两种柳树tubulin与spr1基因家族的序列分析及功能初探

发布时间：2018-04-15 14:51

  本文选题：微管蛋白 + SPR1　； 参考：《中国林业科学研究院》2016年博士论文

【摘要】：微管(microtubule,MT)是由α与β微管蛋白异质二聚体通过非共价键形成的管状结构。在植物的生理活动中,微管通过微管结合蛋白(microtubule-associated proteins,MAPs)调节自身的动态变化和不同细胞时期的列阵,以维持细胞形态与结构、参与胞质流动、调节细胞有丝分裂、控制细胞极性生长、细胞壁构建、细胞分化调控、信号转导等,因此微管蛋白家族和微管结合蛋白家族的研究一直受到广泛的关注。本研究以旱柳及其变种龙爪柳为试验材料,针对α与β微管蛋白家族的基因结构及其表达模式、结合蛋白SPR1家族的表达模式及其可能的功能开展了初步研究,取得的主要结果如下:(1)克隆鉴定了旱柳和龙爪柳的α与β微管蛋白基因,并对其进行了序列相似性、系统发育、染色体定位以及表达模式的分析。结果显示,两种柳树各有8个α微管蛋白基因和20个β微管蛋白基因。(2)柳树种内α微管蛋白两两基因间核苷酸和氨基酸序列相似性分别在78.3%和88.4%以上,种间α微管蛋白氨基酸序列相似性在88.2%以上,而两种柳树与毛果杨、钻天柳、簸箕柳、拟南芥、玉米、水稻、高粱二穗短柄草和狗尾草等其它植物间的α微管蛋白氨基酸序列相似性在85.1%以上。半数α微管蛋白都包含一个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。系统发育表明,α微管蛋白分为两类,说明这些基因可能起源于不同的祖先。C末端为Y型的TUA1基因在柳树一年生枝条基部、中段、伸长区和茎尖中高度表达,推测它们可能参与木质部的发育过程。(3)两种柳树β微管蛋白家族内部成员间核酸和氨基酸序列相似性分别在74%和86.6%以上,种间同源蛋白氨基酸序列相似性在85.8%以上,柳树与其它植物β微管蛋白间的氨基酸序列相似性在81.5%以上。系统发育显示,柳树β微管蛋白家族被分为4个亚组,结合杨树β微管蛋白基因染色体定位,推测柳树β微管蛋白基因家族经历了杨柳科全基因组重复事件和串联重复事件,而柳树TUB11和TUB12可能来源于区段重复或者转座。基因表达模式分析发现,该家族成员的表达具有一定的组织特异性,并且部分重复基因对在所检测组织中表达差异较大。柳树微管蛋白基因家族成员序列的高度相似性、成员数量的进化扩张、以及表达模式的多样性可能赋予了细胞分裂与生长更高的灵活性,这对多年生木本植物的生长发育习性的可塑性可能具有重要意义。(4)共克隆获得旱柳与龙爪柳微管结合蛋白SPR1基因各6个。序列相似性分析发现,两种柳树的SPR1直系同源蛋白氨基酸序列完全一致,6个成员的氨基酸序列C端和N端比较保守,中间连接区域为高变区。系统发育分析显示,植物SPR1基因分为两大类,SmSPR1基因在2个分类中都有分布。RT-PCR发现,6个基因中只有SP1L1与SP1L2表达,特别是SP1L1,该基因在柳树一年生枝条伸长区和中部表达量均比较高,基部和顶端次之。PSPR1+GUS转基因烟草GUS组织化学分析显示,柳树SPR1启动子在烟草根伸长区、茎木质部与形成层中能够启动GUS基因的表达。研究发现,超表达SmSP1L1、SmSP1L4和SmSP1L5基因使拟南芥幼苗子叶发生了逆时针扭曲生长现象,并导致果荚长度缩短,籽粒数减少。对转基因拟南芥黄化苗下胚轴细胞进行微管的免疫荧光标记,发现微管排列方向发生了不同程度的倾斜,这些结果表明柳树SPR1基因主要在伸长生长较快的细胞中表达,可能具有调节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.

【学位授予单位】：中国林业科学研究院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S792.12
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本文编号：1754580