离子束诱导天竺葵花色突变体的变异机理研究
发布时间：2019-05-22 22:54
【摘要】：花色作为观赏花卉重要的经济指标,在植物性状改良中成为首要培育目标。本研究利用碳离子束辐照天竺葵幼嫩枝条,在辐照当代筛选出一株稳定遗传的花色突变体LPM。以揭示离子束诱导天竺葵突变体花色变异的机理为目的,本研究通过对天竺葵野生型与突变体表型观察、花色形成原因分析、类黄酮成分和含量鉴定以及花色形成关键结构基因的差异表达分析,系统开展了离子束辐照后突变体花色变浅的机制研究。通过RNA-Seq高通量测序技术对花瓣转录组进行深度测序,筛选野生型和突变体间差异表达的unigene,预测天竺葵花色形成相关基因,最终阐明离子束辐照引起天竺葵突变体花色改变的分子机理,为后续花色改良和新品种选育提供理论基础。以下是本研究的主要内容和结果:1、离子束30Gy辐照处理筛选到一株花色突变体LPM,与对照相比其花色和生理指标均发生了明显改变。解剖学观察显示RWT花瓣细胞中充满了橙红色花青素,而LPM细胞则表现为极浅的粉色。2、分子标记检测出RWT和LPM基因组多态性条带66条,其中有18条是LPM中独有的条带,另外48条是LPM中缺失的,RWT和LPM间相似性系数为0.868,遗传多态性13.2%。3、利用高效液相法测定了RWT和LPM中类黄酮成分及含量,结果表明RWT花瓣中含有的天竺葵素、飞燕草素及矢车菊素是其主要的成色物质,LPM中仅检测到矢车菊素的积累,天竺葵素和飞燕草素的缺失是其花色变浅的根本原因。Q-PCR结果显示LPM中花青素合成早期基因CHS、CHI表达明显下调,ANS在LPM整个花色发育过程中都保持着极低的转录水平,这些基因,尤其是ANS的低表达可能抑制或阻断了花青素的生物合成,最终导致花瓣颜色的变化。4、通过RNA-Seq高通量测序技术建立了天竺葵花瓣转录组数据库,测序共组装获得89817个unigene,在LPM和RWT文库中检测到差异表达基因1503个,其中在LPM中显著上调表达基因700个,显著下调表达基因803个。Unigene在多个公共数据库中检测到同源蛋白,其中在SignalP库中比对最多。KEGG通路富集分析显示差异基因极显著性富集在类黄酮生物合成通路中。5、根据转录组测序数据预测到177个unigene可能涉及18种天竺葵花色形成基因,包括花青素合成基因,花青素修饰基因,黄酮和黄酮醇合成基因,花青素运输有关的基因;另外,预测到多个花色调节转录因子。Q-PCR验证14个结构基因和3类转录因子的表达量,结果与转录组数据基本一致,基因表达以下调为主。
[Abstract]:Flower color, as an important economic index of ornamental flowers, has become the primary cultivation goal in the improvement of plant traits. In this study, a stable flower color mutant LPM. was screened out by carbon ion beam irradiation on young branches of geranium. In order to reveal the mechanism of flower color variation induced by ion beam, the causes of flower color formation were analyzed by observing the wild type and mutant phenotype of geranium. The composition and content of flavonoids and the differential expression analysis of key structural genes of flower color formation were studied systematically. The mechanism of flower color lightening after ion beam irradiation was studied systematically. The petal transcription group was sequenced deeply by RNA-Seq high throughput sequencing technique, and the differentially expressed unigene, genes between wild type and mutant were screened to predict the flower color formation of gerbils. Finally, the molecular mechanism of flower color change induced by ion beam irradiation was clarified, which provided a theoretical basis for subsequent flower color improvement and new variety selection. The main contents and results of this study are as follows: 1. A flower color mutant LPM, was screened by ion beam 30Gy irradiation and its color and physiological indexes were significantly changed compared with the control. Anatomical observation showed that RWT petal cells were filled with orange anthocyanins, while LPM cells showed very light pink. 2. 66 bands of RWT and LPM genomic polymorphism were detected by molecular markers, of which 18 were unique to LPM. The other 48 were missing from LPM. The similarity coefficient between RWT and LPM was 0.868, and the genetic polymorphism was 13.2%. The components and contents of flavonoids in RWT and LPM were determined by high performance liquid chromatography (HPLC). The results showed that germanium was contained in RWT petals. Elaeagnus angustifolia and Cypermethrin are the main color-forming substances. Only the accumulation of procyanidin was detected in LPM, and the deletion of geranium and permethrin was the root cause of the lighter flower color. Q-PCR results showed that the anthocyanin synthesis early gene CHS, in LPM was found to be the root cause of lighter flower color. Q-PCR results showed that anthocyanin synthesis in LPM was the early gene of anthocyanin synthesis. The expression of CHI was significantly down-regulated, and ANS maintained a very low transcriptional level during the whole floral development of LPM. The low expression of these genes, especially ANS, may inhibit or block anthocyanin biosynthesis and eventually lead to the change of petal color. The database of gerbils petal transcription group was established by RNA-Seq high throughput sequencing. 89817 unigene, differentially expressed genes were detected in LPM and RWT libraries, of which 1503 genes were upregulated in LPM. 803 genes were significantly down-regulated. Unigene was detected in several public databases, among which the most homologous proteins were compared in SignalP library. Kegg pathway enrichment analysis showed that the differentially expressed genes were significantly enriched in the flavonoids biosynthesis pathway. 5, the Kegg pathway enrichment analysis showed that the differentially expressed genes were significantly enriched in the flavonoids biosynthesis pathway. According to the sequencing data of transcriptional group, it was predicted that 17 unigene might be involved in 18 kinds of geranium flower color forming genes, including anthocyanin synthesis gene, anthocyanin modification gene, flavonoids and flavonoids alcohol synthesis gene, anthocyanin transport related genes. In addition, a number of color-regulated transcription factors were predicted. Q-PCR verified the expression of 14 structural genes and 3 kinds of transcription factors, the results were basically consistent with the data of the transcriptional group, and the gene expression was mainly down-regulated.
