PtrmiR164a在楊樹次生細胞壁合成過程中的功能研究
發(fā)布時間：2018-08-16 18:29
【摘要】：楊樹(Populus trichocarpa)是世界范圍內(nèi)種植面積最廣的速生林木之一,其主要用于造紙和建筑,同時也可用作生物燃料的原材料。2006年美國能源部完成了楊樹全基因組測序,使其作為木本模式植物廣泛應用于林木育種研究。闡明楊樹生長發(fā)育的分子調(diào)控機理,對于其分子育種非常重要,但目前楊樹次生生長、不定根發(fā)生等的遺傳分子調(diào)控機制尚不清楚。前人研究發(fā)現(xiàn),植物體內(nèi)的micro RNAs(mi RNAs)參與調(diào)控植物多個生長發(fā)育過程。mi R164是一類植物中保守的小RNA家族,參與調(diào)控擬南芥葉片、側(cè)向器官、側(cè)根發(fā)生等多個發(fā)育過程。但mi R164在木本植物生長發(fā)育中的調(diào)控功能尚未見報道。為了闡明楊樹mi R164的功能和作用機制,本論文進行了一系列研究,得到如下研究結(jié)果:1.Ptrmi R164a的特異性表達分析采用生物信息學方法,發(fā)現(xiàn)楊樹中Ptrmi R164家族存在6個成員,其前體形成不同的莖環(huán)結(jié)構(gòu),但其成熟體序列高度相似。RT-PCR分析發(fā)現(xiàn),6個Ptrmi R164成員表現(xiàn)出相似的組織表達特異性,即在根中表達量最高,其次為莖。進一步選取Ptrmi R164a開展深入研究。在克隆Ptrmi R164a啟動子的基礎上,GUS組織染色Pro Ptrmi R164a::GUS轉(zhuǎn)基因楊樹發(fā)現(xiàn),Ptrmi R164a在楊樹根、莖、葉中均有表達,但莖橫切片染色顯示,其只在莖初生韌皮部和形成層中表達。Pro Ptrmi R164a::GUS在轉(zhuǎn)基因擬南芥中發(fā)現(xiàn),Ptrmi R164a啟動子在花器官的柱頭、萼片和花絲中有顯著活性。2.Ptrmi R164a靶基因的預測運用mi RBASE數(shù)據(jù)庫對Ptrmi R164a靶基因預測發(fā)現(xiàn)Ptr CUC1.1、Ptr CUC1.1、Ptr CUC2、Ptr NAC1、Ptr NAC1.1、Ptr NAC021、Ptr NAC015等7個靶基因得分最高。與Atmi R164a靶基因進化分析發(fā)現(xiàn)其可分為三類,其中Ptr NAC1和Ptr NAC1.1與Ptrmi R164a成熟體序列最為匹配。3.PtrmiR164a表達下調(diào)轉(zhuǎn)基因植株的獲得克隆Ptrmi R164a成熟體序列,將其連接在植物表達載體上,最終獲得抑制表達載體STTM164。將該載體通過根癌農(nóng)桿菌介導轉(zhuǎn)入楊樹中,抗性篩選后獲得大量陽性轉(zhuǎn)基因植株,進一步采用PCR方法鑒定這些轉(zhuǎn)基因植株。實時定量PCR檢測Ptrmi R164a成熟體的表達水平發(fā)現(xiàn),其在轉(zhuǎn)基因植株中均呈不同程度的下降,選取其表達水平顯著下降的株系2和3進行后續(xù)研究。4.STTM164轉(zhuǎn)基因植株表型觀察及次生壁合成相關基因的表達分析與野生型相比STTM164轉(zhuǎn)基因植株不定根的發(fā)生較早,且側(cè)根較野生型發(fā)達。用營養(yǎng)液培養(yǎng)后得相同結(jié)果。組織化學染色、電鏡掃描觀察和定量統(tǒng)計結(jié)果顯示,在STTM164轉(zhuǎn)基因植株的韌皮部處早于野生型出現(xiàn)木質(zhì)素的多圈層沉積,同時STTM164轉(zhuǎn)基因植株的木質(zhì)部細胞層數(shù),細胞壁厚度的明顯增加導致STTM164轉(zhuǎn)基因植株木質(zhì)部加厚。Klason方法和溴化乙錠分析發(fā)現(xiàn),與野生型相比,STTM164轉(zhuǎn)基因植株木質(zhì)素含量增加。對STTM164轉(zhuǎn)基因植株次生壁合成途徑關鍵酶基因熒光定量PCR(q RT-PCR)檢測發(fā)現(xiàn),其表達水平均被上調(diào)。同時發(fā)現(xiàn),與韌皮部發(fā)育相關基因Ptr LBD1的表達量在STTM164轉(zhuǎn)基因植株中也被上調(diào),暗示Ptr LBD1基因參與調(diào)控STTM164轉(zhuǎn)基因植株韌皮部木質(zhì)素的生長發(fā)育。5.Ptrmi R164a靶基因表達量的檢測及其特異性表達分析q RT-PCR檢測發(fā)現(xiàn),STTM164轉(zhuǎn)基因植株中Ptrmi R164a靶基因表達量均被顯著上調(diào),其中Ptr NAC1表達上升最明顯。組織表達分析發(fā)現(xiàn),這些靶基因在根和莖中表達量最高,該結(jié)果與Ptrmi R164a的時空表達模式高度相似。6.Pro Ptrmi R164a::GUS擬南芥材料的生長素處理用生長素類似物NAA處理Pro Ptrmi R164a::GUS擬南芥材料證實,Ptrmi R164a的表達受生長素誘導。另外,STTM164轉(zhuǎn)基因植株中生長素信號轉(zhuǎn)導蛋白PIN基因的表達量也被上調(diào),上述結(jié)果表明,Ptrmi R164a通過生長素信號通路調(diào)控了楊樹次生壁的合成代謝。綜述所述,本論文在克隆楊樹Ptrmi R164a的基礎上,通過轉(zhuǎn)基因、組織化學分析、組織切片等方法,初步證明了Ptrmi R164a通過影響生長素的信號轉(zhuǎn)導,參與了楊樹次生木質(zhì)部和次生韌皮部的合成代謝調(diào)控。本研究為楊樹木材的發(fā)育調(diào)控提供了一定的分子證據(jù)。
[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.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2
本文編號：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.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2
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