籼粳稻杂种不育系统S5的蛋白互作及转录组研究

发布时间：2018-06-24 03:59

本文选题：水稻 + 籼粳杂种不育　；参考：《华中农业大学》2017年博士论文

【摘要】：水稻既是重要的粮食作物,也是单子叶植物基因组研究的模式植物,是生殖隔离研究的模式系统。亚洲栽培稻包括籼稻和粳稻两个亚种,其杂种通常高度不育,阻碍了对亚种间杂种强大杂种优势的利用。S5是控制籼粳杂种胚囊育性的主要位点,包括ORF3,ORF4和ORF5三个连锁基因。典型籼稻的基因型为ORF3+/ORF4-/ORF5+,典型粳稻的基因型为ORF3-/ORF4+/ORF5-。细胞壁蛋白ORF5+与跨膜蛋白ORF4+共同诱导内质网胁迫进而引起细胞程序性死亡从而导致雌配子败育,内质网蛋白ORF3+则能够阻止或者缓解内质网胁迫阻止雌配子被杀死从而恢复正常育性。本研究从分子细胞学和转录组角度对S5作用机理进行了研究。我们构建了粳稻Balilla穗组织的酵母双杂交文库。利用ORF3,ORF4和ORF5的各种全长以及截短的蛋白作为诱饵筛选文库,并利用ORF5-得到了两个候选基因,但是经过进一步验证之后发现是假阳性。根据对S5作用模式的设想,我们研究ORF4+与膜受体蛋白如S1L4、LYP4和LYP6的互作关系,但是发现也不互作。我们构建了ORF5+-GFP与ORF4+-GFP转烟草BY-2悬浮细胞的稳定细胞系,对ORF5+和ORF4+的蛋白性质进行了初步研究。为了研究位于细胞壁的ORF5+是否通过破坏细胞壁释放寡糖信号分子,我们用三种寡糖对ORF4+-GFP的细胞系进行处理,发现这些寡糖不能诱导细胞的死亡。另外我们也对ORF4+是否为ORF5+的底物进行了初步探究,将ORF5+与N端或者C端带荧光蛋白的ORF4+共同转入烟草叶片,观察ORF4+的定位变化,但是并未观测到有改变。在BY-2细胞系和烟草瞬时转化过程中,我们发现融合了荧光蛋白的ORF5+并不定位在细胞壁上,推测融合的标签可能会影响ORF5+的功能。但是,我们发现ORF5+或者ORF5-融合GFP标签的拟南芥稳定转化植株能够降低叶片中PR基因表达量。ORF5+编码天冬氨酸蛋白酶,转基因植株BalillaORF5+不育。为了探究ORF5+酶活功能是否为必需,我们将其两个酶活位点的天冬氨酸D132和D337分别和同时诱变为天冬氨酰并转入Balilla,发现不管是分别诱变还是同时诱变类型都正常可育。因此,我们认为ORF5+的酶活是其发挥功能所必需的。ORF4+编码一个N端在细胞外,C端在细胞内的单次跨膜蛋白。ORF4+-GFP与内吞marker部分重合,暗示其可能参与内吞。我们对ORF4+预测的内吞位点进行诱变,发现这些位点与ORF4+的内吞现象无关。ORF4+超表达植株BalillaORF4+OX株高变矮,分蘖数减少,其叶片会产生假病斑并积累胼胝质,植株对白叶枯病菌PXO341的抗性增强,Balilla ORF4-OX则不会出现这些表型,这说明ORF4+和ORF4-的功能差异很大。另外,我们利用基因敲除技术CRISPR在Balilla中成功敲除了ORF4+,获得了一个S5位点为ORF3-/ORF4-/ORF5-的植株。通过比较Balilla,败育的BalillaORF5+和育性恢复的BalillaORF3+ORF5+胚囊发育过程,我们将胚囊败育过程划分为三个时期(MMC,MEI和AME)并研究了这些时期的转录组数据。通过比较BalillaORF5+和Balilla,发现ORF5+在MMC时期引起大量细胞壁重构基因如EXP,XTH,GH9,PAE,PME,PGase和AGP的上调表达,这些基因可能导致细胞壁完整性的破坏,并诱导了下游的生物和非生物胁迫反应以及内质网胁迫。MEI时期,持续的胁迫反应激活了由TIP2,TDR,OsAP25和OsAP37介导PCD过程。此外胚囊细胞通过上调表达OsSWN1,OsMYB46,OsCesA4,OsCesA7和OsCesA9等控制次生细胞壁合成的基因大量合成纤维素并通过调控胼胝质代谢大量积累胼胝质以稳固细胞壁。AME时期,败育的胚囊中积累了大量的纤维素和胼胝质。通过比较BalillaORF3+ORF5+和Balilla,我们发现MMC时期ORF5+在BalillaORF3+ORF5+依然能够引起部分细胞壁重构基因的上调表达进而引起胁迫反应。由于ORF3+的存在,这些反应没有BalillaORF5+强烈,并且在MEI时期被抑制,胚囊正常发育。ORF3+是一个分子伴侣蛋白,我们推测ORF3+可能通过修饰ORF4+的互作蛋白或者下游的信号分子抑制胁迫信号。通过BalillaORF3+ORF5+与BalillaORF5+的对比发现ORF3+使BalillaORF3+ORF5+中与抗病抗逆相关的基因,如OsMTs,OsLEAs和OsPR10a等的表达量都大幅度上调。本研究对S5位点的三个基因功能做了进一步的研究,揭示了ORF5+的酶活对其功能的重要性,ORF4+与抗病的相关性。结合Balilla,BalillaORF5+和BalillaORF3+ORF5+的转录组分析结果,认为ORF5+可能破坏细胞壁的完整性,被细胞膜上的ORF4+感知,向细胞内传递信号引起生物和非生物胁迫以及内质网胁迫和PCD。ORF3+存在时,内质网胁迫被抑制且抗逆能力增强从而抑制了ORF5+引起的胁迫反应,故胚囊正常发育。本研究有助于我们进一步理解S5的作用机理,为更好在生产上利用S5提供理论依据。
[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.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S511

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