杨树GRF1/2d负调控不定根的发生和发育

doi:10.11707/j.1001-7488.20170304

摘要/Abstract

摘要： [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P_PtGRF1/2d∷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，PtAIL9，PtBBM2，PtPLT1.2和PtWOX11b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。

关键词: PtGRF1/2d, 银腺杨84K, 过量表达, 不定根, 负调控

Abstract: [objective] Growth-regulating factors (GRFs) are plant-specific transcription factors that are involved in various developmental events, especially in root development. It is interesting to investigate the roles of GRFs in growth and development of tissues and organs, rooting in particular, in woody plants, which would not only increase our knowledge on the mechanisms of the regulation of adventitious root formation and also be useful for cutting propagation in woody plants.[Method] PtGRF1/2d gene and its promotor were isolated from Populus alba×P. glandulosa ‘84K’. The overexpression vector of miR396-resistant construct 35S:mPtGRF1/2d-GFP and the promoter-testing vector of P_PtGRF1/2d∷GUS was generated. The miR396-resistant version of mPtGRF1/2d contained six silent mutations within the miR396-complementary domain of the PtGRF1/2d genomic clone, thereby increasing the number of mismatches between miR396 and mPtGRF1/2d without altering the amino acid sequence of the encoded PtGRF1/2d protein. mPtGRF1/2d overexpression transgenic poplars and P_PtGRF1/2d∷GUS transgenic poplars were obtained respectively. Expression characters in P_PtGRF1/2d∷GUS transgenic poplars and time of adventitious root appearance, the number and length of adventitious roots in overexpression transgenic poplars were analyzed. The expression of related transcript factors at early stage of adventitious root formation in overexpression transgenic poplars were analyzed by qRT-PCR.[Result] The results showed that the expression of PtGRF1/2d was mainly in the pericycle and tip of root, suggesting general involvement of PtGRF1/2d in the root development. The development of adventitious roots in mPtGRF1/2d overexpression transgenic poplars was significantly affected in comparison to the non-transgenic poplars used as controls. Overexpression of PtGRF1/2d caused not only the delayed initiation of adventitious roots but also the reduced number and length of adventitious roots, which indicates that PtGRF1/2d exhibits a negative regulation on initiation and development of adventitious root. qRT-PCR analysis indicated that the expression of 5 transcript factors previously shown involvement in adventitious root formation, including PtSCR, PtAIL9, PtBBM2, PtPLT1.2 and PtWOX11b, was down-regulated at early stage of adventitious root formation in overexpression transgenic poplars, which demonstrated that overexpression of PtGRF1/2d suppressed the expression of key factors for adventitious root formation thus resulted in the delayed formation of adventitious roots and the reduced number and length of adventitious roots, and finally influenced the growth of poplar.[Conclusion] PtGRF1/2d is a negative regulator of poplar adventitious root formation, functioning in the upstream of adventitious root formation regulation pathway, down-regulates the expression of adventitious root related factors for adventitious root formation thus suppresses the induction of the root primordium and adventitious root formation, and finally leads to the delayed adventitious roots appearance and the reduced number and length.

Key words: PtGRF1/2d, Populus alba×P. glandulosa ‘84K’, overexpression, adventitious root, negative regulation

中图分类号:

S718.46

周厚君, 魏凯莉, 江成, 赵岩秋, 宋学勤, 卢孟柱. 杨树GRF1/2d负调控不定根的发生和发育[J]. 林业科学, 2017, 53(3): 33-39.

Zhou Houjun, Wei Kaili, Jiang Cheng, Zhao Yanqiu, Song Xueqin, Lu Mengzhu. Negative Regulation of GRF1/2d on the Formation and Development of Adventitious Roots in Populus alba×P. glandulosa ‘84K’[J]. Scientia Silvae Sinicae, 2017, 53(3): 33-39.

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