萝卜花青素合成关键基因的分离克隆与表达分析
毕业论文关键词：萝卜；花青素；生物合成；基因克隆；转录分析
Molecular Analysis and Transcript Profiling of Structural and Regulatory Anthocyanin Biosynthetic Genes in Radish
Student majoring in Horticulture Name Yin Xiaowei
Tutor Professor Liu Liwang
Abstract：Radish,a one or two year old herb, belongs to the family Cruciferae and the genus Raphanus.Some genotypes of radish have colored flesh and /or skin due to pigmentation brought by accumulation of anthocyanin.Anthocyanins are a big group naturally occurring water-soluble pigments referred to as flavonoids.In this study,fifteen genomic sequences were isolated and sequenced from radish taproot .The length of these sequences were between 900 bp to 1,579 bp and the unigene coverage to most of the corresponding cloned sequences was more than 93%. Gene structure analysis revealed that RsF3’H is intronless and anthocyanin biosynthetic genes (ABGs) bear asymmetrical exons, with exception of RsSAM. Anthocyanin accumulation showed a gradual increase in the leaf of the red radish and the taproot of colored cultivars during development, with a rapid increase at 30 DAS and the highest content at maturity. Spatial-temporal transcriptional analysis of 14 genes revealed detectable expressions of twelve ABGs in various tissues at different growth levels. The investigation of anthocyanin accumulation and gene expression in four color variant radish taproot cultivars, at different stages of development indicated that total anthocyanin correlated with transcript levels of ABGs, particularly RsUFGT, RsF3H, RsANS, RsCHS3 and RsDFR. Our results suggest that these candidate genes play key roles in phenotypic and spatial-temporal anthocyanin accumulation in radish through coordinated regulation.
Key words: Radish；Anthocyanin；Biosynthesis；Gene cloning；Transcript profiling
目 录
摘要3
关键词3
Abstract3
Key words3
引言3
1材料与方法4
1.1植物材料 4
1.2萝卜花青素单体含量的测定5
1.3萝卜基因组DNA，RNA的提取与反转录5
1.4花青素生物合成相关基因（ABGs）的确立、分离克隆和表达分析6
1.5花青素生物合成相关基因（ABGs）的转录分析7
2结果与分析7
2.1萝卜中总花青素含量的测定7
2.2萝卜中花青素生物合成基因的分离克隆与序列分析8
2.3萝卜中花青素生物合成基因的实时荧光定量PCR分析10
2.4基因表达和花青素含量的相关性分析14
3讨论 16
3.1四个品种萝卜各组织中花青素含量 16
3.2萝卜中与花青素合成相关的基因 16
3.3萝卜花青素合成上游ABGs基因的表达 17
3.4花青素合成特定途径中ABGs基因的表达 17
3.5与花青素转移和定位有关的ABGs的表达 18
致谢18
参考文献18
图1 不同发育阶段的各品种萝卜5
图2不同品种萝卜在不同发育阶段不同组织中的花青素含量8