【作者】 刘玉博；

【导师】 张宪省；

【作者基本信息】 山东农业大学 ， 发育生物学， 2011， 博士

【摘要】 体细胞胚胎发生是植物发育过程中的特殊现象,指各种器官或组织的体细胞通过组织培养可以再生出类似于合子胚的结构——体细胞胚,进而发育成完整的植株。自从早期利用胡萝卜细胞培养体细胞胚的研究开始,这种独特的发育潜能已经成为细胞离体培养体系再生完整植株的重要手段,也成为研究植物胚胎发生早期形态建成调控机理的替代模型。因此,研究体细胞胚胎发生与发育的机理对于揭示细胞全能性与研究合子胚的形态发生等问题具有重要意义。目前,有关体细胞胚胎发生的分子机理和基因调控研究还很少,有待展开系统的研究。本实验室已有的实验结果指出生长素诱导WUS基因的表达对体细胞胚胎发生过程中茎端分生组织的重建是至关重要的。本文将从体细胞胚根端分生组织建成的机制以及microRNA(miRNA)影响体细胞胚胎发生的机理两个方面进行深入研究,期望研究结果为理解体细胞胚胎发生和发育的分子机理提供新的重要信息。主要研究结果如下:(1)细胞分裂素调控体细胞胚胎发生早期根端分生组织的建成为了研究体细胞胚胎发生过程中根端分生组织的重新建立情况,我们用激光共聚焦显微镜观察了根分生组织特征基因的表达模式。根端静止中心特征基因WUSCHEL RELATED HOMEOBOX 5(WOX5)最早在体细胞胚诱导16小时后开始表达,根端分生组织特征基因PLETHORA 2(PLT2)在诱导后24小时内就已经表达,之后SHORT ROOT(SHR)开始表达,而SCARECROW(SCR)开始表达的时间较晚,直到诱导2天后才检测到。雌激素诱导的pER8-WOX5 anti植株抑制了WOX5基因的表达,其胚性愈伤组织几乎丧失了再生体细胞胚的能力,而plt1-1 plt2-1双突变体的初级体细胞胚茎端虽形态正常并分化出两片子叶,但根端出现了严重的缺陷而且无法伸长。以上结果表明体细胞胚胎发生过程中根端分生组织特征基因的表达决定了体细胞胚根端分生组织的建立。在体细胞胚诱导的早期(1~4天),表征生长素分布的DR5荧光信号无法在WOX5和PLT2表达区域被检测到;诱导5天后,体细胞胚已形成成熟的子叶,此时生长素的信号开始在根端分生组织特征基因的表达位置分布。这说明在茎端分生组织重新建成过程中起重要调节作用的生长素,在体细胞胚诱导早期并不分布在根端分生组织区域。施加细胞分裂素合成的抑制剂洛伐他丁导致胚性愈伤组织的胚性完全丧失而无法再生体细胞胚。进一步的实验发现,A类细胞分裂素响应调节因子ARABIDOPSIS RESPONSE REGULATOR 7(ARR7)基因的表达区域与PLT2一直重叠,细胞分裂素合成基因ISOPENTENYLTRANSFERASE 5(IPT5)的表达也向根端集中。而且利用细胞分裂素受体基因ARABIDOPSIS HISTIDINE KINASE(AHK)的双突变体ahk2 ahk4和ahk3 ahk4以及细胞分裂素响应调节因子35S::ARR7和35S::ARR15过量表达植株,抑制细胞分裂素的信号转导途径,会导致初级体细胞胚出现类似plt1-1 plt2-1双突变体根端缺陷的表型,次级体细胞胚的再生也出现异常。这说明细胞分裂素在体细胞胚胎发生早期特异的分布在根端分生组织区域,对胚的根端分生组织的建立起主要的调控作用。虽然生长素和细胞分裂素共同参与了合子胚顶-基轴的建立,但是与生长素和细胞分裂素协同调控体细胞胚纵向茎端-根端分生组织的建成的模式是不同的。生长素调控体细胞胚胎发生早期WUS的表达和茎端分生组织的形成,而细胞分裂素通过其信号转导途径调控WOX5的表达和根端分生组织的形成。(2)miR167通过其靶基因ARF6和ARF8调控体细胞胚胎发生植物中的miRNA是一类约21个核苷酸组成的内源非编码小分子RNA,通过与其靶基因mRNA互补配对结合,以抑制基因表达或切割mRNA的方式,实现对靶基因的负调控。近年来,越来越多的miRNA在植物生长发育过程中的功能和作用被揭示出来,我们也运用芯片技术检测在体细胞胚胎发生过程中差异表达的miRNA,并结合遗传学实验最终发现miR167在体细胞胚胎发生过程中起着重要作用。首先,我们发现过量表达miR167家族的成员miR167c会显著抑制体细胞胚胎发生。35S::miR167c的愈伤组织在培养过程中逐渐丧失胚性,仅能再生少量的体细胞胚,同时愈伤组织中的LEAFY COTYLEDON 1(LEC1)、LEC2和FUSCA 3(FUS3)等胚胎特征基因的表达水平也明显降低。进一步研究发现过量表达miR167c会导致生长素极性运输蛋白PIN-FORMED 1(PIN1)在愈伤组织中的极性定位消失,导致生长素极性分布模式无法形成,并使WUS基因表达模式紊乱,从而抑制了体细胞胚胎发生。另外,miR167c的靶基因AUXIN RESPONSE FACTOR 6(ARF6)和ARF8的突变体也导致体细胞胚的发生出现异常,arf6-2、arf8-3和arf6-2 arf8-3/+突变体诱导的体细胞胚再生频率明显降低。从表达模式上来看,miR167c及其靶基因ARF6和ARF8均在再生的体细胞胚中特异表达,也说明它们在体细胞胚的诱导过程中起作用。以上结果证明miR167是通过生长素响应因子ARF6和ARF8来调控生长素的极性运输及分布,进而影响体细胞胚茎端分生组织的建成。更多还原

