基于蛋白组学的甘蓝自交不亲和性相关新基因的克隆与功能探索

发布时间：2018-08-18 08:51

【摘要】：显花植物的有性生殖过程是一个包括从花粉接触柱头到受精完成的连续的复杂的过程,能否成功授粉是其中至关重要的一步。大多数雌雄同体植物进化出自交不亲和系统(Self-incompatibility,SI)来阻止自交、促进杂交、保持物种多样性。根据遗传机制的不同,自交不亲和分为孢子体型自交不亲和(Sporophytic self-incompatibility,SSI)与配子体型自交不亲和(Gametophytic self-incompatibilty,GSI)。芸薹属甘蓝由单一S位点基因控制自交不亲和反应,是典型的孢子体型自交不亲和植物,其自交不亲和信号传导途径是研究植物细胞间信号传导的模式系统。蛋白质是生命活动的直接体现者,随着人类和各种生物基因组测序的完成,生物研究进入到后基因组时代,其中蛋白质组学是后基因组时代研究的重要手段之一。蛋白质双向电泳(Two dimensional Electrophoresis,2-DE)技术由于能够同时在一张胶上分离几千甚至上万个蛋白质点,成为了蛋白质组学研究的主要支撑技术之一,质谱技术的发展又使其更广泛的应用于蛋白质组学研究。酵母双杂交技术、pull-down等方法也为在体内、体外研究蛋白质相互作用提供了简单、高效、快捷的途径。本文利用蛋白质双向电泳技术分离、鉴定SI甘蓝自花、异花授粉不同时间点柱头差异表达蛋白质。筛选出自花授粉特异表达和异花授粉上调表达蛋白质,分析它们在花器官、叶片等组织和授粉过程中m RNA表达量变化。利用pull-down和质谱技术分离、鉴定了甘蓝柱头与SI相关新基因CML27相互作用的蛋白质;研究了SI相关基因SRBP1 RNA结合力;利用酵母双杂交技术检测了SI新因子Bo ROH1与Bo Exo70A1之间的相互作用,主要的工作与结果如下:甘蓝柱头差异表达蛋白质的分离与鉴定(1)SI甘蓝柱头差异表达蛋白质的分离与鉴定通过形态学观察和亲和指数测定两种方法鉴定了SI甘蓝A4自交不亲和性和与F1杂交亲和性。优化了双向电泳过程中IPG胶条、上样量以及等电聚焦程序等条件,获得了分辨率高、重复性好的蛋白质双向电泳(2-DE)图像。利用PDQuest软件分析自花授粉0 min、1 h、2 h和异花授粉1 h柱头总蛋白质2-DE图像,发现自花授粉过程中,柱头总蛋白质主要在1 h时发生变化。自花授粉0min和1 h 2-DE图像中发现了26个差异表达蛋白质点,其中6个特异表达,16个上调表达,4个下调表达;自花、异花授粉1 h 2-DE图像中发现了26个差异表达蛋白质点,其中3个特异表达,6个在自花授粉柱头中上调表达,17个下调表达。自花授粉和异花授粉柱头2-DE图像中成功鉴定了44个差异表达蛋白质点。KEGG和GO注释归类到:防御与胁迫反应(15,34%)、蛋白质代谢(7,16%)、碳水化合物与能量代谢(6,14%)、翻译调控(5,11%)、花粉管发育(4,9%)、囊泡运输(4,9%)、细胞骨架蛋白质(2,4.5%)和未知功能蛋白质(1,2.5%)等8个生物学过程。SI甘蓝自花授粉0 min vs 30 min和0 min vs 60 min转录组中分别发现了2870和3029个差异表达基因。GO和KEGG数据库在0 min vs 30 min组中分别注释了2738和815个基因,占总差异表达基因的95%和28%;0 min vs 60 min组中分别注释了2904和961个差异表达基因,占总差异表达基因的96%和32%。综合蛋白质组和转录组数据认为差异表达基因、蛋白质CML27、SRBP1和ROH1是SI相关基因。(2)差异表达蛋白质基因的克隆与表达分析分析了自花授粉柱头特异表达蛋白质C2H2、VPS29和annexin2以及异花授粉柱头上调表达蛋白质CML27、myosin、tubulin和BPS1在花器官与自花、异花授粉过程中m RNA表达量。组织表达特异性分析发现,C2H2、VPS29、annexin2和tubulin在柱头中m RNA表达量高于花粉;myosin和BPS1在花粉和柱头中m RNA表达量相同;CML27在花粉中的m RNA表达量高于柱头。q RT-PCR分析发现自花、异花授粉过程中,C2H2和CML27 m RNA先上调再下调表达;myosin m RNA上调表达;Tubulin和BPS1先下调再上调表达;VPS29在自花授粉过程中先上调再下调表达,异花授粉过程中下调表达;Annexin2在自花授粉过程中下调表达,异花授粉过程中先下调再上调表达。(3)SI相关新基因CML27的表达和柱头相互作用蛋白质鉴定将大肠杆菌中诱导表达的CML27蛋白质与SI甘蓝自花授粉1 h柱头总蛋白质孵育后,电泳分离,在130-180 k Da处发现了一条特异性条带。利用质谱技术成功在该条带中发现了19种蛋白质,KEGG和GO注释归类到:防御与胁迫反应(2,10.5%)、翻译调控(5,26%)、基因沉默(2,10.5%)、蛋白质代谢(4,21%)、钙离子结合(2,10.5%)、染色体组织(2,10.5%)、囊泡运输(1,5.5%)和SI相关基因(1,5.5%)等8种生物学反应过程。在这些蛋白质中5种蛋白质与Ca2+相关(AGP31、EF-2、EF-1α、PLDα1和PLDα2)、1个蛋白质(coatomer subunit alpha-2-like)与囊泡运输相关和1个已知SI相关因子(SLG),说明CML27可能参与到自交不亲和反应中,作用于自花授粉柱头乳突细胞内Ca2+浓度的变化。(4)SI相关新基因SRBP1的基因克隆及功能研究氨基酸序列分析发现拟南芥中SRBP1与甘蓝SRBP1A/2A氨基酸序列相似性达到了98%。生物信息学分析发现SRBP1、SRBP1A/2A在N-端含有一个RNA识别域(RRM)、C-端含有一个富含甘氨酸序列(GRD)。转基因拟南芥GUS表达分析发现,在植株根尖、茎尖、侧根萌发点、叶片以及花器官中都观察到了较强的GUS信号,随着组织发育GUS信号变弱。SRBP1功能研究发现,原核表达SRBP1蛋白质与长链RNA结合,不与mi RNA结合;烟草表达SRBP1蛋白质特异性的与mi RNA166结合。这些结果表明SRBP1能与长链和mi RNA结合,蛋白质的翻译后修饰可能对于SRBP1与miRNA的结合力有决定性作用。(5)SI相关新基因ROH1与Exo70A1的表达与相互作用研究甘蓝中ROH1为单外显子编码基因,编码的蛋白质含有398个氨基酸残基。ROH1在甘蓝花药、柱头、幼茎、幼根及叶片中都有表达。自花授粉过程中,ROH1在柱头中表达量呈现出“上升-下降-上升”的变化趋势,1 h时表达量最高;Exo70A1在柱头中表达量呈现出先下降后上升的变化趋势,授粉1 h时表达量最高。通过酵母双杂交技术验证了ROH1和Exo70A1之间的相互作用。
