PtrmiR164a在杨树次生细胞壁合成过程中的功能研究
发布时间：2018-08-16 18:29
【摘要】：杨树(Populus trichocarpa)是世界范围内种植面积最广的速生林木之一,其主要用于造纸和建筑,同时也可用作生物燃料的原材料。2006年美国能源部完成了杨树全基因组测序,使其作为木本模式植物广泛应用于林木育种研究。阐明杨树生长发育的分子调控机理,对于其分子育种非常重要,但目前杨树次生生长、不定根发生等的遗传分子调控机制尚不清楚。前人研究发现,植物体内的micro RNAs(mi RNAs)参与调控植物多个生长发育过程。mi R164是一类植物中保守的小RNA家族,参与调控拟南芥叶片、侧向器官、侧根发生等多个发育过程。但mi R164在木本植物生长发育中的调控功能尚未见报道。为了阐明杨树mi R164的功能和作用机制,本论文进行了一系列研究,得到如下研究结果:1.Ptrmi R164a的特异性表达分析采用生物信息学方法,发现杨树中Ptrmi R164家族存在6个成员,其前体形成不同的茎环结构,但其成熟体序列高度相似。RT-PCR分析发现,6个Ptrmi R164成员表现出相似的组织表达特异性,即在根中表达量最高,其次为茎。进一步选取Ptrmi R164a开展深入研究。在克隆Ptrmi R164a启动子的基础上,GUS组织染色Pro Ptrmi R164a::GUS转基因杨树发现,Ptrmi R164a在杨树根、茎、叶中均有表达,但茎横切片染色显示,其只在茎初生韧皮部和形成层中表达。Pro Ptrmi R164a::GUS在转基因拟南芥中发现,Ptrmi R164a启动子在花器官的柱头、萼片和花丝中有显著活性。2.Ptrmi R164a靶基因的预测运用mi RBASE数据库对Ptrmi R164a靶基因预测发现Ptr CUC1.1、Ptr CUC1.1、Ptr CUC2、Ptr NAC1、Ptr NAC1.1、Ptr NAC021、Ptr NAC015等7个靶基因得分最高。与Atmi R164a靶基因进化分析发现其可分为三类,其中Ptr NAC1和Ptr NAC1.1与Ptrmi R164a成熟体序列最为匹配。3.PtrmiR164a表达下调转基因植株的获得克隆Ptrmi R164a成熟体序列,将其连接在植物表达载体上,最终获得抑制表达载体STTM164。将该载体通过根癌农杆菌介导转入杨树中,抗性筛选后获得大量阳性转基因植株,进一步采用PCR方法鉴定这些转基因植株。实时定量PCR检测Ptrmi R164a成熟体的表达水平发现,其在转基因植株中均呈不同程度的下降,选取其表达水平显著下降的株系2和3进行后续研究。4.STTM164转基因植株表型观察及次生壁合成相关基因的表达分析与野生型相比STTM164转基因植株不定根的发生较早,且侧根较野生型发达。用营养液培养后得相同结果。组织化学染色、电镜扫描观察和定量统计结果显示,在STTM164转基因植株的韧皮部处早于野生型出现木质素的多圈层沉积,同时STTM164转基因植株的木质部细胞层数,细胞壁厚度的明显增加导致STTM164转基因植株木质部加厚。Klason方法和溴化乙锭分析发现,与野生型相比,STTM164转基因植株木质素含量增加。对STTM164转基因植株次生壁合成途径关键酶基因荧光定量PCR(q RT-PCR)检测发现,其表达水平均被上调。同时发现,与韧皮部发育相关基因Ptr LBD1的表达量在STTM164转基因植株中也被上调,暗示Ptr LBD1基因参与调控STTM164转基因植株韧皮部木质素的生长发育。5.Ptrmi R164a靶基因表达量的检测及其特异性表达分析q RT-PCR检测发现,STTM164转基因植株中Ptrmi R164a靶基因表达量均被显著上调,其中Ptr NAC1表达上升最明显。组织表达分析发现,这些靶基因在根和茎中表达量最高,该结果与Ptrmi R164a的时空表达模式高度相似。6.Pro Ptrmi R164a::GUS拟南芥材料的生长素处理用生长素类似物NAA处理Pro Ptrmi R164a::GUS拟南芥材料证实,Ptrmi R164a的表达受生长素诱导。另外,STTM164转基因植株中生长素信号转导蛋白PIN基因的表达量也被上调,上述结果表明,Ptrmi R164a通过生长素信号通路调控了杨树次生壁的合成代谢。综述所述,本论文在克隆杨树Ptrmi R164a的基础上,通过转基因、组织化学分析、组织切片等方法,初步证明了Ptrmi R164a通过影响生长素的信号转导,参与了杨树次生木质部和次生韧皮部的合成代谢调控。本研究为杨树木材的发育调控提供了一定的分子证据。
[Abstract]:Populus trichocarpa is one of the most widely planted fast-growing trees in the world. Populus trichocarpa is mainly used in papermaking, construction and biofuel. In 2006, the U.S. Department of Energy completed the whole genome sequencing of poplar, making it widely used as a model woody plant in forest breeding. Molecular regulation of development is very important for molecular breeding, but the genetic and molecular mechanisms of poplar secondary growth and adventitious root formation are still unclear. In order to elucidate the function and mechanism of poplar mi R164, a series of studies were carried out in this paper, and the following results were obtained: 1. The specific expression of Ptrmi R164a was analyzed by biology. Informatics analysis showed that there were six members of Ptrmi R164 family in poplar, whose precursors formed different stem ring structures, but their mature body sequences were highly similar. RT-PCR analysis showed that the six Ptrmi R164 members showed similar tissue expression specificity, that is, the highest expression was in root, followed by stem. On the basis of cloning Ptrmi R164a promoter, GUS tissue staining of Pro Ptrmi R164a:: GUS transgenic poplar showed that Ptrmi R164a was expressed in root, stem and leaf of poplar, but stem cross section staining showed that Ptrmi R164a was only expressed in primary phloem and cambium of stem. Ptr CUC1.1, Ptr CUC1.1, Ptr CUC2, Ptr NAC1, Ptr NAC1.1, Ptr NAC021, Ptr NAC015 and other seven target genes scored the highest in the prediction of Ptr mi R164a target genes using the MIRBASE database. Ptr NAC1, Ptr NAC1.1 and Ptr NAC1.1 were most matched with Ptrmi R164a mature body sequence. 