本文選題：水稻 + 秈粳雜種不育�。� 參考：《華中農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：水稻既是重要的糧食作物,也是單子葉植物基因組研究的模式植物,是生殖隔離研究的模式系統(tǒng)。亞洲栽培稻包括秈稻和粳稻兩個(gè)亞種,其雜種通常高度不育,阻礙了對(duì)亞種間雜種強(qiáng)大雜種優(yōu)勢(shì)的利用。S5是控制秈粳雜種胚囊育性的主要位點(diǎn),包括ORF3,ORF4和ORF5三個(gè)連鎖基因。典型秈稻的基因型為ORF3+/ORF4-/ORF5+,典型粳稻的基因型為ORF3-/ORF4+/ORF5-。細(xì)胞壁蛋白ORF5+與跨膜蛋白ORF4+共同誘導(dǎo)內(nèi)質(zhì)網(wǎng)脅迫進(jìn)而引起細(xì)胞程序性死亡從而導(dǎo)致雌配子敗育,內(nèi)質(zhì)網(wǎng)蛋白ORF3+則能夠阻止或者緩解內(nèi)質(zhì)網(wǎng)脅迫阻止雌配子被殺死從而恢復(fù)正常育性。本研究從分子細(xì)胞學(xué)和轉(zhuǎn)錄組角度對(duì)S5作用機(jī)理進(jìn)行了研究。我們構(gòu)建了粳稻Balilla穗組織的酵母雙雜交文庫。利用ORF3,ORF4和ORF5的各種全長(zhǎng)以及截短的蛋白作為誘餌篩選文庫,并利用ORF5-得到了兩個(gè)候選基因,但是經(jīng)過進(jìn)一步驗(yàn)證之后發(fā)現(xiàn)是假陽性。根據(jù)對(duì)S5作用模式的設(shè)想,我們研究ORF4+與膜受體蛋白如S1L4、LYP4和LYP6的互作關(guān)系,但是發(fā)現(xiàn)也不互作。我們構(gòu)建了ORF5+-GFP與ORF4+-GFP轉(zhuǎn)煙草BY-2懸浮細(xì)胞的穩(wěn)定細(xì)胞系,對(duì)ORF5+和ORF4+的蛋白性質(zhì)進(jìn)行了初步研究。為了研究位于細(xì)胞壁的ORF5+是否通過破壞細(xì)胞壁釋放寡糖信號(hào)分子,我們用三種寡糖對(duì)ORF4+-GFP的細(xì)胞系進(jìn)行處理,發(fā)現(xiàn)這些寡糖不能誘導(dǎo)細(xì)胞的死亡。另外我們也對(duì)ORF4+是否為ORF5+的底物進(jìn)行了初步探究,將ORF5+與N端或者C端帶熒光蛋白的ORF4+共同轉(zhuǎn)入煙草葉片,觀察ORF4+的定位變化,但是并未觀測(cè)到有改變。在BY-2細(xì)胞系和煙草瞬時(shí)轉(zhuǎn)化過程中,我們發(fā)現(xiàn)融合了熒光蛋白的ORF5+并不定位在細(xì)胞壁上,推測(cè)融合的標(biāo)簽可能會(huì)影響ORF5+的功能。但是,我們發(fā)現(xiàn)ORF5+或者ORF5-融合GFP標(biāo)簽的擬南芥穩(wěn)定轉(zhuǎn)化植株能夠降低葉片中PR基因表達(dá)量。ORF5+編碼天冬氨酸蛋白酶,轉(zhuǎn)基因植株BalillaORF5+不育。為了探究ORF5+酶活功能是否為必需,我們將其兩個(gè)酶活位點(diǎn)的天冬氨酸D132和D337分別和同時(shí)誘變?yōu)樘於滨２⑥D(zhuǎn)入Balilla,發(fā)現(xiàn)不管是分別誘變還是同時(shí)誘變類型都正�？捎Ｒ虼�,我們認(rèn)為ORF5+的酶活是其發(fā)揮功能所必需的。ORF4+編碼一個(gè)N端在細(xì)胞外,C端在細(xì)胞內(nèi)的單次跨膜蛋白。ORF4+-GFP與內(nèi)吞marker部分重合,暗示其可能參與內(nèi)吞。我們對(duì)ORF4+預(yù)測(cè)的內(nèi)吞位點(diǎn)進(jìn)行誘變,發(fā)現(xiàn)這些位點(diǎn)與ORF4+的內(nèi)吞現(xiàn)象無關(guān)。ORF4+超表達(dá)植株BalillaORF4+OX株高變矮,分蘗數(shù)減少,其葉片會(huì)產(chǎn)生假病斑并積累胼胝質(zhì),植株對(duì)白葉枯病菌PXO341的抗性增強(qiáng),Balilla ORF4-OX則不會(huì)出現(xiàn)這些表型,這說明ORF4+和ORF4-的功能差異很大。另外,我們利用基因敲除技術(shù)CRISPR在Balilla中成功敲除了ORF4+,獲得了一個(gè)S5位點(diǎn)為ORF3-/ORF4-/ORF5-的植株。通過比較Balilla,敗育的BalillaORF5+和育性恢復(fù)的BalillaORF3+ORF5+胚囊發(fā)育過程,我們將胚囊敗育過程劃分為三個(gè)時(shí)期(MMC,MEI和AME)并研究了這些時(shí)期的轉(zhuǎn)錄組數(shù)據(jù)。通過比較BalillaORF5+和Balilla,發(fā)現(xiàn)ORF5+在MMC時(shí)期引起大量細(xì)胞壁重構(gòu)基因如EXP,XTH,GH9,PAE,PME,PGase和AGP的上調(diào)表達(dá),這些基因可能導(dǎo)致細(xì)胞壁完整性的破壞,并誘導(dǎo)了下游的生物和非生物脅迫反應(yīng)以及內(nèi)質(zhì)網(wǎng)脅迫。MEI時(shí)期,持續(xù)的脅迫反應(yīng)激活了由TIP2,TDR,OsAP25和OsAP37介導(dǎo)PCD過程。此外胚囊細(xì)胞通過上調(diào)表達(dá)OsSWN1,OsMYB46,OsCesA4,OsCesA7和OsCesA9等控制次生細(xì)胞壁合成的基因大量合成纖維素并通過調(diào)控胼胝質(zhì)代謝大量積累胼胝質(zhì)以穩(wěn)固細(xì)胞壁。AME時(shí)期,敗育的胚囊中積累了大量的纖維素和胼胝質(zhì)。通過比較BalillaORF3+ORF5+和Balilla,我們發(fā)現(xiàn)MMC時(shí)期ORF5+在BalillaORF3+ORF5+依然能夠引起部分細(xì)胞壁重構(gòu)基因的上調(diào)表達(dá)進(jìn)而引起脅迫反應(yīng)。由于ORF3+的存在,這些反應(yīng)沒有BalillaORF5+強(qiáng)烈,并且在MEI時(shí)期被抑制,胚囊正常發(fā)育。ORF3+是一個(gè)分子伴侶蛋白,我們推測(cè)ORF3+可能通過修飾ORF4+的互作蛋白或者下游的信號(hào)分子抑制脅迫信號(hào)。通過BalillaORF3+ORF5+與BalillaORF5+的對(duì)比發(fā)現(xiàn)ORF3+使BalillaORF3+ORF5+中與抗病抗逆相關(guān)的基因,如OsMTs,OsLEAs和OsPR10a等的表達(dá)量都大幅度上調(diào)。本研究對(duì)S5位點(diǎn)的三個(gè)基因功能做了進(jìn)一步的研究,揭示了ORF5+的酶活對(duì)其功能的重要性,ORF4+與抗病的相關(guān)性。結(jié)合Balilla,BalillaORF5+和BalillaORF3+ORF5+的轉(zhuǎn)錄組分析結(jié)果,認(rèn)為ORF5+可能破壞細(xì)胞壁的完整性,被細(xì)胞膜上的ORF4+感知,向細(xì)胞內(nèi)傳遞信號(hào)引起生物和非生物脅迫以及內(nèi)質(zhì)網(wǎng)脅迫和PCD。ORF3+存在時(shí),內(nèi)質(zhì)網(wǎng)脅迫被抑制且抗逆能力增強(qiáng)從而抑制了ORF5+引起的脅迫反應(yīng),故胚囊正常發(fā)育。本研究有助于我們進(jìn)一步理解S5的作用機(jī)理,為更好在生產(chǎn)上利用S5提供理論依據(jù)。