【学位授予单位】：中国科学院研究生院(近代物理研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S682.19
本文编号：2483347
[Abstract]:Flower color, as an important economic index of ornamental flowers, has become the primary cultivation goal in the improvement of plant traits. In this study, a stable flower color mutant LPM. was screened out by carbon ion beam irradiation on young branches of geranium. In order to reveal the mechanism of flower color variation induced by ion beam, the causes of flower color formation were analyzed by observing the wild type and mutant phenotype of geranium. The composition and content of flavonoids and the differential expression analysis of key structural genes of flower color formation were studied systematically. The mechanism of flower color lightening after ion beam irradiation was studied systematically. The petal transcription group was sequenced deeply by RNA-Seq high throughput sequencing technique, and the differentially expressed unigene, genes between wild type and mutant were screened to predict the flower color formation of gerbils. Finally, the molecular mechanism of flower color change induced by ion beam irradiation was clarified, which provided a theoretical basis for subsequent flower color improvement and new variety selection. The main contents and results of this study are as follows: 1. A flower color mutant LPM, was screened by ion beam 30Gy irradiation and its color and physiological indexes were significantly changed compared with the control. Anatomical observation showed that RWT petal cells were filled with orange anthocyanins, while LPM cells showed very light pink. 2. 66 bands of RWT and LPM genomic polymorphism were detected by molecular markers, of which 18 were unique to LPM. The other 48 were missing from LPM. The similarity coefficient between RWT and LPM was 0.868, and the genetic polymorphism was 13.2%. The components and contents of flavonoids in RWT and LPM were determined by high performance liquid chromatography (HPLC). The results showed that germanium was contained in RWT petals. Elaeagnus angustifolia and Cypermethrin are the main color-forming substances. Only the accumulation of procyanidin was detected in LPM, and the deletion of geranium and permethrin was the root cause of the lighter flower color. Q-PCR results showed that the anthocyanin synthesis early gene CHS, in LPM was found to be the root cause of lighter flower color. Q-PCR results showed that anthocyanin synthesis in LPM was the early gene of anthocyanin synthesis. The expression of CHI was significantly down-regulated, and ANS maintained a very low transcriptional level during the whole floral development of LPM. The low expression of these genes, especially ANS, may inhibit or block anthocyanin biosynthesis and eventually lead to the change of petal color. The database of gerbils petal transcription group was established by RNA-Seq high throughput sequencing. 89817 unigene, differentially expressed genes were detected in LPM and RWT libraries, of which 1503 genes were upregulated in LPM. 803 genes were significantly down-regulated. Unigene was detected in several public databases, among which the most homologous proteins were compared in SignalP library. Kegg pathway enrichment analysis showed that the differentially expressed genes were significantly enriched in the flavonoids biosynthesis pathway. 5, the Kegg pathway enrichment analysis showed that the differentially expressed genes were significantly enriched in the flavonoids biosynthesis pathway. According to the sequencing data of transcriptional group, it was predicted that 17 unigene might be involved in 18 kinds of geranium flower color forming genes, including anthocyanin synthesis gene, anthocyanin modification gene, flavonoids and flavonoids alcohol synthesis gene, anthocyanin transport related genes. In addition, a number of color-regulated transcription factors were predicted. Q-PCR verified the expression of 14 structural genes and 3 kinds of transcription factors, the results were basically consistent with the data of the transcriptional group, and the gene expression was mainly down-regulated.
【学位授予单位】：中国科学院研究生院(近代物理研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S682.19
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