發(fā)布時間：2018-05-25 02:38

  本文選題：水稻 + 擬南芥　； 參考：《沈陽農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：開花期在水稻(Oryza sativa)發(fā)育和產(chǎn)量形成過程中起到非常重要的作用。水稻開花期是內(nèi)源因素和外界的環(huán)境條件相互作用、多基因參與的、復(fù)雜的生命過程。雖然,目前已經(jīng)初步建立了 Heading date 1和Early heading date 1為關(guān)鍵調(diào)節(jié)因子的促進(jìn)或抑制水稻開花的分子調(diào)控網(wǎng)絡(luò)。但是,水稻開花期調(diào)控的分子機(jī)制還遠(yuǎn)不清楚。本項目借助生物信息學(xué)方法,利用擬南芥(Arabidopsis thaniala)中開花時間相關(guān)基因Flowering bHLHs(FBHs)基因家族的序列,篩選獲得了水稻中的同源基因OsFBHs;利用CRISPR-Cas9基因編輯技術(shù)、DNA重組和水稻轉(zhuǎn)基因技術(shù),創(chuàng)制了OFBVHs基因超表達(dá)、下調(diào)表達(dá)或基因定點(diǎn)敲除的水稻或擬南芥轉(zhuǎn)基因材料;經(jīng)過對轉(zhuǎn)基因株系的農(nóng)藝性狀觀察,篩選獲得了開花期等農(nóng)藝性狀發(fā)生變化的OsFBH1和OsFBH3基因的轉(zhuǎn)基因株系。利用這些開花期改變的轉(zhuǎn)基因株系為試材,采用熒光定量RT-PCR,酵母單(雙)雜交,染色質(zhì)免疫共沉淀(Chromatin Immunoprecipitation,ChIP)等技術(shù),本項目深入研究了OsFBH 的表達(dá)模式、亞細(xì)胞定位、調(diào)控開花期的靶基因;揭示了 OsFBHs調(diào)控水稻開花期的分子機(jī)制。為不同生育期的水稻品種改良,提高水稻的適應(yīng)性、產(chǎn)量和品質(zhì)提供理論依據(jù)。本項目的主要研究結(jié)果如下:1.水稻OsFBHs與擬南芥FBHs基因進(jìn)化上保守,而且親緣關(guān)系較近。擬南芥中FBHs基因家族有4個成員(包括:FBH1-4-),編碼包含有HLH保守結(jié)構(gòu)域的蛋白質(zhì)。水稻中有4個FBHs的同源基因(OsFBH1-4)與擬南芥中的FBHs親源關(guān)系較近。其中OsFBH1和OsFBH2與擬南芥的FBH1和FBH2的親緣關(guān)系較近,OsFBH3和OsFBH4與擬南芥中的FBH3和FBH4的親緣關(guān)系較近。2.OsFBH1的表達(dá)模式和亞細(xì)胞定位分析。熒光定量RT-PCR與GUS染色的結(jié)果表明,OsFBH1在水稻的葉片和葉鞘中表達(dá)水平較高,在根部和胚芽鞘組織中也有表達(dá)。OsFBH1的表達(dá)受生物鐘的調(diào)控,其表達(dá)高峰出現(xiàn)在夜間。不同的生長發(fā)育時期,OsFBH1的表達(dá)水平不同。在播種后50天,OsFBH1出現(xiàn)表達(dá)高峰。激光共聚焦顯微鏡觀察結(jié)果表明,OsFBH1所編碼的蛋白質(zhì)定位在細(xì)胞核中。3.超表達(dá)OsFBH1可以明顯延遲擬南芥的開花時間。與野生型(wild-type,WT)相比,擬南芥OsFBH1超表達(dá)轉(zhuǎn)基因株系的蓮座葉數(shù)目顯著增加,現(xiàn)蕾時間及第一朵花開放時間明顯延遲。發(fā)育時期轉(zhuǎn)換的試驗結(jié)果表明,擬南芥OsFBH1的超表達(dá)轉(zhuǎn)基因株系,使得擬南芥幼年葉和成年葉增加,延長了幼年營養(yǎng)生長期和成年營養(yǎng)生長期,從而延長了擬南芥從營養(yǎng)生長向生殖生長過渡的時間,最終引起擬南芥晚花。分子水平檢測結(jié)果表明,OsFBH1表達(dá)上調(diào),使促進(jìn)擬南芥開花的關(guān)鍵基因,例如:Flowering Locus T(FT),Twin sister of FT,Suppressor of Overexpression of CONSTANS1(SOC1)的表達(dá)水平下調(diào),從而延遲擬南芥開花。4.超表達(dá)OsFBH1可以明顯的延遲水稻的開花期。與野生型(沈農(nóng)9816,SN9816)相比,超表達(dá)OsFBH1水稻轉(zhuǎn)基因株系開花期明顯延遲。分子水平檢測結(jié)果表明,OsFBH1表達(dá)上調(diào),導(dǎo)致促進(jìn)水稻開花的關(guān)鍵基因,例如:Heading Date 3a和Rice Flowering locus T1的表達(dá)水平明顯降低,從而,延遲水稻開花時間。此外,超表達(dá)OsFBH1水稻轉(zhuǎn)基因株系還表現(xiàn)為株高降低,水稻籽粒寬度變窄,分蘗數(shù)減少等多種生長發(fā)育異常的表型。5.超表達(dá)OsFBH3促進(jìn)擬南芥開花。與WT相比,超表達(dá)OsFBH3擬南芥轉(zhuǎn)基因株系表現(xiàn)為早花。蓮座葉數(shù)目明顯減少,現(xiàn)蕾時間和開花時間明顯早于WT。發(fā)育時期轉(zhuǎn)換的試驗結(jié)果表明,OsFBH3沒有影響幼年營養(yǎng)生長期向成年營養(yǎng)生長期的時期轉(zhuǎn)換,主要是縮短了成年營養(yǎng)生長期,促進(jìn)了營養(yǎng)生長向生殖生長的時期轉(zhuǎn)換,進(jìn)而導(dǎo)致擬南芥開花提前。RT-PCR的結(jié)果表明,在長日照和短日照條件下,擬南芥OsFBH3超表達(dá)轉(zhuǎn)基因株系提高CONSTANS的表達(dá)。從而提高SOC1和FT的表達(dá)水平,促進(jìn)了擬南芥開花。除了影響開花期外,超表達(dá)OsFBH3還導(dǎo)致擬南芥其它方面的生長發(fā)育異常,表現(xiàn)在株型變小,葉片和花變小,花苔變細(xì),果莢變短,胚胎數(shù)目減少,敗育胚胎數(shù)目增加等方面。6.OsFBH 的表達(dá)模式和亞細(xì)胞定位分析。熒光定量RT-PCR和GUS染色的研究結(jié)果表明,OsFBH3主要在水稻的葉片,葉鞘和莖干中表達(dá);在不同時期OsFBH3的表達(dá)水平不同,在播種后50天左右,OsFBH3出現(xiàn)表達(dá)高峰。OsFBH3的表達(dá)受生物鐘的調(diào)控,表達(dá)最高峰出現(xiàn)在夜間。激光共聚焦顯微鏡觀察結(jié)果表明,OsFBH3所編碼的蛋白質(zhì)定位于細(xì)胞核。