3. Ptrmi R164a mature body sequence was obtained from transgenic plants with down-regulated expression of Ptrmi R164a, and was linked to plant expression vector. Finally, the inhibitory expression vector STTM164 was obtained. A large number of positive transgenic plants were obtained and identified by PCR. The expression of Ptrmi R164a in transgenic plants was detected by real-time quantitative PCR. Lines 2 and 3 with significantly decreased expression were selected for further study. Compared with the wild type, the adventitious roots of STTM164 transgenic plants occurred earlier and the lateral roots were more developed. The same results were obtained after culture in nutrient solution. Histochemical staining, scanning electron microscopy and quantitative analysis showed that the phloem of STTM164 transgenic plants was earlier than that of the wild type. The results of Klason method and EB analysis showed that the lignin content of STTM164 transgenic plants increased compared with wild type. The secondary plants of STTM164 transgenic plants were found to have higher lignin content. Quantitative fluorescence PCR (q RT-PCR) showed that the expression levels of the key enzymes in the wall synthesis pathway were up-regulated, and the expression levels of the phloem development-related gene Ptr LBD1 were up-regulated in STTM164 transgenic plants, suggesting that the Ptr LBD1 gene was involved in the regulation of phloem lignin growth and development in STTM164 transgenic plants.5.Ptrmi R16 Detection of 4A target gene expression and Q RT-PCR analysis showed that the expression of Ptrmi R164a was significantly up-regulated in STTM164 transgenic plants, and the expression of Ptr NAC1 was the most obvious. Pro Ptrmi R164a:: GUS Arabidopsis auxin treatment Pro Ptrmi R164a:: GUS Arabidopsis auxin analogue NAA treatment Pro Ptrmi R164a:: GUS Arabidopsis materials confirmed that the expression of Ptrmi R164a was induced by auxin. In addition, the expression of auxin signal transduction protein PIN gene in STTM164 transgenic plants was also up-regulated, the above results showed that Ptrmi R164a::GUS Arabidopsis materials. R164a regulates the synthesis and metabolism of secondary wall of poplar through auxin signaling pathway. In this review, based on the cloning of poplar Ptrmi R164a, we preliminarily proved that Ptrmi R164a participated in secondary xylem and secondary phloem of poplar through signal transduction affecting auxin through gene transformation, histochemical analysis and histological section. This study provides some molecular evidence for the development and regulation of poplar wood.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2
本文编号：2186818
[Abstract]:Populus trichocarpa is one of the most widely planted fast-growing trees in the world. Populus trichocarpa is mainly used in papermaking, construction and biofuel. In 2006, the U.S. Department of Energy completed the whole genome sequencing of poplar, making it widely used as a model woody plant in forest breeding. Molecular regulation of development is very important for molecular breeding, but the genetic and molecular mechanisms of poplar secondary growth and adventitious root formation are still unclear. In order to elucidate the function and mechanism of poplar mi R164, a series of studies were carried out in this paper, and the following results were obtained: 1. The specific expression of Ptrmi R164a was analyzed by biology. Informatics analysis showed that there were six members of Ptrmi R164 family in