miR在豆科植物苜蓿根中对其菌根化和分生组织活性的影响实用教案

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1、2022-4-231主要(zhyo)内容 INTRODUCTION RESULTS 1、 Mtr-miR396a and mtr-miR396b genes show different tissue-specific expression profiles in Medicago truncatula roots 2、In M. truncatula roots, miR396 regulates six GRF and two bHLH79 transcription factors 3、Characterization of the miR396 regulatory network i

2、n M. truncatula第1页/共36页第一页，共37页。 4、 miR396 negatively affects root growth, RAM size and cell proliferation in the root apex 5、 Silencing of MtGRF genes mimics the miR396-OE root growth phenotype 6、 Hormonal control of miR396 expression 7、miR396 limits mycorrhizal colonization but not nodulation2022-

3、4-232第2页/共36页第二页，共37页。INTRODUCTIONIn plants, root growth is controlled via coordinated cell division and expansion at the root apex, where three regions are observed: (i) the meristematic zone, (ii) the elongation zone, and (iii) the differentiation zoneRoot development is regulated by complex inter

4、actions of hormone signalling pathways2022-4-233第3页/共36页第三页，共37页。In recent years, microRNAs (miRNAs) have emerged as important regulators of the architecture of the root systemin Arabidopsis thaliana, miR165/166 governs the radial patterning of vascular and pericycle tissues via quantitative repress

5、ion of HD-ZIP III transcription factorsA set of auxin-related miRNAs, including miR160, also regulate lateral root initiation and/or root growth2022-4-234第4页/共36页第四页，共37页。However, few miRNAs have been reported to play roles in RAM functionAlthough large sets of mycorrhization or nodulation-responsiv

6、e miRNAs have been identified in legumes, few have been functionally linked to these processes.2022-4-235第5页/共36页第五页，共37页。 In A. thaliana, miR396 is encoded by two loci, post-transcriptionally represses several members of the growth-regulating factor (GRF) family. These plant-specific TFs control th

7、e growth and development of leaves and stems.miR396 may have evolved to play roles in various regulatory networks in plants.In this paper, we describe the roles of the miR396 regulatory network in M. truncatula roots.2022-4-236第6页/共36页第六页，共37页。RESULTS1、Mtr-miR396a and mtr-miR396b genes show differen

8、t tissue-specific expression profiles in Medicago truncatula rootsMtr-miR396a 和 mtr-miR396b 在根中的表达(biod)具有空间特异性2022-4-237第7页/共36页第七页，共37页。2022-4-238miR396 expression in organs and root tips两种miRNAs在整株植物中都有表达，其中(qzhng)miR396b在根、瘤、叶和豆荚中的表达量较高，miR396a在根尖中较多第8页/共36页第八页，共37页。2022-4-239两者在根中的表达(biod)在根尖横切

9、面中的表达(biod)在根尖中的表达(biod)在侧根中的表达第9页/共36页第九页，共37页。2022-4-23102、In M. truncatula roots, miR396 regulates six GRF and two bHLH79 transcription factorsWe found reads corresponding to the miR396-related cleavage products of six GRF mRNAs (MtGRF1, MtGi10-TC183867; MtGRF2, MtGi10-TC183494; MtGRF3, MtGi10 BG

10、454006; MtGRF4, Medtr5g027250; MtGRF5, Medtr8g020560; MtGRF6, Medtr7g126820). These transcripts possess a putative miR396 binding site, and the encoded proteins all contain the conserved domains of GRFs第10页/共36页第十页，共37页。We also observed abundant degradome reads corresponding to specific cleavage of

11、two non-GRF transcripts at miR396 binding sites, Based on tBLASTX analysis, the 2 predicted proteins showed high similarity (43 and 46% respectively) to AtbHLH79/BIGPETAL (At1g59640), a TF involved in the control of petal growth in A. thaliana.2022-4-2311第11页/共36页第十一页，共37页。2022-4-2312In M. truncatul

12、a, six MtGRF and two bHLH79 genes have miR396 binding sites第12页/共36页第十二页，共37页。3、Characterization of the miR396 regulatory network in M. truncatula To investigate miR396-mediated gene regulation in planta, we over-expressed mtr-miR396a and mtr-miR396b precursors in M. truncatula roots (miR396-OE).202

13、2-4-2313左图说明OE突变体能成功(chnggng)过量表达成熟的miRNAs第13页/共36页第十三页，共37页。2022-4-2314Levels of transcripts of miR396 targets weremeasured using quantitative RT-PCR in miR396-OE rootsMtGRF-mRNA 水平和MtbHLH79转录(zhun l)水平都降低，说明miR396可能抑制这些基因的表达水平第14页/共36页第十四页，共37页。To further validate their targets, we generated roots

14、 expressing a target mimicry construct that was successfully designed to hinder miR396 activity in A. thaliana. Mimicry (MIM) transcripts specifically trap members of an miRNA family, and thus prevent their activity on endogenous targets2022-4-2315第15页/共36页第十五页，共37页。2022-4-2316Levels of transcripts

15、of miR396 targets weremeasured using quantitative RT-PCR in roots inactivated for miR396activity (MIM396)除了GRF6，其他(qt)基因表达水平都上升，这里还不能排除有其他(qt)因子对GRF6进行调节的可能，本文中没有深入研究第16页/共36页第十六页，共37页。4、miR396 negatively affects root growth, RAM size and cell proliferation in the root apex To investigate the functi