[Abstract]:The flowering period plays a very important role in the development of rice (Oryza sativa) and the process of yield formation. The flowering period of rice is the interaction of endogenous and external environmental conditions, multi gene participation, and complex life process. Although Heading date 1 and Early heading date 1 have been preliminarily established as the key regulating factors However, the molecular mechanism of Rice Flowering Regulation is far from clear. With the help of bioinformatics, this project uses the sequence of the Flowering bHLHs (FBHs) gene family of the flowering time related gene of Arabidopsis (Arabidopsis thaniala) to select the homologous gene OsFBHs in rice. Using CRISPR-Cas9 gene editing technique, DNA recombination and rice transgenic technology, the overexpression of OFBVHs gene was created, and the transgenic rice or Arabidopsis thaliana was downregulated and the genes were knocked out of the gene. After observing the agronomic characters of the transgenic lines, the transformation of the OsFBH1 and OsFBH3 genes of the agronomic traits, such as the flowering period, was obtained. In this project, we used these transgenic lines as test materials, using fluorescence quantitative RT-PCR, yeast single (double) hybridization, chromatin immunoprecipitation (Chromatin Immunoprecipitation, ChIP) and so on. This project deeply studied the expression pattern of OsFBH, subcellular localization and regulation of the target gene of flowering period, and revealed that OsFBHs regulates rice. The molecular mechanism of flowering period is a theoretical basis for improving rice varieties at different growth stages, improving rice adaptability, yield and quality. The main results of this project are as follows: 1. the FBHs gene of rice OsFBHs and Arabidopsis thaliana are conserved and closely related. There are 4 members of the FBHs gene family in Arabidopsis (including: FBH1-4-). The code contains proteins with HLH conserved domains. 4 FBHs homologous genes (OsFBH1-4) in rice are closely related to FBHs affinity in Arabidopsis. OsFBH1 and OsFBH2 are closely related to FBH1 and FBH2 in Arabidopsis, and OsFBH3 and OsFBH4 are closely related to FBH3 and FBH4 in Arabidopsis. The results of localization analysis. The results of fluorescence quantitative RT-PCR and GUS staining showed that the expression level of OsFBH1 in rice leaves and Ye Qiaozhong was higher. The expression of.OsFBH1 in the root and the coleoptile tissues was also regulated by the biological clock. The expression peak appeared at night. The expression level of OsFBH1 was different. 50 days after sowing, OsFBH1 showed a peak expression. The results of laser confocal microscopy showed that the.3. overexpression of the protein encoded by OsFBH1 in the nucleus could significantly delay the flowering time of Arabidopsis. Compared with the wild type (wild-type, WT), the number of lotus leaves in the transgenic lines of OsFBH1 overexpressed in Arabidopsis was increased significantly, and the bud time and the first bud time were increased. The opening time of a flower was obviously delayed. The experimental results of the