【作者】 刘娜；

【导师】 刘振华；

【作者基本信息】 山东大学 ， 细胞生物学， 2020， 硕士

【摘要】 植物细胞具有全能性,在离体培养条件下可通过“脱分化”和“再分化”两个过程实现植株再生。植物细胞经历“脱分化”后多数形成愈伤组织,进而获得再生植株的能力,因此愈伤组织形成是植株再生的关键步骤。一些愈伤组织形成调控因子(如Auxin Response Factor 7(ARF7))得到鉴定。然而,目前愈伤组织形成调控网络还不够清晰,更多新的调控因子还有待鉴定。MYB转录因子作为最大的转录因子家族之一,广泛参与植物生长发育及逆境胁迫响应,但是其在愈伤组织形成中的功能却鲜有报道。本论文通过对拟南芥不同阶段的离体培养材料表达谱芯片的分析,筛选得到两个特异性在愈伤组织中高表达的MYB家族转录因子基因MYB94及其同源基因MYB96,并对二者在愈伤组织形成中的功能和机制进行了研究。主要研究结果和结论如下:1.MYB94和MYB96抑制愈伤组织的起始和发育愈伤组织的形成包含起始和发育两个步骤,为了探究MYB94与MYB96对愈伤组织形成不同阶段的影响,利用5-Ethyny1-2’-deoxyuridine(EdU)染色观察、组织透明观察、基因功能回补实验等研究发现,myb94与myb96外植体在CIM(Callus Inducing Medium)培养2天时形成的聚集EdU荧光信号数目、CIM培养4天时形成的细胞团突起的数目以及CIM培养18天时形成的愈伤组织体积都较野生型增大,而双突变体myb94 myb96形成愈伤组织的数目较两个单突变体明显增多,体积也明显增大,表明MYB94与MYB96在早期抑制中柱鞘细胞的分裂、愈伤组织的起始及在后期抑制愈伤组织的发育中二者功能冗余。利用GUS染色分析发现,MYB94和MYB96在愈伤组织起始和发育过程中表达,这与二者的功能一致。2.MYB94和MYB96通过直接抑制LBD29的表达抑制愈伤组织起始利用qRT-PCR分析及GUS染色分析发现,与Col-0背景相比,LATERAL ORGAN BOUNDARIES DOMAIN 29(LBD29)在 myb94、myb96 及 myb94myb96愈伤组织中的表达升高,表明在愈伤组织形成中,MYB94和MYB96抑制LBD29的表达。凝胶阻滞实验(EMSA)和染色质免疫沉淀(ChIP)分析发现,MYB94和MYB96直接结合LBD29的启动子区域。利用CRISPR/Cas9技术在myb94 myb96中敲除LBD29,发现LBD29功能的缺失基本恢复了 myb94 myb96的愈伤组织起始的表型,但无法恢复myb94myb96的愈伤组织发育的表型。表明在愈伤组织起始中,LBD29在遗传学上位于MYB94和MYB96的下游。综合以上结果表明,MYB94与MYB96通过直接抑制LBD29的表达抑制愈伤组织起始。3.MYB94和MYB96可能通过促进超长链不饱和脂肪酸的合成抑制愈伤组织发育有报道发现,超长链不饱和脂肪酸(VLCFA)抑制愈伤组织的发育。因此利用qRT-PCR分析发现,与Co1-0相比,myb94 myb96的愈伤组织中超长链不饱和脂肪酸合成酶基因 3-KETOACYL-COA SYNTHASE(KCS1),3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE I(KAS1)和 PASTICCINO 1(PAS1)的表达水平降低。表明MYB94和MYB96可能通过促进VLCFA的生物合成来抑制愈伤组织的发育。4.结论在愈伤组织形成过程中,MYB94和MYB96受到生长素的诱导表达,它们一方面通过直接抑制LBD29的表达抑制愈伤组织的起始,另一方面通过促进超长链不饱和脂肪酸的合成抑制愈伤组织的发育。更多还原

【Abstract】 Plant somatic cells can be reprogrammed during in vitro culture due to their totipotency,and regenerate shoots through "dedifferentiation" and "redifferentiation".Callus induction after somatic cell "dedifferentiation" is the initial step of a typical plant regeneration system.Several callus formation regulators have been identified,such as ARF7.However,the molecular control of callus formation is largely unknown.The MYB transcription factors represent a large family of transcription factors in all eukaryotes.In plants,MYB transcription factors are shown to be involved in the regulation of plant development and responses to biotic and abiotic stresses.However,participation of MYB transcription factors in the regulation of callus formation has not yet been described.In this study,analysis of microarray data showed that two MYB transcription factors,MY1394 and MYB96 were specifically expressed at high levels during callus formation.The function and regulatory mechanisms of them in callus formation were studied and the results were as follows:1.MYB94 and MYB96 inhibited both callus initiation and growthEthynyl deoxyuridine(EdU)staining showed that after 2 d on CIM,myb94,myb966 generated larger numbers of cell clusters than the WT,and myb94 myb96 produced the largest.Tissue clearing showed that the frequencies of outgrowth initiation were higher from root explants of myb94,myb96,and myb94 myb96 than from that of the WT..After 18 days’ culture on CIM,myb94,myb96,and myb94 myb96 generated more and larger calli than the WT,with myh94 myb96 producing the most.MYB94 and MYB96 were expressed in the newly formed callus.The results show that MYB94 and MYB96 redundantly inhibit both callus initiation and growth.2.MYB94 and MYB96 inhibit callus initiation via directly repressing the transcription of LBD29qRT-PCR and GUS staining showed that LBD29 expression was increased in myb94,myb96,and myb94 myb96 background compared to that in Col-0.EMSA and ChIP anslysis showed that MYB94 and MYB96 could directly bind to the promoter of LBD29.Mutation of LBD29 in myb94 myb96 rescued its callus initiation phenotype,but not callus growth phenotype,suggesting that MYB94 and MYB96 act upstream of LBD29 during callus initiation,but not during callus growth.The above results show that MYB94 and MYB96 inhibit callus initiation via directly repressing the transcription of LBD29.3.MYB94 and MYB96 might inhibit callus growth via regulating VLCFA biosynthesisCompared to Col-0,the expression of KCS1,KAS1 and PAS1 were decreased in myb94 myb96.Thus,MYB94 and MYB96 might inhibit callus growth via regulating VLCFA biosynthesis.4.ConclusionsMYB94 and MYB96 are up-regulated during CIM incubation and expressed in newly formed callus.They play inhibitory roles in callus initiation via directly repressing the transcription of LBD29 and also inhibit callus growth via regulating VLCFA biosynthesis.更多还原