本文選題：微管蛋白 + SPR1　； 參考：《中國(guó)林業(yè)科學(xué)研究院》2016年博士論文

【摘要】：微管(microtubule,MT)是由α與β微管蛋白異質(zhì)二聚體通過非共價(jià)鍵形成的管狀結(jié)構(gòu)。在植物的生理活動(dòng)中,微管通過微管結(jié)合蛋白(microtubule-associated proteins,MAPs)調(diào)節(jié)自身的動(dòng)態(tài)變化和不同細(xì)胞時(shí)期的列陣,以維持細(xì)胞形態(tài)與結(jié)構(gòu)、參與胞質(zhì)流動(dòng)、調(diào)節(jié)細(xì)胞有絲分裂、控制細(xì)胞極性生長(zhǎng)、細(xì)胞壁構(gòu)建、細(xì)胞分化調(diào)控、信號(hào)轉(zhuǎn)導(dǎo)等,因此微管蛋白家族和微管結(jié)合蛋白家族的研究一直受到廣泛的關(guān)注。本研究以旱柳及其變種龍爪柳為試驗(yàn)材料,針對(duì)α與β微管蛋白家族的基因結(jié)構(gòu)及其表達(dá)模式、結(jié)合蛋白SPR1家族的表達(dá)模式及其可能的功能開展了初步研究,取得的主要結(jié)果如下:(1)克隆鑒定了旱柳和龍爪柳的α與β微管蛋白基因,并對(duì)其進(jìn)行了序列相似性、系統(tǒng)發(fā)育、染色體定位以及表達(dá)模式的分析。結(jié)果顯示,兩種柳樹各有8個(gè)α微管蛋白基因和20個(gè)β微管蛋白基因。(2)柳樹種內(nèi)α微管蛋白兩兩基因間核苷酸和氨基酸序列相似性分別在78.3%和88.4%以上,種間α微管蛋白氨基酸序列相似性在88.2%以上,而兩種柳樹與毛果楊、鉆天柳、簸箕柳、擬南芥、玉米、水稻、高粱二穗短柄草和狗尾草等其它植物間的α微管蛋白氨基酸序列相似性在85.1%以上。半數(shù)α微管蛋白都包含一個(gè)C末端甲硫氨酸、亮氨酸、谷氨酸或谷氨酰胺,而不是典型的酪氨酸。系統(tǒng)發(fā)育表明,α微管蛋白分為兩類,說明這些基因可能起源于不同的祖先。C末端為Y型的TUA1基因在柳樹一年生枝條基部、中段、伸長(zhǎng)區(qū)和莖尖中高度表達(dá),推測(cè)它們可能參與木質(zhì)部的發(fā)育過程。(3)兩種柳樹β微管蛋白家族內(nèi)部成員間核酸和氨基酸序列相似性分別在74%和86.6%以上,種間同源蛋白氨基酸序列相似性在85.8%以上,柳樹與其它植物β微管蛋白間的氨基酸序列相似性在81.5%以上。系統(tǒng)發(fā)育顯示,柳樹β微管蛋白家族被分為4個(gè)亞組,結(jié)合楊樹β微管蛋白基因染色體定位,推測(cè)柳樹β微管蛋白基因家族經(jīng)歷了楊柳科全基因組重復(fù)事件和串聯(lián)重復(fù)事件,而柳樹TUB11和TUB12可能來源于區(qū)段重復(fù)或者轉(zhuǎn)座�；虮磉_(dá)模式分析發(fā)現(xiàn),該家族成員的表達(dá)具有一定的組織特異性,并且部分重復(fù)基因?qū)υ谒鶛z測(cè)組織中表達(dá)差異較大。柳樹微管蛋白基因家族成員序列的高度相似性、成員數(shù)量的進(jìn)化擴(kuò)張、以及表達(dá)模式的多樣性可能賦予了細(xì)胞分裂與生長(zhǎng)更高的靈活性,這對(duì)多年生木本植物的生長(zhǎng)發(fā)育習(xí)性的可塑性可能具有重要意義。(4)共克隆獲得旱柳與龍爪柳微管結(jié)合蛋白SPR1基因各6個(gè)。序列相似性分析發(fā)現(xiàn),兩種柳樹的SPR1直系同源蛋白氨基酸序列完全一致,6個(gè)成員的氨基酸序列C端和N端比較保守,中間連接區(qū)域?yàn)楦咦儏^(qū)。系統(tǒng)發(fā)育分析顯示,植物SPR1基因分為兩大類,SmSPR1基因在2個(gè)分類中都有分布。RT-PCR發(fā)現(xiàn),6個(gè)基因中只有SP1L1與SP1L2表達(dá),特別是SP1L1,該基因在柳樹一年生枝條伸長(zhǎng)區(qū)和中部表達(dá)量均比較高,基部和頂端次之。PSPR1+GUS轉(zhuǎn)基因煙草GUS組織化學(xué)分析顯示,柳樹SPR1啟動(dòng)子在煙草根伸長(zhǎng)區(qū)、莖木質(zhì)部與形成層中能夠啟動(dòng)GUS基因的表達(dá)。研究發(fā)現(xiàn),超表達(dá)SmSP1L1、SmSP1L4和SmSP1L5基因使擬南芥幼苗子葉發(fā)生了逆時(shí)針扭曲生長(zhǎng)現(xiàn)象,并導(dǎo)致果莢長(zhǎng)度縮短,籽粒數(shù)減少。對(duì)轉(zhuǎn)基因擬南芥黃化苗下胚軸細(xì)胞進(jìn)行微管的免疫熒光標(biāo)記,發(fā)現(xiàn)微管排列方向發(fā)生了不同程度的傾斜,這些結(jié)果表明柳樹SPR1基因主要在伸長(zhǎng)生長(zhǎng)較快的細(xì)胞中表達(dá),可能具有調(diào)節(jié)植物微管延伸方向的功能。
[Abstract]:Microtubules (microtubule, MT) is through the tubular structure non covalent bond formed by alpha and beta tubulin dimer. In two different physiological activities of plants, the microtubule microtubule binding protein (microtubule-associated proteins, MAPs) dynamic regulation changes and different period of the cell array, to maintain cell morphology and structure, participate in cytoplasmic flow, regulating mitosis, growth control cell polarity, cell wall formation, cell differentiation, signal transduction etc. Therefore, tubulin and microtubule binding protein family of the family has received widespread concern. In this study, Salix matsudana willow and its variants as experimental materials, the pattern of gene structure and expression of alpha with the beta tubulin binding protein family, the expression pattern of SPR1 family and its possible function to carry out a preliminary study, the main results are as follows: (1) cloning and identification of Salix Liu and Longzhua alpha and beta tubulin gene, and analyzed its sequence similarity, phylogeny, chromosomal localization and expression pattern analysis. The results showed that two kinds of willows each with 8 alpha tubulin gene and 20 beta tubulin gene. (2) a willow alpha tubulin protein 22 gene nucleotide