本文選題：棉花 + 全基因組復制��； 參考：《南京農(nóng)業(yè)大學》2016年博士論文

【摘要】：棉花是世界上重要的經(jīng)濟作物,棉纖維是純天然的紡織原料,成熟的棉纖維主要由次生壁纖維素組成。進化研究表明,與可可從進化上分歧后,棉屬祖先經(jīng)歷了一次多倍化事件,然而該古多倍化事件發(fā)生的具體時間及程度目前還有爭議。在此次多倍化事件后棉花經(jīng)歷了二倍體化過程,染色體發(fā)生重排,大量重復基因丟失。到目前為止,在棉花中還沒有任何重復基因保留傾向性的報道。本研究以擬南芥次生壁合成相關(guān)基因為參照,鑒定出棉花中次生壁合成相關(guān)基因,系統(tǒng)分析了這些基因在棉花二倍體化過程中的保留與丟失、在陸地棉不同組織器官的表達模式、表達分化以及受選擇壓等特征。研究結(jié)果初步闡明次生壁合成相關(guān)基因在棉花多倍化事件后的進化模式,首次提出次生壁合成相關(guān)基因在棉花進化過程中被優(yōu)先保留,發(fā)現(xiàn)次生壁合成轉(zhuǎn)錄調(diào)控第三級 NAC ( NAM, ATAF1,2 and CUC2 )轉(zhuǎn)錄因子基因 NST1 ( NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 ) 和 SND1 ( SECONDARY WALL-ASSOCIATEDNAC DOMAIN PROTEIN1 )的重復基因發(fā)生表達分化,推測棉纖維次生壁合成轉(zhuǎn)錄調(diào)控與擬南芥維管束纖維次生壁合成轉(zhuǎn)錄調(diào)控相似,為深入研究棉纖維次生壁物質(zhì)合成的轉(zhuǎn)錄調(diào)控奠定了基礎。在棉纖維細胞伸長末期、次生壁加厚初期,細胞壁中胼胝質(zhì)大量沉積,最大能占到細胞壁質(zhì)量分數(shù)的10.5%。隨后胼胝質(zhì)被降解,在纖維成熟時其含量下降到1%。然而,胼胝質(zhì)在纖維發(fā)育過渡時期的積累對棉纖維發(fā)育,特別是對次生壁發(fā)育的影響到目前為止還未有報道。纖維細胞壁中胼胝質(zhì)的降解受到β-1,3-葡聚糖酶基因的控制。本研究對棉花β-1,3-葡聚糖酶基因家族成員進行結(jié)構(gòu)、聚類、擴增特征、表達模式及進化速率等系統(tǒng)分析,發(fā)現(xiàn)Subfamily E β-1,3-葡聚糖酶基因經(jīng)歷了功能分化,明確該類基因不含有C端信號序列,具有組織器官表達�；�,進而發(fā)現(xiàn)GhGLU18,只在棉纖維中表達,而且在纖維發(fā)育過渡時期表達量最高。主要研究結(jié)果如下:1.次生壁合成相關(guān)基因在棉花多倍化事件后的分子進化以擬南芥(Arabidopsis thaliana)次生壁合成基因為參照,利用OrthoMCL軟件及聚類分析的方法,對雷蒙德氏棉(Gossypium raimondii)、楊樹(Poplus trichocarpa )、葡萄(Vitis vinifera )和可可(Theobroma cacao)基因組中的次生壁合成相關(guān)基因進行鑒定。在雷蒙德氏棉中,共鑒定出141個與次生壁合成相關(guān)的基因。棉花中次生壁合成基因的拷貝數(shù)分布從0至6,其與擬南芥次生壁合成基因拷貝數(shù)的比例平均為1.9:1。進一步研究發(fā)現(xiàn)棉花次生壁合成基因廣泛分布于13條染色體上,未發(fā)現(xiàn)集中分布的區(qū)域。為研究次生壁合成基因在棉屬古多倍化事件后二倍體化過程中的保留與丟失情況,以可可的基因數(shù)為參考,計算了其他物種總基因數(shù)及次生壁合成相關(guān)基因數(shù)與可可相應基因數(shù)的比例。棉花和可可分歧后,棉花經(jīng)歷了 一次全基因組5-6倍化事件,可可沒有經(jīng)歷全基因組重復(Whole genome duplication, WGD )事件,然而棉花(37505 )和可可(29452)的總基因數(shù)目比僅為1.27,說明在棉花的二倍體化過程中大部分重復基因都丟失了。相對于總基因的數(shù)目比例,棉花次生壁合成相關(guān)基因是可可的兩倍,高出總基因數(shù)目比56.7%,表明次生壁合成相關(guān)基因在棉花二倍體化過程中被優(yōu)先保留下來。四倍體棉花在很大程度上保留了來自于兩個二倍體供體棉花的次生壁合成相關(guān)基因。利用陸地棉TM-1的RNA-seq數(shù)據(jù)對棉花次生壁合成相關(guān)基因進行不同組織器官表達分析,發(fā)現(xiàn)這些基因在富含次生壁物質(zhì)的組織器官中高表達。在陸地棉的次生壁合成轉(zhuǎn)錄調(diào)控網(wǎng)絡中,第三級NAC轉(zhuǎn)錄因子NST1和SND1的重復基因發(fā)生表達分化,部分重復基因在次生壁加厚時期的纖維中表達。我們推測棉纖維次生壁合成轉(zhuǎn)錄調(diào)控與維管束纖維次生壁合成轉(zhuǎn)錄調(diào)控相似。進一步研究發(fā)現(xiàn),結(jié)構(gòu)基因,特別是次生壁纖維素合成的CesA基因,重復基因間的表達模式幾乎一致,并且受到了強烈的純化選擇。部分同源基因表達分化的程度小于重復基因間的表達分化程度。2.棉花β-1,3-葡聚糖酶家族基因的比較分析利用β-1,3-葡聚糖酶基因的種子文件PF00332通過HMMER程序檢索蛋白數(shù)據(jù)庫,將得到的候選β-1,3-葡聚糖酶基因經(jīng)BLASTP驗證,獲得全基因組覆蓋的β-1,3-葡聚糖酶基因。在雷蒙德氏棉(G. raimondii)、亞洲棉(G.arboreu )、陸地棉(G.hirsutum)TM-1和海島棉(G. barbadense) 3-79中分別鑒定出67、68、130和158個β-1,3-葡聚糖酶基因。根據(jù)蛋白結(jié)構(gòu),棉花β-1,3-葡聚糖酶基因存在5種類型(type Ⅰ to type Ⅴ)。聚類分析揭示棉花(β-1,3-葡聚糖酶基因可以分為8個亞家族(Subfamily A-H),同一亞家族內(nèi)的β-1,3--葡聚糖酶基因具有相似的蛋白結(jié)構(gòu)及外顯子-內(nèi)含子結(jié)構(gòu)。不同亞家族的β-1,3-葡聚糖酶基因具有不同的基因擴增模式,Subfamily A, B, C,G和Hβ-1,3-葡聚糖酶基因的擴增模式為片段重復(全基因組重復),Subfamily E為串聯(lián)重復,而Subfamily D和F的β-1,3-葡聚糖酶基因通過以上兩種模式進行擴增。利用陸地棉TM-1的RNA-seq數(shù)據(jù)對β-1,3-葡聚糖酶基因進行表達分析,發(fā)現(xiàn)陸地棉β-1,3-葡聚糖酶基因的表達在不同組織及發(fā)育不同時期存在多樣性表達特征,而且同屬一類的β-1,3--葡聚糖酶基因也有著不同的表達模式。Subfamily E β-1,3-葡聚糖酶基因的表達比較特殊,這類基因不含有C端信號序列,只在某一類組織器官中表達,在其他部位不表達,如GhGLU45只在花器官中表達,GhGLU18只在纖維中表達。進一步分析β-1,3-葡聚糖酶基因的進化速率和亞組表達的偏向性,發(fā)現(xiàn)Subfamily Eβ-1,3-葡聚糖酶基因在二倍體和四倍體中都經(jīng)歷了較快的蛋白進化,而且表現(xiàn)出很高比例的亞組表達偏好性。其中GhGLU18,只在棉纖維中表達,而且在纖維發(fā)育過渡時期表達量最高,推測GhGLU18可能和過渡時期胼胝質(zhì)的降解相關(guān)。通過轉(zhuǎn)基因技術(shù)將GhGLU18基因轉(zhuǎn)入棉花,目前已經(jīng)獲得轉(zhuǎn)基因過量和抑制表達材料,為研究GhGLU18及過渡時期胼胝質(zhì)的積累和降解對棉纖維發(fā)育的影響奠定基礎。
