调控拟南芥表皮毛发生的核心转录因子的水稻同源蛋白的功能研究

发布时间：2018-07-04 12:54

本文选题：OsTCL1 + OsGL1　；参考：《东北师范大学》2017年博士论文

【摘要】：细胞命运决定的调控一直是发育生物学的核心问题之一。拟南芥表皮毛和根毛的发生被当作植物细胞命运决定的重要研究模型。表皮毛是植物地上部分表皮组织特有的延伸出的毛状附属物,它能增加表皮厚度、减少水分和热量的散失、减轻昆虫和病原体侵害、减轻机械损伤等。拟南芥的表皮毛是一种特化的单细胞,广泛分布在莲座叶、茎生叶、茎和花瓣上。水稻的表皮毛分为三种:长毛(macro hair)、微毛(micro hair)和腺毛(glandular hair)。无表皮毛是一种非常好的农艺性状,利于增加包装效率并且减少收割、晾晒、包装过程中粉尘给农民带来的过敏症状,因此无毛性状被广泛应用到水稻种植当中。根毛是植物根表皮细胞上特化的管状突起。它能够增加根与土壤的接触面积,利于植物吸收土壤中的水分和营养,并对植物与土壤中微生物的相互作用起着重要作用。在拟南芥中,R2R3 MYB转录因子GLABRA1(GL1)、bHLH转录因子GLABRA3/ENHANCER OF GLABRA3(GL3/EGL3)和WDR蛋白TRANSPARENTTESTAGLABRA1(TTG1)构成MBW转录激活复合体,激活GLABROUS2(GL2)的表达,进而诱导了表皮毛的发生。MBW转录激活复合体同时可以诱导包括TRICHOMELESS1(TCL1)、TCL2、TRYPTICHON(TRY)、CAPRICE(CPC)、ENHANCEROFTRYANDCPC1(ETC1)、ETC2和ETC3在内的R3 MYB基因的表达。这些转录因子可以进入邻近的细胞当中与GL1竞争结合GL3,抑制转录激活复合体的功能,从而抑制了表皮毛的发生。调控拟南芥根毛发生的机制与调控表皮毛发生的机制是类似的。只是在根细胞中,GL1被另一个R2R3 MYB转录因子WEREWOLF(WER)所替代。WER、GL3/EGL3和TTG1共同构成了MBW转录激活复合体,启动下游GL2的表达,进而抑制根表皮细胞分化成根毛细胞。当WER被R3 MYBs转录因子所代替,这种复合体不会激活GL2的表达,使根表皮细胞分化成根毛细胞。然而,水稻表皮毛和根毛发生是否受到类似转录复合体的调控尚不清楚。本研究中,我们用拟南芥GL1、GL3、TTG1和TCL1的氨基酸全序列BLAST水稻的蛋白数据库,分别寻找到它们在水稻中的同源蛋白OsGL1(OsGL1A、OsGL1B、OsGL1C、OsGL1D、OsGL1E)、OsGL3(OsGL3A、OsGL3B、OsGL3C)、OsTTG1(OsTTG1A、OsTTG1B)、OsTCL1以及OsTCL2。我们的研究表明,OsGL1E、OsGL3B、OsTTG1A以及OsTCL1都能够定位在细胞核中。此外,OsTTG1A和OsTCL1还能够分别定位在细胞质和细胞膜上。通过原生质体实验证实OsGL3B是转录激活子,而OsTTG1A和OsTCL1有微弱的转录激活活性,OsGL1E没有转录激活活性。在拟南芥原生质体中,GL1、TTG1和TCL1可以与OsGL3B互作,OsGL1E、OsTTG1A和OsTCL1都能与GL3互作,也能与OsGL3B互作。以上结果暗示,水稻中的相应转录因子也可以形成MBW复合体。我们以OsTCL1为例,研究其对拟南芥表皮毛和根毛发生的调控。用双35S启动子调控OsTCL1的表达能够抑制表皮毛的发生,同时还促进根毛的发生。此外,利用tcl1、try和cpc突变体为背景的表型恢复实验的结果说明,OsTCL1与TCL1的功能是类似的,但不完全一致。并且qRT-PCR结果显示,在过量表达OsTCL1的转基因拟南芥中,GL1的表达量大幅下降。以上结果表明,OsTCL1调控拟南芥的表皮毛和根毛发生,很可能与拟南芥R3 MYB转录因子调控表皮毛和根毛发生的机制类似。另外,我们在ttg1突变体背景下过量表达OsTTG1A使突变体无表皮毛的表型得到恢复,这证明了OsTTG1A与TTG1对拟南芥表皮毛发生有类似的调控作用。在水稻中,我们同样通过过量表达OsTCL1,研究其对于水稻表皮毛和根毛发生的调控作用。但是我们并没有发现明显的表皮毛和根毛的表型变化。qRT-PCR结果显示,与拟南芥中GL2的表达被明显抑制不同,过量表达OsTCL1的转基因水稻中OsGL2的表达量明显升高。并且,OsGL3C的表达量也有明显升高,这与过量表达TCL1或OsTCL1的拟南芥中,GL3的表达量没有明显变化也是不同的。以上结果暗示水稻很可能通过与拟南芥不同的机制调控表皮毛和根毛的发生。
[Abstract]:The regulation of cell fate determination is one of the core problems in developmental biology. The occurrence of the surface fur and root hairs of Arabidopsis is regarded as an important research model for the fate of plant cells. The surface fur is a special hair like appendage of the epidermal tissue of the plant. It can increase the thickness of the epidermis and reduce the loss of water and heat. The epidermis of Arabidopsis is a special single cell, widely distributed in the lotus leaves, stems, stems and petals. The epidermis of rice is divided into three kinds: long hair (macro hair), micro hair and glandular hair. No surface fur is a very good agronomic character. Increasing the efficiency of packaging and reducing the allergic symptoms caused by dust in the process of harvesting, drying and packing, so the hairless characters are widely used in rice planting. The root hair is a special tubular protuberance on the root epidermal cells of plants. It can increase the contact area between the root and the soil, and benefit the plant to absorb water and nutrition in the soil. In Arabidopsis, the R2R3 MYB transcription factor GLABRA1 (GL1), the bHLH transcription factor GLABRA3/ENHANCER OF GLABRA3 (GL3/EGL3) and WDR protein TRANSPARENTTESTAGLABRA1 (TTG1) constitute the transcriptional activation complex, which activates the expression of the transcription factor, thus inducing the hair of the epidermis. The transcriptional activation complex of.MBW can also induce the expression of TRICHOMELESS1 (TCL1), TCL2, TRYPTICHON (TRY), CAPRICE (CPC), ENHANCEROFTRYANDCPC1 (ETC1), ETC2 