and amino acid sequence similarity were above 78.3% and 88.4%, between alpha tubulin amino acid sequence similarity in more than 88.2%, while the two willow and poplar, willow dustpan, Chosenia arbutifolia, Arabidopsis, maize, rice, alpha tubulin amino acid sequences of two sorghum distachyon and Setaria viridis and other plants the similarity between the above 85.1%. Half of alpha tubulin contains a C terminal methionine, leucine, glutamine or glutamic acid, tyrosine. Instead of the typical phylogeny suggests that alpha tubulin is divided into two categories, suggesting that these genes can be It originated in the middle end of different ancestor.C Y type of TUA1 gene in the annual branches of willow base, elongation zone and shoot tip in high expression, speculated that they may be involved in xylem development. (3) two members of willow beta tubulin family between nucleic acid and amino acid sequence similarity respectively in the above 74% and 86.6% orthologous protein amino acid sequence similarity is above 85.8%, the amino acid sequence of willow and beta tubulin similarity between other plants in more than 81.5%. Phylogeny showed that willow beta tubulin family was divided into 4 subgroups, combination of beta tubulin genes in poplar, willow that beta tubulin protein gene family has experienced Salicaceous whole genome duplication events and tandem duplication events, while willow TUB11 and TUB12 might come from the duplicated or transposition. Model analysis found that the gene expression, members of the family The expression is tissue specific, and some duplicate gene pairs was detected in different tissues. The expression of high similarity member willow tubulin gene family sequences, the number of members of the evolutionary expansion, and diversity of expression patterns may confer cell division and growth of greater flexibility, the perennial woody plant the growth habit of plasticity may have important significance. (4) obtained with Salix microtubule binding protein SPR1 gene of Salix matsudana 6 were cloned. Sequence similarity analysis showed that two kinds of willow SPR1 orthologous identical amino acid sequences of amino acid sequences of 6 members of the C and N ends is conservative. The middle connecting region is highly variable region. Phylogenetic analysis showed that SPR1 gene is divided into two categories, SmSPR1 gene in 2 categories are distributed in the.RT-PCR found that 6 genes had SP1L1 and The expression of SP1L2, especially SP1L1, the gene expression was relatively high in willow annual branch elongation zone and central analysis base and the top of the.PSPR1+GUS transgenic tobacco GUS histochemistry showed that willow SPR1 promoter in tobacco root elongation zone, stem xylem and cambium can start the expression of the GUS gene. Found that over expression of SmSP1L1, SmSP1L4 and SmSP1L5 genes of Arabidopsis cotyledons showed an anticlockwise twisted growth phenomenon, and lead to shorten the length of pod, seed number was reduced. Immunofluorescence of microtubules of transgenic Arabidopsis etiolated hypocotyl cells, found various degrees of tilt microtubule orientation, these results suggest that willow SPR1 gene expression in cells mainly grow faster in the elongation, may regulate plant microtubules extend the function of direction.

【學(xué)位授予單位】：中國(guó)林業(yè)科學(xué)研究院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S792.12
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本文編號(hào)：1754580