression of transgenic plant senescence.5,35S-OsSAPP2 transgenic plants gene, whereas the expression of OsTZF1 decreased; 35S-OsSAPP2 transgenic Arabidopsis leaf senescence associated genes (S4G2), a key transcription factor of aging (NAC1NAC2, AN4CP, WRKY6) and encoding key enzymes of chlorophyll degradation gene (ACD1) expression and increased faster than the wild type; photosynthesis related genes (RbcL, RbcS) the expression volume decreased significantly, in the artificial dark induced senescence was also observed under similar results of.6,35S-OsSAPP2 transgenic plants on plant hormone response promoting plant senescence hormone ethylene and abscisic acid can accelerate 35S-OsSAPP2 transgenic rice leaves in vitro senescence, and inhibit growth hormone and plant senescence It can effectively alleviate premature senescence of rice leaves caused by overexpression of OsSAPP2 gene. In conclusion, this study found that overexpressing OsS4PP2 transgenic plants had premature decay phenotype, so OsSAPP2 gene is a positive regulator of leaf senescence in rice.

【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S511
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本文編號(hào)：1666640