and ETC3, which can enter the adjacent cells to compete with the cells to inhibit the function of the transcriptional activation complex and thus inhibit the function of the transcriptional activation complex. The mechanism of regulation of the root hairs of Arabidopsis is similar to that in the regulatory table. Only in the root cells, GL1 is replaced by another R2R3 MYB transcription factor WEREWOLF (WER), and GL3/EGL3 and TTG1 co constitute the MBW transcriptional activation complex, initiating the expression of the downstream GL2 and inhibiting the root epidermal cells. It is transformed into root hair cells. When WER is replaced by R3 MYBs transcription factors, this complex does not activate GL2 expression and causes root epidermal cells to differentiate into root hair cells. However, it is not clear whether the hair and root hair of rice is regulated by similar transcriptional complexes. In this study, we used the amino acid full order of Arabidopsis GL1, GL3, TTG1, and TCL1. The protein database of BLAST rice is listed to find the homologous protein OsGL1 (OsGL1A, OsGL1B, OsGL1C, OsGL1D, OsGL1E) in rice, OsGL3 (OsGL3A, OsGL3B, OsGL3C), and our research shows that all of them can be located in the nucleus. TCL1 can also be located on the cytoplasm and cell membrane respectively. Through protoplast experiments, OsGL3B is a transcriptional activator, and OsTTG1A and OsTCL1 have weak transcriptional activation activity, and OsGL1E has no transcriptional activation activity. In Arabidopsis protoplasts, GL1, TTG1 and TCL1 can interact with OsGL3B, OsGL1E, OsTTG1A, and OsTCL1. The above results suggest that the corresponding transcription factors in rice can also form a MBW complex. We take OsTCL1 as an example to study the regulation of the hair and root hair of Arabidopsis. The expression of OsTCL1 with double 35S promoter can inhibit the occurrence of hair and also promote the occurrence of root hair. In addition, TCL1, try and C are used. The results of the phenotypic recovery experiment with the PC mutant showed that the function of OsTCL1 and TCL1 was similar, but not exactly consistent. And qRT-PCR results showed that the expression of GL1 in the transgenic Arabidopsis thaliana with excessive expression of OsTCL1 decreased significantly. The results showed that OsTCL1 regulates the hair and root hair of the Arabidopsis thaliana, which is likely to be with the pseudo south. The mechanism of R3 MYB transcription factor of mustard regulates the mechanism of epidermal hair and root hair. In addition, our overexpression of OsTTG1A under the TTG1 mutant background restores the phenotype of the mutant without the epidermis, which proves that OsTTG1A and TTG1 have similar regulatory effects on the formation of the Arabidopsis thaliana fur. In rice, we also overexpress OsTCL1, We studied the regulation of the hair and root hair of rice. But we did not find the obvious phenotypic changes of the surface fur and root hair..qRT-PCR results showed that the expression of GL2 in the Arabidopsis was significantly inhibited, and the amount of OsGL2 in the transgenic rice with excessive expression of OsTCL1 was significantly increased. And the expression of OsGL3C was also expressed. There was a significant increase in the expression of GL3 in Arabidopsis thaliana, which overexpressed TCL1 or OsTCL1. The results suggested that rice is likely to regulate the formation of fur and root hairs by different mechanisms of Arabidopsis.
【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2

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