[Abstract]:Sexual reproduction in flowering plants is a continuous and complex process from pollen contact to fertilization. Successful pollination is a crucial step. Most hermaphrodites have evolved self-incompatibility (SI) systems to prevent self-fertilization, promote hybridization, and maintain species diversity. According to different genetic mechanisms, self-incompatibility can be divided into sporophytic self-incompatibility (SSI) and gametophytic self-incompatibility (GSI). Brassica napus is a typical sporophytic self-incompatibility plant, which is controlled by a single S locus gene. The incompatibility signal transduction pathway is a model system for studying plant intercellular signal transduction. Protein is the direct embodiment of life activities. With the completion of human and various biological genome sequencing, biological research has entered the post-genome era, in which proteomics is one of the important means of post-genome era research. Two-dimensional electrophoresis (2-DE) technology has become one of the main supporting technologies in proteomics because it can separate thousands or even tens of thousands of protein spots on a single gel at the same time. The development of mass spectrometry technology has made it more widely used in proteomics. Yeast two-hybrid technology, pull-down and other methods. In this paper, two-dimensional protein electrophoresis (2-DE) was used to isolate and identify the differentially expressed proteins in stigma of self-flowering and cross-pollination of SI cabbage. The protein interacting with CML27 was identified by pull-down and mass spectrometry. The binding capacity of SRBP1 RNA was studied. The interaction between Bo ROH1 and Bo Exo 70A1 was detected by yeast two-hybrid technique. The main work and results were as follows: Isolation and identification of differentially expressed proteins in stigma of Brassica oleracea L. (1) Isolation and identification of differentially expressed proteins in stigma of SI cabbage L. The self-incompatibility and hybridization affinity of SI cabbage A4 with F1 were identified by morphological observation and affinity index determination. Two-dimensional electrophoresis (2-DE) images with high resolution and good repeatability were obtained under the conditions of sample size and isoelectric focusing program. The 2-DE images of total stigma proteins at 0 min, 1 h, 2 h of self-pollination and 1 h of cross-pollination were analyzed by PDQuest software. It was found that the total stigma proteins changed mainly at 1 h of self-pollination. 26 differentially expressed protein spots were found in 1H 2-DE and 1H 2-DE images, of which 6 were specific, 16 were up-regulated and 4 were down-regulated. 26 differentially expressed protein spots were found in 1H 2-DE images of self-pollination and cross-pollination, of which 3 were specific, 6 were up-regulated and 17 were down-regulated in stigma of self-pollination and cross-pollination. 44 differentially expressed protein spots were identified in 2-DE images of pollen stigma. KEGG and GO annotations were classified as defense and stress responses (15,34%), protein metabolism (7,16%), carbohydrate and energy metabolism (6,14%), translation regulation (5,11%), pollen tube development (4,9%), vesicle transport (4,9%), cytoskeleton protein (2,4.5%) and unknown functional protein (1). Two hundred and eighty-nine differentially expressed genes were found in the 0 min vs 30 min and 0 min vs 60 min transcription groups of SI cabbage. 