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

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1、2022/12/291miR396 affects mycorrhization and root meristem activity inthe legume Medicago truncatulaThe Plant Journal(2013)doi:10.1111/tpj.121782022/12/292主要内容nINTRODUCTIONnRESULTSn 1、Mtr-miR396a and mtr-miR396b genes show different tissue-specific expression profiles in Medicago truncatula rootsn 2

2、、In M.truncatula roots,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 gr

3、owth phenotypen 6、Hormonal control of miR396 expressionn 7、miR396 limits mycorrhizal colonization but not nodulation2022/12/293INTRODUCTIONnIn 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)t

4、he elongation zone,and(iii)the differentiation zonenRoot development is regulated by complex interactions of hormone signalling pathways2022/12/294nIn recent years,microRNAs(miRNAs)have emerged as important regulators of the architecture of the root systemnin Arabidopsis thaliana,miR165/166 governs

5、the radial patterning 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 growth2022/12/295nHowever,few miRNAs have been reported to play roles in RAM functi

6、onnAlthough large sets of mycorrhization or nodulation-responsive miRNAs have been identified in legumes,few have been functionally linked to these processes.2022/12/296nIn A.thaliana,miR396 is encoded by two loci,post-transcriptionally represses several members of the growth-regulating factor(GRF)f

7、amily.These plant-specific TFs control 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.2022/12/297RESULTSn1、Mtr-miR396a and mtr-m

8、iR396b genes show different tissue-specific expression profiles in Medicago truncatula rootsnMtr-miR396a 和 mtr-miR396b 在根中的表达具有空间特异性2022/12/2982022/12/299miR396 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2022/12/2910两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202

9、2/12/2911n2、In M.truncatula roots,miR396 regulates six GRF and two bHLH79 transcription 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,

10、Medtr7g126820).These transcripts possess a putative miR396 binding site,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 p

11、redicted proteins showed high similarity(43 and 46%respectively)to AtbHLH79/BIGPETAL(At1g59640),a TF involved in the control of petal growth in A.thaliana.2022/12/29122022/12/2913In M.truncatula,six MtGRF and two bHLH79 genes have miR396 binding sitesn3、Characterization of the miR396 regulatory netw

12、ork in M.truncatulanTo investigate miR396-mediated gene regulation in planta,we over-expressed mtr-miR396a and mtr-miR396b precursors in M.truncatula roots(miR396-OE).2022/12/2914左图说明OE突变体能成功过量表达成熟的miRNAs2022/12/2915Levels of transcripts of miR396 targets weremeasured using quantitative RT-PCR in mi

13、R396-OE rootsMtGRF-mRNA 水平和MtbHLH79转录水平都降低，说明miR396可能抑制这些基因的表达水平nTo 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 members of an miRNA family,and

14、thus prevent their activity on endogenous targets2022/12/29162022/12/2917Levels of transcripts of miR396 targets weremeasured using quantitative RT-PCR in roots inactivated for miR396activity(MIM396)除了GRF6，其他基因表达水平都上升，这里还不能排除有其他因子对GRF6进行调节的可能，本文中没有深入研究n4、miR396 negatively affects root growth,RAM siz

15、e and cell proliferation in the root apexnTo 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).2022/12/29182022/12/2919miR396-OE 中主根长度和干重明显减少，而MIM396

16、中根长稍微增加，而干重却大量增加。说明miR396过表达能抑制根的生长2022/12/2920Root meristem size is reduced inmiR396-OE composite plants2022/12/2921两者之间没有大的变化，说明miR396对细胞从细胞周期进入分裂周期的开关没有大的影响2022/12/2922The expression of mitotic cell cycle(CYCB1;1,CYCB1;3,CYCB2;1,CYCD3;1,histone H4,CDC16)or endocycle(CCS52B)markers was measured us

17、ing 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 experimentsnTogether,these data strongly suggest that miR396-dependent modulation of root growth may essentially be due to a re

18、striction of cell division activity.2022/12/2923n5、Silencing of MtGRF genes mimics the miR396-OE root growth phenotypenTo study the role of the miR396 targets on root growth,we inactivated them using an RNAi strategy.nFirst,we generated composite plants expressing three RNAi constructs targeting a c

19、onserved region of various GRF genes(MtGRF2,MtGRF4 and MtGRF6).nIn addition,we prepared an RNAi vector targeting both MtbHLH79 genes and confirmed their efficient silencing2022/12/29242022/12/2925Silencing of 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

20、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),and measured pre-miR396 and target mRNA levels in roots.2022/12/29262022/12/2927Gibberellin,cytokinin and auxin t

21、reatments did not affectexpression of any of these genes2022/12/2928miR396b对ABA和BR 有明显的反应，并且GRF5在BR处理下下调，说明BR和miR396b,GRF5之间有某种作用关系2022/12/2929Levels 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

22、 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n7、miR396 limits mycorrhizal colonization but not nodulationnWe next decided to study the potential role of miR396 in root symbioses.nWe analysed mtr

23、-miR396a and mtr-miR396b expression profiles in roots inoculated with the symbiotic bacteria Sinorhizobium meliloti.2022/12/29302022/12/2931miR396a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2022/12/2932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nWe also analysed the expression of miR396 in res

24、ponse to Myc-LCOs,the lipochitooligosaccharide signal molecules produced by AM fungi.nThe 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.2022/12/29332022/12/2934miR396 affects f

25、ungal colonization during AM symbiosis.菌根的丛枝量和形态没变化，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.nHere,we showed that,in the model legume M.truncatula,miR396 represses not only MtGRF but also two MtbHLH79 TF-encoding genes,and that its de-regulation affects root growth and the ability to be colonized by mycorrhizal fungi.2022/12/29352022/12/2936