[Abstract]:Rice is not only an important grain crop, but also a model plant for the study of mono cotyledon genome. It is a model system for the study of reproductive isolation. The Asian cultivated rice, including two subspecies of Indica and japonica rice, is usually highly sterile and hinders the use of.S5 to control the fertility of the indica japonica hybrid embryo sac. The loci include three linkage genes of ORF3, ORF4 and ORF5. The genotype of typical indica rice is ORF3+/ORF4-/ORF5+. The genotype of typical japonica rice is ORF3-/ORF4+/ORF5-. cell wall protein ORF5+ and transmembrane protein ORF4+ co induced endoplasmic reticulum stress and programmed cell death to induce female gamete abortion, and endoplasmic reticulin ORF3+ can be blocked. To stop or alleviate endoplasmic reticulum stress and prevent the female gamete from being killed to restore normal fertility. This study has studied the mechanism of S5's action from the angle of molecular cytology and transcriptome. We constructed a yeast two hybrid library of Balilla Panicle in japonica rice. The various full length of ORF3, ORF4 and ORF5 and the truncated protein were used as bait Shai Xuanwen Two candidate genes were obtained by ORF5-, but after further verification, we found false positive. According to the assumption of S5 action pattern, we studied the interaction between ORF4+ and membrane receptor proteins such as S1L4, LYP4 and LYP6, but found no mutual action. We constructed the stability of ORF5 +-GFP and ORF4+-GFP conversion tobacco BY-2 suspension cells. The protein properties of ORF5+ and ORF4+ were preliminarily studied. In order to study whether ORF5+ located in the cell wall could release oligosaccharide molecules by destroying the cell wall, we treated the cell lines of ORF4+-GFP with three oligosaccharides and found that these oligosaccharides did not induce cell death. In addition, we also used ORF4+ as a substrate for ORF5+. A preliminary study was carried out to transfer the ORF5+ to the N end or C end with ORF4+ to tobacco leaves and observe the changes in the location of ORF4+, but no change was observed. In the BY-2 cell line and the transient transformation of tobacco, we found that the ORF5+ fusion of the fluorescent protein was not located on the cell wall, and the fusion label might be presumed. The function of ORF5+ is affected. However, we have found that ORF5+ or ORF5- fusion GFP tagged Arabidopsis stable transformation plants can reduce the PR gene expression of PR gene,.ORF5+ encoding aspartic protease, and genetically modified plant BalillaORF5+ sterility. In order to explore whether ORF5+ enzyme activity is necessary, we will take the aspartate D1 at its two enzyme active sites. 32 and D337, respectively, mutated into aspartic and turned into Balilla at the same time, and found that both the mutagenesis and the simultaneous mutagenesis were normally fertile. Therefore, we think that the enzyme activity of ORF5+ is the.ORF4+ encoding a N terminal of its function, the single transmembrane protein.ORF4+-GFP in the C terminal and the partial weight of endocytosis marker in the C terminal. It suggests that it may be involved in endocytosis. We mutagenic the endocytosis sites predicted by ORF4+, and found that these sites are not related to the