[Abstract]:Cotton is an important economic crop in the world. Cotton fiber is a pure natural textile raw material, and mature cotton fiber is mainly composed of secondary wall cellulose. Evolutionary studies show that after the evolution of cocoa, the ancestors of the genus cotton have experienced a polyploidy event, but the time and degree of the occurrence of the polyploidy event are still controversial. After this polyploidy event, cotton experienced diploid process, rearrangement of chromosomes, and repeated gene loss. Up to now, there has not been any duplication of gene retention tendency in cotton. In this study, the related genes of secondary wall synthesis in cotton were identified as the related genes of secondary wall synthesis of Arabidopsis thaliana, and the system of secondary wall synthesis in cotton was identified. The retention and loss of these genes in the diploid process of cotton, expression patterns, expression differentiation and selective pressure in different tissues of upland cotton were analyzed. The results preliminarily elucidated the evolution pattern of the secondary wall synthesis related genes after the polyploidy of cotton, and first proposed the secondary wall synthesis related genes in cotton. In the process of evolution, it was preferred to find that the secondary wall synthesis and transcription regulation third NAC (NAM, ATAF1,2 and CUC2) transcription factor gene NST1 (NAC SECONDARY WALL THICKENING PROMOTING FACTOR1) and reduplication genes were expressed and differentiated, and the secondary wall of cotton fibers was conjectured. The transcriptional regulation is similar to the transcriptional regulation of the secondary wall of the vascular bundle of Arabidopsis thaliana. It lays the foundation for the deep study of the transcription regulation of the material synthesis of the secondary wall of cotton fibers. At the end of the extension of the cotton fiber cells, the callose deposited in the cell wall at the early stage of the secondary wall thickening, and the maximum of the cell wall mass fraction was 10.5%. then callose. However, the accumulation of callose in the transition period of fiber development has not yet been reported on the development of cotton fiber, especially the secondary wall development. The degradation of callose in the fibrous cell wall is controlled by the beta -1,3- glucan enzyme gene in the fibrous cell wall. This study on cotton beta -1,3- glucan. The family members of the enzyme gene family carried out a systematic analysis of structure, clustering, amplification characteristics, expression pattern and evolution rate. It was found that the Subfamily E beta -1,3- glucan enzyme gene had undergone functional