基于蛋白組學(xué)的甘藍(lán)自交不親和性相關(guān)新基因的克隆與功能探索
發(fā)布時(shí)間：2018-08-18 08:51
【摘要】：顯花植物的有性生殖過程是一個(gè)包括從花粉接觸柱頭到受精完成的連續(xù)的復(fù)雜的過程,能否成功授粉是其中至關(guān)重要的一步。大多數(shù)雌雄同體植物進(jìn)化出自交不親和系統(tǒng)(Self-incompatibility,SI)來阻止自交、促進(jìn)雜交、保持物種多樣性。根據(jù)遺傳機(jī)制的不同,自交不親和分為孢子體型自交不親和(Sporophytic self-incompatibility,SSI)與配子體型自交不親和(Gametophytic self-incompatibilty,GSI)。蕓薹屬甘藍(lán)由單一S位點(diǎn)基因控制自交不親和反應(yīng),是典型的孢子體型自交不親和植物,其自交不親和信號(hào)傳導(dǎo)途徑是研究植物細(xì)胞間信號(hào)傳導(dǎo)的模式系統(tǒng)。蛋白質(zhì)是生命活動(dòng)的直接體現(xiàn)者,隨著人類和各種生物基因組測序的完成,生物研究進(jìn)入到后基因組時(shí)代,其中蛋白質(zhì)組學(xué)是后基因組時(shí)代研究的重要手段之一。蛋白質(zhì)雙向電泳(Two dimensional Electrophoresis,2-DE)技術(shù)由于能夠同時(shí)在一張膠上分離幾千甚至上萬個(gè)蛋白質(zhì)點(diǎn),成為了蛋白質(zhì)組學(xué)研究的主要支撐技術(shù)之一,質(zhì)譜技術(shù)的發(fā)展又使其更廣泛的應(yīng)用于蛋白質(zhì)組學(xué)研究。酵母雙雜交技術(shù)、pull-down等方法也為在體內(nèi)、體外研究蛋白質(zhì)相互作用提供了簡單、高效、快捷的途徑。本文利用蛋白質(zhì)雙向電泳技術(shù)分離、鑒定SI甘藍(lán)自花、異花授粉不同時(shí)間點(diǎn)柱頭差異表達(dá)蛋白質(zhì)。篩選出自花授粉特異表達(dá)和異花授粉上調(diào)表達(dá)蛋白質(zhì),分析它們在花器官、葉片等組織和授粉過程中m RNA表達(dá)量變化。利用pull-down和質(zhì)譜技術(shù)分離、鑒定了甘藍(lán)柱頭與SI相關(guān)新基因CML27相互作用的蛋白質(zhì);研究了SI相關(guān)基因SRBP1 RNA結(jié)合力;利用酵母雙雜交技術(shù)檢測了SI新因子Bo ROH1與Bo Exo70A1之間的相互作用,主要的工作與結(jié)果如下:甘藍(lán)柱頭差異表達(dá)蛋白質(zhì)的分離與鑒定(1)SI甘藍(lán)柱頭差異表達(dá)蛋白質(zhì)的分離與鑒定通過形態(tài)學(xué)觀察和親和指數(shù)測定兩種方法鑒定了SI甘藍(lán)A4自交不親和性和與F1雜交親和性。優(yōu)化了雙向電泳過程中IPG膠條、上樣量以及等電聚焦程序等條件,獲得了分辨率高、重復(fù)性好的蛋白質(zhì)雙向電泳(2-DE)圖像。利用PDQuest軟件分析自花授粉0 min、1 h、2 h和異花授粉1 h柱頭總蛋白質(zhì)2-DE圖像,發(fā)現(xiàn)自花授粉過程中,柱頭總蛋白質(zhì)主要在1 h時(shí)發(fā)生變化。自花授粉0min和1 h 2-DE圖像中發(fā)現(xiàn)了26個(gè)差異表達(dá)蛋白質(zhì)點(diǎn),其中6個(gè)特異表達(dá),16個(gè)上調(diào)表達(dá),4個(gè)下調(diào)表達(dá);自花、異花授粉1 h 2-DE圖像中發(fā)現(xiàn)了26個(gè)差異表達(dá)蛋白質(zhì)點(diǎn),其中3個(gè)特異表達(dá),6個(gè)在自花授粉柱頭中上調(diào)表達(dá),17個(gè)下調(diào)表達(dá)。自花授粉和異花授粉柱頭2-DE圖像中成功鑒定了44個(gè)差異表達(dá)蛋白質(zhì)點(diǎn)。KEGG和GO注釋歸類到:防御與脅迫反應(yīng)(15,34%)、蛋白質(zhì)代謝(7,16%)、碳水化合物與能量代謝(6,14%)、翻譯調(diào)控(5,11%)、花粉管發(fā)育(4,9%)、囊泡運(yùn)輸(4,9%)、細(xì)胞骨架蛋白質(zhì)(2,4.5%)和未知功能蛋白質(zhì)(1,2.5%)等8個(gè)生物學(xué)過程。SI甘藍(lán)自花授粉0 min vs 30 min和0 min vs 60 min轉(zhuǎn)錄組中分別發(fā)現(xiàn)了2870和3029個(gè)差異表達(dá)基因。GO和KEGG數(shù)據(jù)庫在0 min vs 30 min組中分別注釋了2738和815個(gè)基因,占總差異表達(dá)基因的95%和28%;0 min vs 60 min組中分別注釋了2904和961個(gè)差異表達(dá)基因,占總差異表達(dá)基因的96%和32%。