【作者】 王丹；

【导师】 施季森； 陈金慧；

【作者基本信息】 南京林业大学 ， 林木遗传育种， 2021， 博士

【摘要】 植物体细胞胚胎发生(体胚发生)是在离体条件下体细胞通过与合子胚发生相似的途径产生新个体的过程,其不仅是研究高等植物早期合子胚发生的理想模型,同时也是研究人工离体培养条件下植物体细胞胚胎高效发生和调控的重要技术平台。植物microRNAs(miRNAs)是一类长度约21～25nt的非编码单链RNA,其在植物体胚发生过程中具有重要的调控功能。本研究利用降解组测序,并结合实验室前期杂交鹅掌楸体胚发生过程中miRNA的研究,深入探讨miRNA通过调控其靶基因表达,进而深入了解杂交鹅掌楸体胚发生这一过程中的分子调控机制。在此基础上,进一步研究了 LhmiR397a及其靶基因LhLAC11在杂交鹅掌楸体胚发生过程中的生物学功能。1、杂交鹅掌楸体胚发生过程中miRNA靶基因的鉴定通过降解组测序,我们获得了 619个靶基因,其中419个是呈现动态表达模式miRNA的靶基因。对靶基因进行GO注释及KEGG通路分析,我们发现其功能主要体现在胁迫应答、信号转导及植物生长发育方面。利用Cleaveland软件对降解组数据进行分析,预测miRNAs介导的靶基因裂解位点。实时定量PCR(qRT-PCR)检测miRNAs及靶基因在杂交鹅掌楸体胚发生过程中的表达模式,相关性分析表明,miRNAs表达量与其靶基因表达量都在不同程度呈现负相关性。在原生质体中共转miRNAs与靶基因或共转miRNAs与靶基因突变体,验证miRNAs与靶基因的互作。2、LhMIR397a的克隆及生物信息学分析高通量测序发现杂交鹅掌楸中miR397有4个成员,LhmiR397a在胚性愈伤阶段表达量最高,且早期体胚发生过程中表达量逐渐降低,变化极显著。通过qRT-PCR验证LhmiR397在杂交鹅掌楸体胚发生各阶段的表达模式,结果与芯片数据趋势相符。启动子分析发现,LhmiR397启动子区域存在胚发育相关转录因子。且LhmiR397a响应ABA及PEG的处理。表明LhmiR397a在杂交鹅掌楸体胚发生早期发挥重要作用。以杂交鹅掌楸叶片为材料,克隆LhmiR397a前体,并对其前体和成熟体序列进行比对和聚类分析。3、LhLAC11的克隆及生物信息学分析降解组数据分析结果显示LhmiR397a的靶基因分别为LhLAC2、LhLAC10、LhLAC11,对其进行生物信息学和基因结构域分析。qRT-PCR验证其在杂交鹅掌楸体胚发生过程中动态表达;相关性分析显示LhmiR397a与其靶基因LhLAC2、LhLAC10、LhLAC11表达量在不同程度呈现负相关相关性,其中LhmiR397a与LhLAC11表达量相关性最高,相关系数为-0.987,因此我们选择LhLAC11做后期克隆表达研究,且原生质体瞬时表达系统及荧光素酶报告基因验证了 LhmiR397a对LhLAC11的剪切。4、LhmiR397a/LhLAC11参与调控杂交鹅掌楸体胚发生过程通过农杆菌介导的遗传转化分别过表达LhMIR397a、靶基因LhLAC11及靶基因突变体mLhLAC11。经30天愈伤诱导培养后,过表达LhMIR397a愈伤增殖率较低;而过表达LhLAC11及其突变体愈伤增殖率较高。qRT-PCR发现,在过表达LhMIR397a愈伤中,细胞周期相关基因CYCA、CYCB表达量较低,而过表达靶基因及靶基因突变体愈伤中,CYCA、CYCB基因表达量较高。表明过表达LhMIR397a降低了细胞分裂速度,从而降低了愈伤增值率。将转基因阳性愈伤组织培养于体胚诱导培养基上23天后,发现过表达LhMIR397a愈伤中出现早期子叶胚,而对照组及其他组转基因愈伤组织中没有出现早期子叶胚。继续观察发现,过表达LhMIR397a愈伤诱导体胚发生率较高。我们检测了转基因愈伤的生理生化状态,发现过表达LhMIR397a愈伤组织中可溶性糖、淀粉、可溶性蛋白含量较高,而过表达LhLAC11及mLhLAC11愈伤组织中可溶性糖、淀粉、可溶性蛋白含量较低。并且观察了转基因愈伤组织中淀粉粒的含量,发现过表达LhMIR397a愈伤组织中淀粉粒含量较高,而过表达LhLAC11及wLhLAC11愈伤组织中淀粉粒含量较低。表明过表达LhMIR397a促进了细胞营养物质的积累,从而促进了体胚诱导进程。更多还原