endocytosis of ORF4+, and that the.ORF4+ overexpression plant BalillaORF4+OX strain is high and the number of tillers decreases, and the leaves will produce false disease spots and accumulate callose, and the resistance of the plant to the bacterial leaf blight pathogen PXO341 is enhanced, Balilla ORF4-OX The phenotypes did not appear, which indicated that the function of ORF4+ and ORF4- was very different. In addition, we successfully knocked out ORF4+ by gene knockout technique CRISPR in Balilla, and obtained a ORF3-/ORF4-/ORF5- plant with S5 site. By comparing Balilla, abortive BalillaORF5+ and fertility recovering BalillaORF3+ORF5+ embryo sac development process, We divided the embryo sac abortion process into three periods (MMC, MEI and AME) and studied the transcriptional data of these periods. By comparing BalillaORF5+ and Balilla, we found that ORF5+ in MMC caused a large number of cell wall remodeling genes such as EXP, XTH, GH9, PAE, PME, and the expression, which may cause damage to cell wall integrity. The downstream biological and abiotic stress reaction and the endoplasmic reticulum stress.MEI period were induced. The persistent stress reaction activated the PCD process, which was mediated by TIP2, TDR, OsAP25 and OsAP37. In addition, the genes that regulate the secondary cell wall by up regulation of OsSWN1, OsMYB46, OsCesA4, OsCesA7 and OsCesA9 are used to synthesize a large amount of cellulose. Excessive accumulation of callose in the callose metabolism to stabilize the cell wall.AME period, a large amount of cellulose and callose accumulated in the aborted embryo sac. By comparing BalillaORF3+ORF5+ and Balilla, we found that ORF5+ in the MMC period still can cause the up-regulated expression of some cell wall remodeling genes in BalillaORF3+ORF5+ and then cause the stress reaction. Due to the presence of ORF3+, these reactions were not BalillaORF5+ strong, and were suppressed in the MEI period. The normal development of the embryo sac was a molecular chaperone. We speculate that ORF3+ may inhibit the stress signal by modifying the interaction protein of the ORF4+ or the downstream signal molecules. Through the comparison of BalillaORF3+ORF5+ to BalillaORF5+, we found ORF. 3+ increased the expression of genes associated with resistance to disease and resistance in BalillaORF3+ORF5+, such as OsMTs, OsLEAs and OsPR10a. This study further studied the three gene functions of the S5 site, which revealed the importance of the enzyme activity of ORF5+ to its function, the correlation of ORF4+ to disease resistance, and Balilla, BalillaORF5+, and BalillaORF3+OR. The results of F5+ transcriptional analysis suggest that ORF5+ may destroy the integrity of the cell wall, be perceived by ORF4+ on the cell membrane, and transmit signals from the cell to biological and abiotic stress, as well as endoplasmic reticulum stress and the presence of PCD.ORF3+, the endoplasmic reticulum stress is suppressed and the anti inversion ability is enhanced to inhibit the stress response caused by ORF5+, so the embryo sac is positive. This study will help us further understand the mechanism of S5 and provide a theoretical basis for better utilization of S5 in production.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S511

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