16、on of miR396 in roots, we measured the length and dry weight of roots that either overexpressed the mtr-miR396b precursor (miR396-OE) or were inactivated for miR396 activity (MIM396).2022-4-2317第17页/共36页第十七页，共37页。2022-4-2318miR396-OE 中主根长度和干重明显减少，而MIM396中根长稍微增加，而干重却大量增加。说明miR396过表达能抑制(yzh)根的生长第18页/共

17、36页第十八页，共37页。2022-4-2319Root meristem size is reduced inmiR396-OE composite plants第19页/共36页第十九页，共37页。2022-4-2320两者之间没有大的变化，说明miR396对细胞从细胞周期进入(jnr)分裂周期的开关没有大的影响第20页/共36页第二十页，共37页。2022-4-2321The expression of mitotic cell cycle (CYCB1;1, CYCB1;3, CYCB2;1, CYCD3;1, histone H4, CDC16) or endocycle (CCS5

18、2B) markers was measured using quantitativeRT-PCR in miR396-OE and control rootsThe percentage of cells replicating (i.e. EdU-positive nuclei) obtained from 20 root tips per constructin four independent experiments第21页/共36页第二十一页，共37页。Together, these data strongly suggest that miR396-dependent modula

19、tion of root growth may essentially be due to a restriction of cell division activity.2022-4-2322第22页/共36页第二十二页，共37页。5、Silencing of MtGRF genes mimics the miR396-OE root growth phenotype To study the role of the miR396 targets on root growth, we inactivated them using an RNAi strategy. First, we gen

20、erated composite plants expressing three RNAi constructs targeting a conserved region of various GRF genes ( MtGRF2, MtGRF4 and MtGRF6). In addition, we prepared an RNAi vector targeting both MtbHLH79 genes and confirmed their efficient silencing2022-4-2323第23页/共36页第二十三页，共37页。2022-4-2324Silencing of

21、 MtGRF genes mimics miR396 over-expression in roots.iGRF表达减少而ibHLH79表达不变，说明很可能是miR396对GRF基因的负调控使得(sh de)miR396-OE和MIM396植株中根的生长表型不同第24页/共36页第二十四页，共37页。6、Hormonal control of miR396 expression We treated wild-type plants for 1, 3 or 5 h with auxin, cytokinin, gibberellin, abscisic acid or brassinoster

22、oids (BR), and measured pre-miR396 and target mRNA levels in roots.2022-4-2325第25页/共36页第二十五页，共37页。2022-4-2326Gibberellin, cytokinin and auxin treatments did not affectexpression of any of these genes第26页/共36页第二十六页，共37页。2022-4-2327miR396b对ABA和BR 有明显的反应，并且GRF5在BR处理(chl)下下调，说明BR和miR396b, GRF5之间有某种作用关系第

23、27页/共36页第二十七页，共37页。2022-4-2328Levels of GUS transcript in roots expressing the prom-miR396b:GUS construct (grey bars) or the prom-GRF5:GUS construct (black bars) treated or not treated with 1 nM BR for 3 h were measured using quantitative RT-PCR.BR可以活化miR396b的启动子活性，但prom-GRF5:GUS并没有明显减少，说明BR可能是在转录(z

24、hun l)后水平抑制GRF5基因的表达，而且BR和miR396b存在潜在联系，但本文中没有对其研究第28页/共36页第二十八页，共37页。7、 miR396 limits mycorrhizal colonization but not nodulation We next decided to study the potential role of miR396 in root symbioses. We analysed mtr-miR396a and mtr-miR396b expression profiles in roots inoculated with the symbiot

25、ic bacteria Sinorhizobium meliloti.2022-4-2329第29页/共36页第二十九页，共37页。2022-4-2330miR396a启动子首先在根瘤(gnli)导管组织中发现，miR396b启动子在维管组织，感染区（）和氮固定区（）含量很高第30页/共36页第三十页，共37页。2022-4-2331虽然miR396存在于根瘤中，但miR396-OE和MIM396对根瘤密度(md)都没有明显影响第31页/共36页第三十一页，共37页。 We also analysed the expression of miR396 in response to Myc-LC

26、Os, the lipochitooligosaccharide signal molecules produced by AM fungi. The involvement of miR396 during mycorrhization was then analysed at 5 weeks after colonization with R. irregularis (formerly Glomus intraradices) in miR396-OE and MIM396 roots.2022-4-2332第32页/共36页第三十二页，共37页。2022-4-2333miR396 af

27、fects fungal colonization during AM symbiosis.菌根的丛枝量和形态(xngti)没变化，但AM定植在miR396-OE根中的量减少，而在MIM396中的量增加Hence, the action of miR396 limits mycorrhizalcolonization.第33页/共36页第三十三页，共37页。Here, we showed that, in the model legume M. truncatula, miR396 represses not only MtGRF but also two MtbHLH79 TF-encodi

28、ng genes, and that its de-regulation affects root growth and the ability to be colonized by mycorrhizal fungi.2022-4-2334第34页/共36页第三十四页，共37页。2022-4-2335Thank you!第35页/共36页第三十五页，共37页。感谢您的观看(gunkn)！第36页/共36页第三十六页，共37页。NoImage内容(nirng)总结2021/11/13。MtGRF-mRNA 水平和MtbHLH79转录水平都降低(jingd)，说明miR396可能抑制这些基因的表达水平。虽然miR396存在于根瘤中，但miR396-OE和MIM396对根瘤密度都没有明显影响。第35页/共36页。感谢您的观看第三十七页，共37页。