窄冠型杨树分枝相关基因的筛选和功能验证

发布时间：2018-09-05 09:58

【摘要】：植物的分枝发育是一个复杂的生物学过程,受到遗传、激素、环境和营养等多种因素的调控,在植物的形态建成中具有重要的的意义。在禾本科植物中例如水稻和玉米,分蘖数的多少与作物的产量有着直接的关系;在木本植物中,分枝发育影响着木本植物的经济效益和木材材性,在林木的株型发育和生态建成中具有重要的作用。本研究针对窄冠黑白杨冠幅小,分枝多的特点,构建了窄冠黑白杨和中林2025杨早春萌动期腋芽的转录组,寻找影响窄冠和宽冠杨树的差异表达基因,揭示窄冠和宽冠杨树的分枝相关基因的表达模式,构建分枝发育的激素调控通路;同时克隆独脚金内酯信号转导的关键酶-D14α,β-水解酶和生长素极性运输通路的关键运输载体蛋白-PIN蛋白,并构建超表达载体,研究PopD14和PopPIN1在分枝发育中的作用。为进一步研究杨树分枝发育的相关基因及激素调控机理提供一定的分子基础,为揭示木本植物的分枝发育的调控机制奠定理论基础。主要结论如下:1、转录组测序得到198,200,000条clean read。利用BLAST工具将每个文库的reads与毛果杨参考基因组进行比对,112900000条reads可以与480M的参考基因组匹配,占总reads的56.96%;105700000条(53.30%)reads与毛果杨基因组有单一位置匹配。2、对转录组测序得到的差异表达基因进行功能注释,共有3353个差异表达基因在数据库中有注释,其中有3353个差异基因被注释到Nr数据库中,在GO数据库中,2805个差异基因被注释到GO数据库;1389个差异基因被注释到COG数据库;在KEGG数据库中,520个基因注释到101个代谢或信号通路中。其中激素调控相关的KEGG通路是植物激素信号转导(plant hormone signal transduction ko04075)(26 5.00%)、萜烯类代谢(tryptophan metabolism ko00500)(8,1.54%)和ABC转运(ABC transport ko02010)(1,0.19%)。3、通过高通量测序,获得了调控分枝发育的差异表达基因,发现参与生长素响应、激素信号通路、生长素极性运输和生长素生物合成的相关基因参与杨树分枝发育的调控,构建了一个杨树分枝发育的激素调控网络,促进我们对于杨树分枝发育机制的理解,并为杨树分枝发育的研究提供一定的分子基础。4、本研究利用qRT-PCR技术,对随机挑选的参与杨树分枝发育的激素调控的11条基因进行表达量验证,结果表明有10条基因与转录组的RPKM值相符;同时挑选了参与分枝发育的5个关键基因进行不同组织的表达研究,结果发现除了PIN1其它4个基因均在腋芽中高度表达。5、克隆了独脚金内酯信号转导D14和生长素极性运输载体PIN1基因。结果发现,PopD14的全长1169 bp,包含一个1122 bp的ORF区,编码374个氨基酸,同源比对和进化树分析表明,PopD14基因与麻疯树和蓖麻的同源性最高;PopPIN1的全长为1920 bp,包含一个1860 bp的ORF,编码了620个氨基酸残基;进化树分析表明,PopPIN1在进化上与麻疯树和蓖麻的同源关系最近。
[Abstract]:The branching and development of plants is a complex biological process, which is regulated by many factors, such as heredity, hormone, environment and nutrition, etc., which plays an important role in plant morphogenesis. In grasses such as rice and corn, the number of tillers is directly related to the yield of the crop; in woody plants, branch development affects the economic efficiency and wood properties of woody plants, It plays an important role in plant type development and ecological establishment of forest trees. Based on the characteristics of narrow crown black and white poplar with small width and many branches, the transcriptional groups of axillary buds of narrow crown black and white poplar and Zhonglin 2025 poplar during early spring germination were constructed, and the differentially expressed genes affecting narrow crown and wide crown poplar were found. To reveal the expression pattern of branching-related genes in narrow crown and wide crown poplar and to construct the hormone regulation pathway for branch development. At the same time, the key transduction enzymes -D14 伪, 尾 -hydrolase and auxin polar transport pathway were cloned, and the overexpression vector was constructed to study the role of PopD14 and PopPIN1 in branching development. It provides a molecular basis for the further study of the related genes and hormone regulation mechanism of poplar branch development, and lays a theoretical foundation for revealing the regulation mechanism of woody plant branching development. The main conclusions are as follows: 1: 1, 198200000 clean read. were obtained by transcriptome sequencing. Using the BLAST tool to compare the reference genome of each library with the reference genome of Populus tomentosa, 112900000 reads can be matched with the reference genome of 480m. 56.96% (53.30%) of the total reads contained 105700000 pieces (53.30%) of reads matched with the genome of Populus tomentosa. The functional annotation of differentially expressed genes obtained by transcriptome sequencing was carried out. A total of 3353 differentially expressed genes were annotated in the database. 3353 genes were annotated into Nr database, 2805 genes were annotated into GO database in GO database, 1389 genes were annotated into COG database. In the KEGG database, 520 genes were annotated into 101 metabolic or signaling pathways. The KEGG pathway related to hormone regulation was plant hormone signal transduction (plant hormone signal transduction ko04075 (265.00%), terpene metabolized (tryptophan metabolism ko00500 (81.54%) and ABC transport (ABC transport ko02010 (10.19%) .Through high-throughput sequencing, differentially expressed genes regulating branching development were obtained, and the differentially expressed genes were found to be involved in auxin response. Hormone signaling pathway, auxin polar transport and auxin biosynthesis are involved in the regulation of poplar branch development. This study also provides a molecular basis for the study of poplar branching development. In this study, qRT-PCR technique was used to verify the expression of 11 genes randomly selected by hormones involved in poplar branch development. The results showed that there were 10 genes consistent with the RPKM value of transcriptome, and 5 key genes involved in branching development were selected to study the expression of different tissues. The results showed that the other four genes except PIN1 were highly expressed in axillary buds. The gene of Hippogolide signal transduction D14 and auxin polarity transport vector PIN1 were cloned. The results showed that the total length of 1169 bp, of PopD14 contained a ORF region of 1122 bp, encoding 374 amino acids. Homology alignment and phylogenetic tree analysis showed that the homology of PopD14 gene with Jatropha curcas and Castor communis was the highest. The total length of PopPIN1 was 1920 bp, which contained a 1860 bp ORF, encoding 620-amino acid residues. Phylogenetic tree analysis showed that PopPIN1 had the closest phylogenetic relationship with Jatropha curcas and castor.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S792.11

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