poplar, whose precursors formed different stem ring structures, but their mature body sequences were highly similar. RT-PCR analysis showed that the six Ptrmi R164 members showed similar tissue expression specificity, that is, the highest expression was in root, followed by stem. On the basis of cloning Ptrmi R164a promoter, GUS tissue staining of Pro Ptrmi R164a:: GUS transgenic poplar showed that Ptrmi R164a was expressed in root, stem and leaf of poplar, but stem cross section staining showed that Ptrmi R164a was only expressed in primary phloem and cambium of stem. Ptr CUC1.1, Ptr CUC1.1, Ptr CUC2, Ptr NAC1, Ptr NAC1.1, Ptr NAC021, Ptr NAC015 and other seven target genes scored the highest in the prediction of Ptr mi R164a target genes using the MIRBASE database. Ptr NAC1, Ptr NAC1.1 and Ptr NAC1.1 were most matched with Ptrmi R164a mature body sequence. 3. Ptrmi R164a mature body sequence was obtained from transgenic plants with down-regulated expression of Ptrmi R164a, and was linked to plant expression vector. Finally, the inhibitory expression vector STTM164 was obtained. A large number of positive transgenic plants were obtained and identified by PCR. The expression of Ptrmi R164a in transgenic plants was detected by real-time quantitative PCR. Lines 2 and 3 with significantly decreased expression were selected for further study. Compared with the wild type, the adventitious roots of STTM164 transgenic plants occurred earlier and the lateral roots were more developed. The same results were obtained after culture in nutrient solution. Histochemical staining, scanning electron microscopy and quantitative analysis showed that the phloem of STTM164 transgenic plants was earlier than that of the wild type. The results of Klason method and EB analysis showed that the lignin content of STTM164 transgenic plants increased compared with wild type. The secondary plants of STTM164 transgenic plants were found to have higher lignin content. Quantitative fluorescence PCR (q RT-PCR) showed that the expression levels of the key enzymes in the wall synthesis pathway were up-regulated, and the expression levels of the phloem development-related gene Ptr LBD1 were up-regulated in STTM164 transgenic plants, suggesting that the Ptr LBD1 gene was involved in the regulation of phloem lignin growth and development in STTM164 transgenic plants.5.Ptrmi R16 Detection of 4A target gene expression and Q RT-PCR analysis showed that the expression of Ptrmi R164a was significantly up-regulated in STTM164 transgenic plants, and the expression of Ptr NAC1 was the most obvious. Pro Ptrmi R164a:: GUS Arabidopsis auxin treatment Pro Ptrmi R164a:: GUS Arabidopsis auxin analogue NAA treatment Pro Ptrmi R164a:: GUS Arabidopsis materials confirmed that the expression of Ptrmi R164a was induced by auxin. In addition, the expression of auxin signal transduction protein PIN gene in STTM164 transgenic plants was also up-regulated, the above results showed that Ptrmi R164a::GUS Arabidopsis materials. R164a regulates the synthesis and metabolism of secondary wall of poplar through auxin signaling pathway. In this review, based on the cloning of poplar Ptrmi R164a, we preliminarily proved that Ptrmi R164a participated in secondary xylem and secondary phloem of poplar through signal transduction affecting auxin through gene transformation, histochemical analysis and histological section. This study provides some molecular evidence for the development and regulation of poplar wood.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2
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