綜合蛋白質(zhì)組和轉(zhuǎn)錄組數(shù)據(jù)認(rèn)為差異表達(dá)基因、蛋白質(zhì)CML27、SRBP1和ROH1是SI相關(guān)基因。(2)差異表達(dá)蛋白質(zhì)基因的克隆與表達(dá)分析分析了自花授粉柱頭特異表達(dá)蛋白質(zhì)C2H2、VPS29和annexin2以及異花授粉柱頭上調(diào)表達(dá)蛋白質(zhì)CML27、myosin、tubulin和BPS1在花器官與自花、異花授粉過程中m RNA表達(dá)量。組織表達(dá)特異性分析發(fā)現(xiàn),C2H2、VPS29、annexin2和tubulin在柱頭中m RNA表達(dá)量高于花粉;myosin和BPS1在花粉和柱頭中m RNA表達(dá)量相同;CML27在花粉中的m RNA表達(dá)量高于柱頭。q RT-PCR分析發(fā)現(xiàn)自花、異花授粉過程中,C2H2和CML27 m RNA先上調(diào)再下調(diào)表達(dá);myosin m RNA上調(diào)表達(dá);Tubulin和BPS1先下調(diào)再上調(diào)表達(dá);VPS29在自花授粉過程中先上調(diào)再下調(diào)表達(dá),異花授粉過程中下調(diào)表達(dá);Annexin2在自花授粉過程中下調(diào)表達(dá),異花授粉過程中先下調(diào)再上調(diào)表達(dá)。(3)SI相關(guān)新基因CML27的表達(dá)和柱頭相互作用蛋白質(zhì)鑒定將大腸桿菌中誘導(dǎo)表達(dá)的CML27蛋白質(zhì)與SI甘藍(lán)自花授粉1 h柱頭總蛋白質(zhì)孵育后,電泳分離,在130-180 k Da處發(fā)現(xiàn)了一條特異性條帶。利用質(zhì)譜技術(shù)成功在該條帶中發(fā)現(xiàn)了19種蛋白質(zhì),KEGG和GO注釋歸類到:防御與脅迫反應(yīng)(2,10.5%)、翻譯調(diào)控(5,26%)、基因沉默(2,10.5%)、蛋白質(zhì)代謝(4,21%)、鈣離子結(jié)合(2,10.5%)、染色體組織(2,10.5%)、囊泡運(yùn)輸(1,5.5%)和SI相關(guān)基因(1,5.5%)等8種生物學(xué)反應(yīng)過程。在這些蛋白質(zhì)中5種蛋白質(zhì)與Ca2+相關(guān)(AGP31、EF-2、EF-1α、PLDα1和PLDα2)、1個(gè)蛋白質(zhì)(coatomer subunit alpha-2-like)與囊泡運(yùn)輸相關(guān)和1個(gè)已知SI相關(guān)因子(SLG),說明CML27可能參與到自交不親和反應(yīng)中,作用于自花授粉柱頭乳突細(xì)胞內(nèi)Ca2+濃度的變化。(4)SI相關(guān)新基因SRBP1的基因克隆及功能研究氨基酸序列分析發(fā)現(xiàn)擬南芥中SRBP1與甘藍(lán)SRBP1A/2A氨基酸序列相似性達(dá)到了98%。生物信息學(xué)分析發(fā)現(xiàn)SRBP1、SRBP1A/2A在N-端含有一個(gè)RNA識(shí)別域(RRM)、C-端含有一個(gè)富含甘氨酸序列(GRD)。轉(zhuǎn)基因擬南芥GUS表達(dá)分析發(fā)現(xiàn),在植株根尖、莖尖、側(cè)根萌發(fā)點(diǎn)、葉片以及花器官中都觀察到了較強(qiáng)的GUS信號(hào),隨著組織發(fā)育GUS信號(hào)變?nèi)�。SRBP1功能研究發(fā)現(xiàn),原核表達(dá)SRBP1蛋白質(zhì)與長鏈RNA結(jié)合,不與mi RNA結(jié)合;煙草表達(dá)SRBP1蛋白質(zhì)特異性的與mi RNA166結(jié)合。這些結(jié)果表明SRBP1能與長鏈和mi RNA結(jié)合,蛋白質(zhì)的翻譯后修飾可能對于SRBP1與miRNA的結(jié)合力有決定性作用。(5)SI相關(guān)新基因ROH1與Exo70A1的表達(dá)與相互作用研究甘藍(lán)中ROH1為單外顯子編碼基因,編碼的蛋白質(zhì)含有398個(gè)氨基酸殘基。ROH1在甘藍(lán)花藥、柱頭、幼莖、幼根及葉片中都有表達(dá)。自花授粉過程中,ROH1在柱頭中表達(dá)量呈現(xiàn)出“上升-下降-上升”的變化趨勢,1 h時(shí)表達(dá)量最高;Exo70A1在柱頭中表達(dá)量呈現(xiàn)出先下降后上升的變化趨勢,授粉1 h時(shí)表達(dá)量最高。通過酵母雙雜交技術(shù)驗(yàn)證了ROH1和Exo70A1之間的相互作用。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2;S635
，
本文編號(hào)：2188977
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2;S635
，
本文編號(hào)：2188977
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