肇东苜蓿抗寒过程中关键miRNA基因的筛选及其功能分析

Alfalfa (Medicago sativa L.) is a crucial leguminous forage crop, which is widely distributed in northern climates in China. Although this perennial crop is always compromised freezing stress, which would reduce alfalfa yield remarkable. Therefore, identification of freezing response genes is important for breeding for greater freezing tolerance in alfalfa. The alfalfa, Medicago sativa cultivar Zhaodong, is investigated for its good freezing tolerance trait. The microRNA genes, which are playing important roles in response to freezing stress, are identified by small RNA sequence using Solexa/Illumina sequencing. Their expressional profiles in response to freezing stress are characterized by real-time PCR. Meanwhile, miRNA mediated cleavage products in alfalfa are also sequenced by Solexa/Illumina sequencing, which is called degradome sequencing. The targets of miRNA genes are identified by bioinformatics analysis of target cleavage sites. And novel targets of key miRNA genes response to freezing stress are selected for confirmation by RLM 5'-RACE method. These miRNA-mRNA pairs are integrated with gene's interactions from Arabidopsis, and genetic regulation network controlling freezing tolerance in alfalfa is constructed, which is regulated by miRNA genes. These genes response to freezing tolerance identification in Medicago sativa cultivar Zhaodong are useful for improvement of freezing tolerance in alfalfa. And characterization of molecular genetic mechanism that miRNA genes create freezing tolerance in M.sativa cv Zhaodong is valuable for alfalfa genetic breeding.

紫花苜蓿是多年生优质牧草，在我国，苜蓿生长主要集中在北方地区，这些地区气候寒冷，容易发生低温冻害，造成苜蓿减产，大大降低苜蓿的生产效益。因此，如何发掘紫花苜蓿的抗寒基因资源，培育抗寒苜蓿新品种，已成为我国苜蓿产业高产优质目标亟待解决的问题。本课题将以紫花苜蓿中优异的抗寒品种：肇东苜蓿为材料，通过小RNA测序，发掘肇东苜蓿抗寒过程中的关键microRNA基因，并鉴定它们在肇东苜蓿抗寒过程中的表达模式。同时，根据降解组测序结果，发掘microRNA基因调控的功能靶基因，并采用RACE方法进行靶基因切割验证，确定microRNA-mRNA间的调控关系。整合发掘miRNA-mRNA调控关系和拟南芥的基因调控网络数据，构建肇东苜蓿抗寒过程中关键miRNA基因介导的基因调控网络，解析miRNA基因在肇东苜蓿抗寒过程中的分子调控机制，为紫花苜蓿的抗寒品种培育提供基因资源和科学依据。

紫花苜蓿是多年生优质牧草，富含蛋白质等营养成分，具有“牧草之王”的美誉。在我国，紫花苜蓿生长主要集中在北方地区，这些地区气候寒冷，容易发生低温冻害，造成苜蓿减产，大大降低苜蓿的生产效益。因此，如何发掘紫花苜蓿的抗寒基因资源，培育抗寒苜蓿新品种，已成为我国苜蓿产业高产优质目标亟待解决的问题。肇东苜蓿广泛种植于我国东北地区，适应当地严寒气候，是紫花苜蓿中优异的抗寒品种。因此，本项目以肇东苜蓿为研究材料，进行寒冷转录组测序、miRNA测序以及降解组测序，发掘紫花苜蓿中寒冷胁迫相关的miRNA基因、功能基因以及它们的调控模式。同时，整合模式植物的基因调控网络信息，构建紫花苜蓿基因调控网络，解析紫花苜蓿抗寒分子调控机制。主要研究结果如下：.1. 通过转录组测序，获得了紫花苜蓿75551条UniGene序列，发掘寒冷胁迫响应基因5767个，主要集中在胁迫响应、激素调节和抗氧化调控等过程。进一步分析发现，AP2/ERF、WRKY、MYB等转录因子家族基因在紫花苜蓿寒冷胁迫过程中起重要调控作用。其中，10个AP2/ERF转录因子的表达模式得到了qRT-PCR的验证，证实转录组测序平台结果可靠。.2. 通过小RNA测序，鉴定了197个紫花苜蓿miRNA基因在寒冷胁迫下的表达模式，其中，新的miRNA基因24个。发掘寒冷胁迫响应miRNA基因35个，其中12个miRNA基因表达模式得到qRT-PCR验证。通过降解组测序，鉴定了105个miRNA调控的靶基因，建立了miRNA与mRNA间的调控模式。.3. 通过系统生物学方法，挖掘模式植物基因调控网络数据，构建紫花苜蓿基因调控网络，整合本研究发掘的miRNA与mRNA间调控模式，建立紫花苜蓿寒冷胁迫基因调控网络，系统地解析紫花苜蓿抗寒分子调控机制，为紫花苜蓿的抗寒品种培育提供基因资源和科学依据。