miR396在豆科植物苜蓿根中对其菌根化和分生组织活性的ppt课件

1、2022/7/181miR396 affects mycorrhization and root meristem activity inthe legume Medicago truncatulaThe Plant Journal (2021)doi: 10.1111/tpj.121782022/7/182主要内容INTRODUCTIONRESULTS 1、 Mtr-miR396a and mtr-miR396b genes show different tissue-specific expression profiles in Medicago truncatula roots 2、In

2、 M. truncatula roots, miR396 regulates six GRF and two bHLH79 transcription factors 3、Characterization of the miR396 regulatory network in M. truncatula 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 gr

3、owth phenotype 6、 Hormonal control of miR396 expression 7、miR396 limits mycorrhizal colonization but not nodulation2022/7/183INTRODUCTIONIn 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

4、) the elongation zone, and (iii) the differentiation zoneRoot development is regulated by complex interactions of hormone signalling pathways2022/7/184In recent years, microRNAs (miRNAs) have emerged as important regulators of the architecture of the root systemin Arabidopsis thaliana, miR165/166 go

5、verns the radial patterning of vascular and pericycle tissues via quantitative repression of HD-ZIP III transcription factorsA set of auxin-related miRNAs, including miR160, also regulate lateral root initiation and/or root growth2022/7/185However, few miRNAs have been reported to play roles in RAM

6、functionAlthough large sets of mycorrhization or nodulation-responsive miRNAs have been identified in legumes, few have been functionally linked to these processes.2022/7/186In A. thaliana, miR396 is encoded by two loci, post-transcriptionally represses several members of the growth-regulating facto

7、r (GRF) family. These plant-specific TFs control the 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.2022/7/187RESULTS1、Mtr-miR396a a

8、nd mtr-miR396b genes show different tissue-specific expression profiles in Medicago truncatula rootsMtr-miR396a 和 mtr-miR396b 在根中的表达具有空间特异性2022/7/1882022/7/189miR396 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2022/7/1810两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

9、达2022/7/18112、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 BG454006; MtGRF4, Medtr5g027250; MtGRF5, Medtr8g0

10、20560; MtGRF6, Medtr7g126820). These transcripts possess a putative miR396 binding site, and the encoded proteins all contain the conserved domains of GRFsWe also observed abundant degradome reads corresponding to specific cleavage of two non-GRF transcripts at miR396 binding sites, Based on tBLASTX

11、 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.2022/7/18122022/7/1813In M. truncatula, six MtGRF and two bHLH79 genes have miR396 binding sites3、Characterization of the

12、 miR396 regulatory network in M. truncatulaTo investigate miR396-mediated gene regulation in planta, we over-expressed mtr-miR396a and mtr-miR396b precursors in M. truncatula roots (miR396-OE).2022/7/1814左图阐明OE突变体能胜利过量表达成熟的miRNAs2022/7/1815Levels of transcripts of miR396 targets weremeasured using q

13、uantitative 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To further validate their targets, we generated roots expressing a target mimicry construct that was successfully designed to hinder miR396 activity in A. thaliana. Mimicry (MIM) transcripts specifically trap mem

14、bers of an miRNA family, and thus prevent their activity on endogenous targets2022/7/18162022/7/1817Levels of transcripts of miR396 targets weremeasured 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4、miR396 negatively

15、affects root growth, RAM size and cell proliferation in the root apexTo investigate the function 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).2022/7/18182022/7/1819m

16、iR396-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2022/7/1820Root meristem size is reduced inmiR396-OE composite plants2022/7/1821两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2022/7/1822The expression of mitotic cell cycle (CYCB1;1, CYCB1;3, CYCB2;1, CYCD3;1, histone H4, CDC16) or endocyc

17、le (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 independent experimentsTogether, these data strongly suggest that miR396-dependent modulation of root

18、 growth may essentially be due to a restriction of cell division activity.2022/7/18235、Silencing of MtGRF genes mimics the miR396-OE root growth phenotypeTo study the role of the miR396 targets on root growth, we inactivated them using an RNAi strategy.First, we generated composite plants expressing

19、 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 silencing2022/7/18242022/7/1825Silencing of MtGRF genes mimics miR396 over-expression in roots.iGR

20、F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6、Hormonal control of miR396 expressionWe treated wild-type plants for 1, 3 or 5 h with auxin, cytokinin, gibberellin, abscisic acid or brassinosteroids (BR), and measured pre-miR396 and target mRNA levels in roots.2022/7/18262022/

21、7/1827Gibberellin, cytokinin and auxin treatments did not affectexpression of any of these genes2022/7/1828miR396b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2022/7/1829Levels of GUS transcript in roots expressing the prom-miR396b:GUS construct (grey bars) or the prom-GRF5:GUS construct

22、 (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7、 miR396 limits mycorrhizal colonization but not nodulationWe 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 symbiotic bacteria Sinorhizobium meliloti.2022/7/18302022/7/1831miR396a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2022/7/1832虽然miR396存在于根瘤中，但miR396-OE和MIM396对根瘤密度