differentiation. It was clear that the gene did not contain the C terminal signal sequence and had the specificity of tissue and organ expression, and then found that GhGLU18 was expressed only in cotton fiber and in fiber. The main results were as follows: 1. the molecular evolution of the 1. secondary wall synthesis related genes was based on the secondary wall synthesis gene of Arabidopsis (Arabidopsis thaliana) after the polyploidy of cotton. Using OrthoMCL software and cluster analysis, the Gossypium raimondii (Gossypium raimondii) and the poplar (Poplus TR) were used. Ichocarpa) identification of secondary wall synthesis related genes in the genome of Vitis vinifera and Theobroma cacao. In Raymond's cotton, a total of 141 genes related to secondary wall synthesis were identified. The copy number of the secondary wall synthesis gene in cotton was from 0 to 6, and the ratio of the copy number to the secondary wall synthesis of Arabidopsis thaliana was compared. The average 1.9:1. further study found that the cotton secondary wall synthesis gene was widely distributed on 13 chromosomes and did not find the area of concentrated distribution. In order to study the retention and loss of the secondary biosynthesis gene in the diploid process after the polyploidy of the genus Gossypium, the total genes of other species were calculated with the basis of the basis factor of cocoa. The number and ratio of the number of related genes to the secondary wall synthesis and the number of corresponding cocoa genes. After the difference between cotton and cocoa, the cotton had undergone a whole genome 5-6 fold event, and the cocoa did not experience the Whole genome duplication, WGD, but the total gene number of cotton (37505) and cocoa (29452) was only 1.27, indicating that In the diploid process of cotton, most of the repetitive genes were lost. Relative genes of the secondary wall of cotton were two times as high as the proportion of the total genes, and the number of total genes was 56.7%, indicating that the secondary wall synthesis related genes were first preserved in the diploid process of cotton. The tetraploid cotton was greatly improved. The secondary wall synthesis related genes from two diploid donor cotton were retained. Using RNA-seq data from upland cotton TM-1, the expression of different tissues and organs of the cotton secondary wall synthesis related genes was analyzed. It was found that these genes were highly expressed in the tissues and organs rich in secondary wall material. The transcriptional regulation network of the secondary wall of upland cotton was synthesized. In the collaterals, the repeat genes of the third level NAC transcription factors NST1 and SND1 are expressed and differentiated, and some of the repetitive genes are expressed in the fibers of the secondary wall thickening. We speculate that the transcription regulation of the secondary wall of cotton fibers is similar to the regulation of the secondary wall synthesis and transcription of vascular bundles. The expression patterns of the