961 differentially expressed genes, accounting for 96% and 32% of the total differentially expressed genes. According to proteomic and transcriptome data, the differentially expressed genes, including CML27, SRBP1 and ROH1, are SI-related genes. (2) Cloning and expression analysis of differentially expressed proteins, including C2H2, VPS29, annexin 2 and annexin 2, were analyzed. The expression of M RNA in stigma of cross-pollination was higher than that in pollen, and the expression of M RNA in pollen and stigma of myosin, myosin, tubulin and BPS1 was the same. Q-RT-PCR analysis showed that C2H2 and CML27 m RNA were up-regulated and down-regulated during cross-pollination, myosin m RNA was up-regulated, Tubulin and BPS1 were down-regulated and up-regulated, VPS29 was up-regulated and down-regulated during self-pollination and Annexin2 was down-regulated during cross-pollination. (3) Expression of a novel SI-related gene, CML27, and identification of stigma-interacting proteins. After incubation of the inducibly expressed CML27 protein in E. coli with total stigma proteins of SI cabbage after 1 h of self-pollination, a specific strip was found at 130-180 K Da. 19 proteins, KEGG and GO annotations were successfully classified into 8 biological categories: defense and stress responses (2,10.5%), translation regulation (5,26%), gene silencing (2,10.5%), protein metabolism (4,21%), calcium binding (2,10.5%), chromosomal tissue (2,10.5%), vesicle transport (1,5.5%) and SI-related genes (1,5.5%). Reaction Processes. Among these proteins, five proteins were associated with Ca2 + (AGP31, EF-2, EF-1 alpha, PLD alpha-1 and PLD alpha-2), one protein (coatmer subunit alpha-2-like) was associated with vesicle transport and one known SI-related factor (SLG), indicating that CML27 may be involved in self-incompatibility and act on Ca2+ concentration in self-pollinating styloid papilla cells. (4) Cloning and functional analysis of a novel SI-related gene SRBP1. Amino acid sequence analysis showed that the amino acid sequence similarity of SRBP1 and SRBP1A/2A in Arabidopsis reached 98%. Bioinformatics analysis showed that SRBP1 and SRBP1A/2A contained an RNA recognition domain (RRM) at N-terminal and a glycine-rich sequence (GRD) at C-terminal. The analysis of GUS expression in Arabidopsis showed that strong GUS signals were observed in root tips, shoot tips, lateral root germination sites, leaves and flower organs, and weakened with tissue development. These results suggest that SRBP1 can bind to long-chain and miRNA, and post-translational modification of protein may play a decisive role in binding ability of SRBP1 to microRNAs. (5) Expression and interaction of SI-related novel gene ROH1 and Exo70A1 in cabbage. ROH1 is a single exon-encoded gene encoding a protein containing 398 amino acid residues. ROH1 was expressed in the stigma, stigma, young stem, young root and leaf of Brassica napus L. The expression of ROH1 in stigma showed a trend of "up-down-up" during self-pollination, and reached the highest level at 1 h. Exo70A1 expression in stigma showed a trend of first decreasing and then rising, and reached the highest level at 1 h after pollination. Cross validation verified the interaction between ROH1 and Exo70A1.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2;S635
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本文编号：2188977