developmental period transformation showed that the overexpression transgenic line of Arabidopsis OsFBH1 made the young leaf of Arabidopsis and adult Ye Zengjia prolonged the vegetative growth period and the adult vegetative growth period, thus prolonging the time of the Arabidopsis thaliana transition from the camps to the reproductive growth. The results of molecular level detection showed that the expression of OsFBH1 was up-regulated, which made the key genes promoting the flowering of Arabidopsis, such as Flowering Locus T (FT), Twin sister of FT, Suppressor of Overexpression. Compared with the wild type (Shen Nong 9816, SN9816), the flowering period of the transgenic lines of OsFBH1 rice was obviously delayed. The results of molecular level detection showed that the expression of OsFBH1 was up, leading to the key genes promoting the flowering of rice, such as the expression level of Heading Date 3a and Rice Flowering locus T1, so that the rice flowering was delayed. In addition, the overexpression of OsFBH1 transgenic lines also showed that the height of the plant decreased, the width of the rice grain was narrowed, the number of tillers decreased and the number of.5. overexpressed OsFBH3 to promote the flowering of Arabidopsis. Compared with WT, the overexpression of OsFBH3 in the transgenic plants of Arabidopsis thaliana was shown to be early flowers. The experimental results of the flowering time, which was earlier than the WT. development period, showed that OsFBH3 did not affect the period of young vegetative growth period to the period of adult vegetative growth, which mainly shortened the adult vegetative growth period, promoted the period of vegetative growth to the period of reproductive growth, and then led to the result of early flowering of Arabidopsis.RT-PCR. Under the condition of long sunshine and short sunshine, the OsFBH3 overexpression of Arabidopsis thaliana can increase the expression of CONSTANS, thus improving the expression level of SOC1 and FT and promoting the flowering of Arabidopsis. In addition to the flowering period, the overexpression of OsFBH3 also leads to the growth and development of Arabidopsis, which shows the smaller plant type, the smaller leaves and flowers, and the flower moss. The expression pattern and subcellular location analysis of.6.OsFBH, the results of fluorescence quantitative RT-PCR and GUS staining showed that OsFBH3 was mainly expressed in the leaves, leaf sheath and stem of rice, and the expression level of OsFBH3 was different at different times, 50 days after sowing, OsF The expression of the expression peak of BH3 was regulated by the biological clock, and the peak expression appeared at night. The results of laser confocal microscopy showed that the protein encoded by OsFBH3 was located in the nucleus.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S511

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1 閆志強(qiáng);水稻OsFBHs的功能及其分子機(jī)理研究[D];沈陽農(nóng)業(yè)大學(xué);2017年

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本文編號：1931722