synthetic CesA gene are almost identical with the repeated genes and are strongly purified. The degree of differentiation of some homologous genes is less than the degree of expression and differentiation between the duplicated genes.2. cotton beta -1,3- glucan enzyme family gene comparison analysis using the seed file of beta -1,3- glucan enzyme gene PF00332 through HMME The R program retrieved the protein database, and the candidate beta -1,3- glucan enzyme gene was tested by BLASTP to obtain the whole genome covered beta -1,3- glucan enzyme gene. The 67,68130 and 158 beta Portuguese were identified in Raymond's Cotton (G. raimondii), Asian cotton (G.arboreu), land cotton (G.hirsutum) TM-1 and island cotton (G. barbadense) 3-79. According to the protein structure, there are 5 types of cotton beta -1,3- glucanase gene (type I to type V). The cluster analysis reveals that the cotton (beta -1,3- glucanase gene can be divided into 8 subfamilies (Subfamily A-H), and the beta -1,3-- glucanase gene in the same subfamily has similar protein structure and exon intron structure. The beta -1,3- glucanase gene of different subfamilies has different gene amplification patterns. The amplification mode of Subfamily A, B, C, G and H beta -1,3- glucanase gene is fragment repetition (whole genome duplication), Subfamily E is repeated in series, and Subfamily D and beta glucanase genes are amplified by the above two modes. The RNA-seq data of cotton TM-1 expressed the expression of beta -1,3- glucan enzyme gene. It was found that the expression of the gene expression of beta -1,3- glucan enzyme in Upland Cotton existed in different tissues and different stages of development, and the gene of beta -1,3-- glucan enzyme of the same category also had different expression patterns of.Subfamily E beta -1,3- glucan enzyme gene. The expression is special. This kind of gene does not contain C terminal signal sequence. It is expressed only in one kind of tissue and organ, not expressed in other parts. For example, GhGLU45 is only expressed in the flower organs, and GhGLU18 is only expressed in the fiber. The evolution rate of the beta -1,3- glucan enzyme gene and the bias of subgroup expression are further analyzed, and Subfamily E beta -1,3- Portuguese cohesion is found. The glucose enzyme gene has undergone rapid protein evolution in diploid and tetraploid, and shows a high proportion of subgroup preference. Among them, GhGLU18 is expressed only in cotton fibers, and is the highest in the transition period of fiber development. It is presumed that GhGLU18 may be related to the degradation of callose in the transitional period. By transgene technology, GhG The transfer of LU18 gene into cotton has now obtained transgenic overexpression and inhibition of expression material, which lays the foundation for the study of the effect of accumulation and degradation of callose in GhGLU18 and transition period on cotton fiber development.

【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S562

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