次生壁合成相关基因在棉花多倍化事件后的分子进化及棉花β-1,3-葡聚糖酶基因家族的比较分析

发布时间：2018-04-29 22:36

本文选题：棉花 + 全基因组复制　；参考：《南京农业大学》2016年博士论文

【摘要】：棉花是世界上重要的经济作物,棉纤维是纯天然的纺织原料,成熟的棉纤维主要由次生壁纤维素组成。进化研究表明,与可可从进化上分歧后,棉属祖先经历了一次多倍化事件,然而该古多倍化事件发生的具体时间及程度目前还有争议。在此次多倍化事件后棉花经历了二倍体化过程,染色体发生重排,大量重复基因丢失。到目前为止,在棉花中还没有任何重复基因保留倾向性的报道。本研究以拟南芥次生壁合成相关基因为参照,鉴定出棉花中次生壁合成相关基因,系统分析了这些基因在棉花二倍体化过程中的保留与丢失、在陆地棉不同组织器官的表达模式、表达分化以及受选择压等特征。研究结果初步阐明次生壁合成相关基因在棉花多倍化事件后的进化模式,首次提出次生壁合成相关基因在棉花进化过程中被优先保留,发现次生壁合成转录调控第三级 NAC ( NAM, ATAF1,2 and CUC2 )转录因子基因 NST1 ( NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 ) 和 SND1 ( SECONDARY WALL-ASSOCIATEDNAC DOMAIN PROTEIN1 )的重复基因发生表达分化,推测棉纤维次生壁合成转录调控与拟南芥维管束纤维次生壁合成转录调控相似,为深入研究棉纤维次生壁物质合成的转录调控奠定了基础。在棉纤维细胞伸长末期、次生壁加厚初期,细胞壁中胼胝质大量沉积,最大能占到细胞壁质量分数的10.5%。随后胼胝质被降解,在纤维成熟时其含量下降到1%。然而,胼胝质在纤维发育过渡时期的积累对棉纤维发育,特别是对次生壁发育的影响到目前为止还未有报道。纤维细胞壁中胼胝质的降解受到β-1,3-葡聚糖酶基因的控制。本研究对棉花β-1,3-葡聚糖酶基因家族成员进行结构、聚类、扩增特征、表达模式及进化速率等系统分析,发现Subfamily E β-1,3-葡聚糖酶基因经历了功能分化,明确该类基因不含有C端信号序列,具有组织器官表达专化性,进而发现GhGLU18,只在棉纤维中表达,而且在纤维发育过渡时期表达量最高。主要研究结果如下:1.次生壁合成相关基因在棉花多倍化事件后的分子进化以拟南芥(Arabidopsis thaliana)次生壁合成基因为参照,利用OrthoMCL软件及聚类分析的方法,对雷蒙德氏棉(Gossypium raimondii)、杨树(Poplus trichocarpa )、葡萄(Vitis vinifera )和可可(Theobroma cacao)基因组中的次生壁合成相关基因进行鉴定。在雷蒙德氏棉中,共鉴定出141个与次生壁合成相关的基因。棉花中次生壁合成基因的拷贝数分布从0至6,其与拟南芥次生壁合成基因拷贝数的比例平均为1.9:1。进一步研究发现棉花次生壁合成基因广泛分布于13条染色体上,未发现集中分布的区域。为研究次生壁合成基因在棉属古多倍化事件后二倍体化过程中的保留与丢失情况,以可可的基因数为参考,计算了其他物种总基因数及次生壁合成相关基因数与可可相应基因数的比例。棉花和可可分歧后,棉花经历了一次全基因组5-6倍化事件,可可没有经历全基因组重复(Whole genome duplication, WGD )事件,然而棉花(37505 )和可可(29452)的总基因数目比仅为1.27,说明在棉花的二倍体化过程中大部分重复基因都丢失了。相对于总基因的数目比例,棉花次生壁合成相关基因是可可的两倍,高出总基因数目比56.7%,表明次生壁合成相关基因在棉花二倍体化过程中被优先保留下来。四倍体棉花在很大程度上保留了来自于两个二倍体供体棉花的次生壁合成相关基因。利用陆地棉TM-1的RNA-seq数据对棉花次生壁合成相关基因进行不同组织器官表达分析,发现这些基因在富含次生壁物质的组织器官中高表达。在陆地棉的次生壁合成转录调控网络中,第三级NAC转录因子NST1和SND1的重复基因发生表达分化,部分重复基因在次生壁加厚时期的纤维中表达。我们推测棉纤维次生壁合成转录调控与维管束纤维次生壁合成转录调控相似。进一步研究发现,结构基因,特别是次生壁纤维素合成的CesA基因,重复基因间的表达模式几乎一致,并且受到了强烈的纯化选择。部分同源基因表达分化的程度小于重复基因间的表达分化程度。2.棉花β-1,3-葡聚糖酶家族基因的比较分析利用β-1,3-葡聚糖酶基因的种子文件PF00332通过HMMER程序检索蛋白数据库,将得到的候选β-1,3-葡聚糖酶基因经BLASTP验证,获得全基因组覆盖的β-1,3-葡聚糖酶基因。在雷蒙德氏棉(G. raimondii)、亚洲棉(G.arboreu )、陆地棉(G.hirsutum)TM-1和海岛棉(G. barbadense) 3-79中分别鉴定出67、68、130和158个β-1,3-葡聚糖酶基因。根据蛋白结构,棉花β-1,3-葡聚糖酶基因存在5种类型(type Ⅰ to type Ⅴ)。聚类分析揭示棉花(β-1,3-葡聚糖酶基因可以分为8个亚家族(Subfamily A-H),同一亚家族内的β-1,3--葡聚糖酶基因具有相似的蛋白结构及外显子-内含子结构。不同亚家族的β-1,3-葡聚糖酶基因具有不同的基因扩增模式,Subfamily A, B, C,G和Hβ-1,3-葡聚糖酶基因的扩增模式为片段重复(全基因组重复),Subfamily E为串联重复,而Subfamily D和F的β-1,3-葡聚糖酶基因通过以上两种模式进行扩增。利用陆地棉TM-1的RNA-seq数据对β-1,3-葡聚糖酶基因进行表达分析,发现陆地棉β-1,3-葡聚糖酶基因的表达在不同组织及发育不同时期存在多样性表达特征,而且同属一类的β-1,3--葡聚糖酶基因也有着不同的表达模式。Subfamily E β-1,3-葡聚糖酶基因的表达比较特殊,这类基因不含有C端信号序列,只在某一类组织器官中表达,在其他部位不表达,如GhGLU45只在花器官中表达,GhGLU18只在纤维中表达。进一步分析β-1,3-葡聚糖酶基因的进化速率和亚组表达的偏向性,发现Subfamily Eβ-1,3-葡聚糖酶基因在二倍体和四倍体中都经历了较快的蛋白进化,而且表现出很高比例的亚组表达偏好性。其中GhGLU18,只在棉纤维中表达,而且在纤维发育过渡时期表达量最高,推测GhGLU18可能和过渡时期胼胝质的降解相关。通过转基因技术将GhGLU18基因转入棉花,目前已经获得转基因过量和抑制表达材料,为研究GhGLU18及过渡时期胼胝质的积累和降解对棉纤维发育的影响奠定基础。
[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.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S562

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