【Abstract】 Somatic embryos (SE) have been defined as structures that arise from somatic cells but resemble zygotic embryos. Somatic embryogenesis is an important way to generate SE and whole plants. Since the initial description of SE formation from carrot callus cells, this unique developmental process has been recognized both as an important way for plants regeneration from cell culture systems and as a potential model for studying early regulatory and morphogenetic events in plant embryogenesis. Thus, somatic embryogenesis has been used for studying the plant cell totipotency and the developmental events in zygotic embryogenesis. However, molecular mechanisms of somatic embryogenesis remain largely unknown. Previous studies in our laboratory have demonstrated that auxin-induced WUS expression is essential for shoot apical meristem (SAM) formation during Arabidopsis somatic embryogenesis. Herein, we focus on studying the mechanism of root apical meristem (RAM) establishment and effects of microRNA (miRNA) in Arabidopsis somatic embryogenesis. Our results shed new light on molecular mechanisms regulating somatic embryogenesis in Arabidopsis. The main results are as follows:(1) RAM establishment was regulated by cytokinin during early somatic embryogenesisIn order to study the initiation of RAM stem cells during somatic embryogenesis in Arabidopsis, we analyzed the expression pattern of genes which were reported to express in the RAM during embryogenesis using laser scanning confocal microscope. The localization of root quiescent center-specific gene WUSCHEL RELATED HOMEOBOX 5 (WOX5) was analyzed by pWOX5::GFP expression. WOX5 gene expression was detected in embryonic calli within 16 hours after SE induction. Then, signals of other genes involved in root stem cell specification during early embryogenesis such as PLETHORA 2 (PLT2), SHORT ROOT (SHR) and SCARECROW (SCR), were detected sequentially within two days after SE induction. More importantly, repressing the expression of WOX5 or PLT resulted in abnormal somatic embryogenesis. Embryonic callus of pER8-WOX5-anti almost lost the ability to reproduce SE after induction by 17β-estradiol. Primary SE of the plt1-1 plt2-1 double mutant could form normal shoot apex with cotyledons, but defected in root apex and SE elongation. These results indicated that RAM-specific genes play important roles in SE root development during early somatic embryogenesis.Auxin that is important in SAM renewal only concentrated around shoot apex during early somatic embryogenesis, but not in RAM region, because the genes related to auxin biosynthesis and transport only expressed in shoot apex. Fluorescence signal of DR5rev::YFP or DR5rev::GFP, which mark auxin distribution, did not overlap with the signal of pWOX5::GFP or pPLT2::RFP during early somatic embryogenesis. Only after 5 days induction, auxin response signal could be detected in the root apex.Application of cytokinin synthesis inhibitor lovastatin led to complete loss of embryonic ability in the callus, and failure to induce SE. Further experiments showed that the expression region of ARABIDOPSIS RESPONSE REGULATOR 7 (ARR7), which is used to mark cytokinin response, could overlap the expression region of PLT2 during early somatic embryogenesis, and the expression of cytokinin biosynthetic gene IPT5 was detected in the root apex. Furthermore, repressing the cytokinin signal transduction resulted in defective somatic embryogenesis. The double mutants of cytokinin receptor genes ARABIDOPSIS HISTIDINE KINASE (AHK), ahk2 ahk4 and ahk3 ahk4, showed similar phenotype to that of the double mutant plt1-1 plt2-1 in the root apex. Overexpression of ARR7 and ARR15 both resulted in abnormal primary and secondary SE. These results indicated that cytokinin specifically distributed in root apex, and regulated the establishment of RAM through its signal transduction.Our results suggest that although the interaction between auxin and cytokinin plays an important role in the establishment of apical-basal polarity in zygotic embryo, there is a different pattern in the SE shoot-root apex polarity. Auxin regulates WUS expression and SAM renewal in shoot apex while cytokinin controls WOX5 expression and RAM formation in root apex.(2) MiR167 regulated somatic embryogenesis through its target genes ARF6 and ARF8 MiRNAs are small, endogenous RNAs that regulate gene expression in plants and animals. In plants, these ~21 nucleotide RNAs are processed from stem-loop regions of long primary transcripts and are loaded into silencing complexes, where they generally direct cleavage of complementary mRNAs of their target genes. In recent years, more and more functions of miRNA were revealed at various stages of plant growth and development. We detected a large number of miRNA differentially expressed during SE induction by microarray technology, and found that miR167 played an important role in Arabidopsis somatic embryogenesis.First, we showed that overexpression of miR167 family members decreased somatic embryogenesis frequency. Moreover, the expression of WUS and embryo-specific genes, such as LEAFY COTYLEDON 1 (LEC1), LEC2 and FUSCA 3 (FUS3), were repressed. Further experiments indicated that the polar localization of PIN-FORMED 1 (PIN1) and normal expression pattern of WUS were not detected in 35S::miR167c, resulting in disrupted pattern of SE shoot stem cell renewal.Both miR167c and its target genes AUXIN RESPONSE FACTOR 6 (ARF6)/ARF8 expressed specifically in embryonic callus where SE was produced. In addition, mutants of ARF6 and ARF8 also showed defects in somatic embryogenesis. Primary SE of arf6-2, arf8-3 and arf6-2 arf8-3/+ were morphologically normal, but the frequency of secondary SE induction decreased obviously.Our results suggested that miR167 regulated auxin polar transport and gradient distribution through auxin response factor ARF6 and ARF8, thereby affecting RAM establishment in somatic embryogenesis.更多还原