miR396在豆科植物苜蓿根中对其菌根化和分生组织活性的影响ppt课件

1、miR396 affects mycorrhization and root meristem activity inthe legume Medicago truncatulaThe Plant Journal (2021)doi: 10.1111/tpj.12178主要内容nINTRODUCTIONnRESULTSn 1、 Mtr-miR396a and mtr-miR396b genes show different tissue-specific expression profiles in Medicago truncatula rootsn 2、In M. truncatula r

2、oots, miR396 regulates six GRF and two bHLH79 transcription factorsn 3、Characterization of the miR396 regulatory network in M. truncatulan 4、 miR396 negatively affects root growth, RAM size and cell proliferation in the root apexn 5、 Silencing of MtGRF genes mimics the miR396-OE root growth phenotyp

3、en 6、 Hormonal control of miR396 expressionn 7、miR396 limits mycorrhizal colonization but not nodulationINTRODUCTIONnIn 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 zon

4、e, and (iii) the differentiation zonenRoot development is regulated by complex interactions of hormone signalling pathwaysnIn recent years, microRNAs (miRNAs) have emerged as important regulators of the architecture of the root systemnin Arabidopsis thaliana, miR165/166 governs the radial patterning

5、 of vascular and pericycle tissues via quantitative repression of HD-ZIP III transcription factorsnA set of auxin-related miRNAs, including miR160, also regulate lateral root initiation and/or root growthnHowever, few miRNAs have been reported to play roles in RAM functionnAlthough large sets of myc

6、orrhization or nodulation-responsive miRNAs have been identified in legumes, few have been functionally linked to these processes.nIn A. thaliana, miR396 is encoded by two loci, post-transcriptionally represses several members of the growth-regulating factor (GRF) family. These plant-specific TFs co

7、ntrol the growth and development of leaves and stems.nmiR396 may have evolved to play roles in various regulatory networks in plants.nIn this paper, we describe the roles of the miR396 regulatory network in M. truncatula roots.RESULTSn1、Mtr-miR396a and mtr-miR396b genes show different tissue-specifi

8、c expression profiles in Medicago truncatula rootsnMtr-miR396a 和 mtr-miR396b 在根中的表达具有空间特异性miR396 expression in organs and root tips两种miRNAs在整株植物中都有表达，其中miR396b在根、瘤、叶和豆荚中的表达量较高，miR396a在根尖中较多两者在根中的表达在根尖横切面中的表达在根尖中的表达在侧根中的表达n2、In M. truncatula roots, miR396 regulates six GRF and two bHLH79 transcriptio

9、n factorsnWe found reads corresponding to the miR396-related cleavage products of six GRF mRNAs (MtGRF1, MtGi10-TC183867; MtGRF2, MtGi10-TC183494; MtGRF3, MtGi10 BG454006; MtGRF4, Medtr5g027250; MtGRF5, Medtr8g020560; MtGRF6, Medtr7g126820). These transcripts possess a putative miR396 binding site,

10、and the encoded proteins all contain the conserved domains of GRFsnWe also observed abundant degradome reads corresponding to specific cleavage of two non-GRF transcripts at miR396 binding sites, Based on tBLASTX analysis, the 2 predicted proteins showed high similarity (43 and 46% respectively) to

11、AtbHLH79/BIGPETAL (At1g59640), a TF involved in the control of petal growth in A. thaliana.In M. truncatula, six MtGRF and two bHLH79 genes have miR396 binding sitesn3、Characterization of the miR396 regulatory network in M. truncatulanTo investigate miR396-mediated gene regulation in planta, we over

12、-expressed mtr-miR396a and mtr-miR396b precursors in M. truncatula roots (miR396-OE).左图阐明OE突变体能胜利过量表达成熟的miRNAsLevels of transcripts of miR396 targets weremeasured using quantitative RT-PCR in miR396-OE rootsMtGRF-mRNA 程度和MtbHLH79转录程度都降低，阐明miR396能够抑制这些基因的表达程度nTo further validate their targets, we gen

13、erated roots 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 targetsLevels of transcripts of miR396 targets weremeasure

14、d using quantitative RT-PCR in roots inactivated for miR396activity (MIM396)除了GRF6，其他基因表达程度都上升，这里还不能排除有其他因子对GRF6进展调理的能够，本文中没有深化研讨n4、miR396 negatively affects root growth, RAM size and cell proliferation in the root apexnTo investigate the function of miR396 in roots, we measured the length and dry w

15、eight of roots that either overexpressed the mtr-miR396b precursor (miR396-OE) or were inactivated for miR396 activity (MIM396).miR396-OE 中主根长度和干重明显减少，而MIM396中根长略微添加，而干重却大量添加。阐明miR396过表达能抑制根的生长Root meristem size is reduced inmiR396-OE composite plants两者之间没有大的变化，阐明miR396对细胞从细胞周期进入分裂周期的开关没有大的影响The exp

16、ression of mitotic cell cycle (CYCB1;1, CYCB1;3, CYCB2;1, CYCD3;1, histone H4, CDC16) or endocycle (CCS52B) 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 indep

17、endent experimentsnTogether, these data strongly suggest that miR396-dependent modulation of root growth may essentially be due to a restriction of cell division activity.n5、Silencing of MtGRF genes mimics the miR396-OE root growth phenotypenTo study the role of the miR396 targets on root growth, we

18、 inactivated them using an RNAi strategy.nFirst, we generated composite plants expressing three RNAi constructs targeting a conserved region of various GRF genes ( MtGRF2, MtGRF4 and MtGRF6).nIn addition, we prepared an RNAi vector targeting both MtbHLH79 genes and confirmed their efficient silencin

19、gSilencing of MtGRF genes mimics miR396 over-expression in roots.iGRF表达减少而ibHLH79表达不变，阐明很能够是miR396对GRF基因的负调控使得miR396-OE和MIM396植株中根的生长表型不同n6、Hormonal control of miR396 expressionnWe treated wild-type plants for 1, 3 or 5 h with auxin, cytokinin, gibberellin, abscisic acid or brassinosteroids (BR), an

20、d measured pre-miR396 and target mRNA levels in roots.Gibberellin, cytokinin and auxin treatments did not affectexpression of any of these genesmiR396b对ABA和BR 有明显的反响，并且GRF5在BR处置下下调，阐明BR和miR396b, GRF5之间有某种作用关系Levels of GUS transcript in roots expressing the prom-miR396b:GUS construct (grey bars) or t

21、he 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，阐明BR能够是在转录后程度抑制GRF5基因的表达，而且BR和miR396b存在潜在联络，但本文中没有对其研讨n7、 miR396 limits mycorrhizal colonization but not nodulationnWe next decided to study the potential role of miR396 in root symbioses.nWe analysed mtr-miR396a and mtr-miR396b expression profiles in roots inoculated with the symbiotic bacteria Sinorhizobium meliloti.miR396a启动子首先在根瘤导管组织中发现，miR396b启动子在维管组织，感染区和氮固定区含量很高虽然miR396存在于根瘤中，但miR396-OE和MIM39