miR396在落叶松体细胞胚胎中的功能研究

[目的]探讨miR396在落叶松体细胞胚胎发生中的功能.以期该研究能为解决体胚发育中子叶畸形胚与生根率低的问题提供新的视角,同时也为进一步揭示体胚发育的分子调控网络做基础铺垫.[方法]构建pre-miR396超表达载体,通过农杆菌介导侵染,将pre-miR396转入落叶松胚性细胞中,筛选稳定抗性细胞系.在DNA与RNA水平上,对抗性细胞系进行的鉴定、检测,同时统计比对其与野生细胞系间发芽率、叶片数目以及生根率的差异.[结果]在抗性细胞系基因组中,扩增出HTP与miR396的特异性片段,且无农杆菌Virg基因片段.RNA表达水平上,抗性系pre-miR396与miR396表达皆增加,而靶基因LaGRFs的表达量下调.统计表明抗性系胚的发芽率、生根率、叶片数目畸形率分别为70.60%、0.60%、37%,而野生型分别为92.50%、10.55%、4%,抗性系胚的发芽率、生根率显著降低(Sig.=0.000),叶片数目畸形率显著增加(Sig.=0.000).[结论]miR396在落叶松体细胞胚胎发生过程中,可能通过负调节靶基因LaGRFs的表达或者通过LaGRFs影响与它相互作用的基因,参与体胚子叶原基细胞代谢,从而影响了子叶与叶片的发育;通过负调节LaGRFs或者其它与根部发育相关的靶基因,影响了根端分生区细胞的活动,参与调控根的发育.

Function Analysis of miR396 in Somatic Embryos of Larix leptolepis

[Objective]To study the function of miR396 in the process of somatic embryogenesis in Larix leptolepis, and provide a new perspective for solve the problem of abnormal cotyledon embryos occurring in somatic embryo development and low rooting rate. It is also proposed to lay a foundation for further study on molecular regulatory network of somatic embryo development. [Method]A pre-miR396 expression vector was constructed and inserted into the somatic cells of L. leptolepis through agrobacterium infection. Then the cells infected were screened until stable resistant lines were obtained. Molecular detection and verification were carried out at the DNA and RNA levels of resistant cell lines. The rate of germination and rooting and the number of leaves were also collected on resistant cell lines and wild lines. [Result]In resistant cell lines, the fragments of HTP and miR396 were amplified while the virg gene fragment was not obtained; the expression levels of the pre-miR396 and miR396 increased, and the expression of target genes LaGRFs decreased. The germination rate, rooting rate and leaf numbers abnormality rate of positive somatic embryos were 70.60%, 0.60% and 37% respectively, contrast to 92.50%, 10.55% and 4% in the wild types. The somatic embryos of positive cell line had significantly lower germination rate and rooting rate (Sig.=0.000), and greatly higher leaf numbers abnormality rate (Sig.=0.000) than the wild types. [Conclusion]During the process of L. leptolepis somatic embryogenesis, miR396 might be involved in the metabolism of somatic embryo cotyledon primordial cell by regulating negatively the expression of target genes LaGRFs or through LaGRFs to influence the genes interacted, so as to make effect on the development of cotyledon and blade, and also by regulating negatively the LaGRFs or other target genes related to root development to influence the cell activity of apical root and participating in regulating the root development.