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【目的】生长调节因子(GRFs)是一类植物特有的转录因子,调控植物生长发育的多个生物学过程。研究杨树组织和器官发育中GRFs的作用,尤其是对不定根形成的调控,不仅可以丰富根发育的理论,而且对于杨树的扦插繁殖具有实际应用价值。【方法】从银腺杨84K中分离了PtGRF1/2d基因和其启动子,通过对其miR396靶位点核苷酸进行同义突变,获得不受miR396调控的突变形式的mPtGRF1/2d,并将启动子和该突变形式分别构建至含有GUS报告基因的植物表达载体和过量表达载体,通过遗传转化分别获得PPtGRF1/2d∷GUS启动子驱动GUS转基因杨树和mPtGRF1/2d过表达转基因杨树。通过GUS染色分析PtGRF1/2d杨树启动子的表达特性,并对过表达mPtGRF1/2d杨树不定根的发生时间、数目和长度进行统计,利用qRT-PCR分析不定根发育早期相关转录因子的表达。【结果】PtGRF1/2d主要在根的中柱鞘和根尖位置表达,说明其参与了根的形成;过量表达mPtGRF1/2d基因影响了杨树不定根的发生、发育,导致了不定根发生延迟、数目和长度均减少,且差异均达到显著水平或极显著水平,表明PtGRF1/2d对不定根的发生和发育具有负调控作用。qRT-PCR分析显示,过表达mPtGRF1/2d杨树的不定根发育相关基因PtSCR,Pt AIL9,Pt BBM2,Pt PLT1.2和Pt WOX11b的表达量均被下调,表明PtGRF1/2d的过量表达抑制了根原基发生和不定根发育相关的关键调控因子的表达,影响了根原基的发生和不定根的形成,导致不定根数目和长度的变化,最终影响了杨树的生长。【结论】PtGRF1/2d作为不定根形成的负调控因子,位于不定根调控途径的上游,通过下调促进不定根形成的相关转录因子的表达来抑制根原基形成和不定根发育,导致不定根发生延迟、数目和长度减少。
【Objective】 Growth regulators (GRFs) are a group of plant-specific transcription factors that regulate multiple biological processes in plant growth and development. Studying the function of GRFs in poplar tissue and organ development, especially the regulation of adventitious root formation, not only enriches the theory of root development, but also has practical application value for poplar cutting propagation. 【Method】 The PtGRF1 / 2d gene and its promoter were isolated from the Ginkgo biloba 84K and the mutated mPtGRF1 / 2d, which is not regulated by miR396, was obtained by synonymous mutation of its miR396 target site. The promoter and the mutant form were respectively constructed into plant expression vector containing GUS reporter gene and overexpression vector. The PPtGRF1 / 2d :: GUS promoter driven GUS transgenic poplar and mPtGRF1 / 2d overexpressing transgenic poplar were obtained by genetic transformation respectively. The expression characteristics of the promoter of PtGRF1 / 2d poplar were analyzed by GUS staining. The time, number and length of adventitious roots of poplar mPtGRF1 / 2d were counted. The expression of transcription factors related to early adventitious root development was analyzed by qRT-PCR. 【Result】 PtGRF1 / 2d was mainly expressed in the mesocapillaries and apices of the roots, indicating that it is involved in root formation. Overexpression of mPtGRF1 / 2d gene affected the occurrence and development of adventitious roots in poplars, resulting in the delay of adventitious roots. The number of And length decreased, and the difference reached a significant level or extremely significant level, indicating that PtGRF1 / 2d negative adventitious root formation and development have a negative regulatory role. qRT-PCR analysis showed that expression of adventitious root development-related genes PtSCR, Pt AIL9, Pt BBM2, Pt PLT1.2 and Pt WOX11b were down-regulated in over-expressed mPtGRF1 / 2d poplar trees, indicating that overexpression of PtGRF1 / The expression of key regulators involved in adventitious root development affected root primordia and adventitious root formation, resulting in changes in the number and length of adventitious roots, eventually affecting poplar growth. 【Conclusion】 As a negative regulator of adventitious root formation, PtGRF1 / 2d locates upstream of the adventitious root regulators and inhibits root primordium formation and adventitious root development through down-regulation of transcription factors involved in adventitious root formation, resulting in delayed adventitious root growth and reduced number and length .