芹菜中芹菜素积累关键酶基因分析及分子标记开发与验证

Apigenin is an important flavonoid in celery, which has high medicinal value. The breeding of new celery varieties with high apigenin content is in accordance with the development trend of breeding. Now, there are still many controversies in transgenic food. Molecular marker assisted breeding can speed up the process of conventional breeding. At present, there has not been a report on the molecular marker assisted breeding of celery. Based on the previous research, this project will clone FSI gene full-length and analyzed FSI gene sequence. Fluorescence quantitative PCR was used to detect the FSI gene expression characteristic in different apigenin content celery accessions to reveal the role of FSI gene in apigenin accumulation of celery. The FSI gene sequence and promoter sequence were cloned in high apigenin content celery accessions and low apigenin content celery accessions, and then mutation sites of FSI were found by comparative analysis of sequence. Then functional markers are designed according to the mutation sites of FSI, and verified in a large number of celery accessions, which have different apigenin content. These functional markers will provide technical innovation for the cultivation of high apigenin content celery varieties. This project has important theoretical value and practical significance.

芹菜素是芹菜中的主要黄酮类物质，具有很高的药用价值。高芹菜素含量芹菜新品种的培育符合当今育种的发展趋势，目前转基因食品还存在争议，采用分子标记辅助育种可以加快常规育种的进程。目前尚未见到关于芹菜分子标记辅助育种的报道。本项目在前期研究的基础上，通过RACE技术克隆芹菜FSI基因的全长并进行序列分析，利用荧光定量PCR技术对FSI基因在不同芹菜素含量芹菜资源中进行表达特性分析，揭示FSI基因在芹菜中芹菜素积累中的作用；克隆高芹菜素含量芹菜和低芹菜素含量芹菜FSI基因序列和启动子序列，通过比较分析发掘FSI基因的突变位点，设计功能标记，并在大量芹菜素含量存在差异的芹菜资源中进行验证，为培育高芹菜素含量且高品质的芹菜品种提供技术创新，具有重要的理论价值和现实意义。

芹菜素是芹菜中的主要黄酮类物质，具有很高的药用价值。高芹菜素含量芹菜新品种的培育符合当今育种的发展趋势。本项目在前期研究的基础上，克隆芹菜FSI基因组序列和启动子序列，对FSI基因进行表达特性分析，通过比较高芹菜素含量芹菜和低芹菜素含量芹菜FSI基因组序列和启动子序列的差异，开发功能标记，并进行验证。结果表明：1）AgFSI基因全长为1701 bp，包含3个内含子区域和1065 bp的开放阅读框，编码355个氨基酸。AgFSI 具有20G-FeII_Oxy 保守结构域，属于20G-FeII_Oxy超级家族，其他伞型科植物中的FSI具有很高的相似性，相似度达到93.66%。2）荧光定量PCR分析表明AgFSI与芹菜资源叶片中芹菜素含量正相关，AgFSI与不同逆境（高温、低温、UV-B胁迫、盐胁迫）处理后芹菜中芹菜素的变化正相关。3）克隆得到AgFSI 起始密码子前1511 bp 启动子序列，该序列含有真核生物启动子的典型元件、光反应元件、厌氧或缺氧诱导顺式作用元件、茉莉酸甲酯相关的顺式作用元件、干旱诱导MYB转录因子的结合位点和一些未知功能的元件。4）分别扩增获得5份高芹菜素含量资源（HAA）和8份低芹菜素含量资源（LAA）基因组及启动子序列，序列比对获得4个等位变异序列。这4种等位变异在启动子区域存在29个单核苷酸多态性（SNP）位点和8个插入缺失（In/Del）位点，在基因组区域存在25个单核苷酸多态性（SNP）位点和3个插入缺失（In/Del）位点。5）依据芹菜资源的HAA型和LAA型AgFSI基因组序列的SNP和启动子序列的SNP，分别设计3对分子标记来区分两种类型芹菜资源。为了验证标记与芹菜素含量的相关性，将上述3对标记分别在112份芹菜资源进行扩增，结果表明标记AFPA1/AFPB1与芹菜中芹菜素含量相关，可以有效预测芹菜资源中的芹菜素含量。