【Abstract】 Somatic cells response to considerable stress,and go through a series of hormone pathway,then format an embryo.The developmental process is recorded as somatic embryogenesis(SE).SE is not only an ideal model of early zygotic embryogenesis in higher plants,but also an important platform to study the efficient embryogenesis and regulation of plant somatic cells in vitro culture.microRNA(miRNA)in plant is a class of non-coding single-stranded RNA with a length of 21-25nt,and plays an important regulatory role during the process of somatic embryogenesis.In this study,we combined degradome sequencing with previous research on miRNAs to lucubrate the regulation of miRNAs during somatic embryogenesis in Liriodendron sino-americanum through cleavage of their targets.And then make further research on the biological function of LhmiR397a and its target LhLAC11.1、Identification of miRNAs targets during Liriodendron sino-americanum SEWe identified 619 targets of miRNAs through degradome sequencing,and 419 transcripts are targets of differentially expressed miRNAs.GO annotation and KEGG analysis showed that target transcripts are involved in various processes,including plant metabolism,signal transduction and stress response.We used the Cleaveland software to analysis the data of degradome sequencing and identified the cleavage site of targets.To confirm the dynamic expression of miRNA-target modules,a real-time quantitative PCR(qRT-PCR)analysis was performed.The correlative analysis showed significant negative correlation in the expression pattern between the miRNAs and their targets.And the regulation between miRNAs and their targets was identified by miRNA contransfected into protoplast with their targets or target mutant.2、Cloning and bioinformatics analysis of LhmiR397aFour members of miR397 were identified through high-throughput sequencing.The highest expression level of LhmiR397a was at embryogenic stage,and it decreased gradually during early somatic embryogenesis.A real-time quantitative PCR(qRT-PCR)analysis was performed to confirm the dynamic expression of LhmiR397a.The result showed that the expression level was in line with the microarray data.Various cis-elements involved in embryo development were identified in the promoter of LhmiR397.Afterwards,we cloned the precursor of LhmiR397a and then bioinformatics analysis was performed.3、Cloning and bioinformatics analysis of LhLAC11Three targets were identified for LhmiR397a by degradome sequencing,and they were LhLAC2、LhLAC10、LhLAC11 respectively.Then bioinformatics analysis and domain analysis were performed.To identify the dynamic expression pattern of the three targets,we conducted qRT-PCR.And the correlative analysis showed negative correlation in the expression pattern between the LhmiR397aand its targets.The expression of LhLAC11 had the most significant correlation with the expression of LhmiR397a,and the correlation coefficient was-0.987.As a result,we cloned LhLAC11 subsequently.And then we confirmed the cleavage on LhLAC11 mediated by LhmiR397a using transient expression system of protoplast and luciferase analysis.4、LhmiR397a/LhLAC11 module was involved in regulation of Liriodendron sino-americanum SETo identify the function of LhmiR397a/LhLAC11 module in Liriodendron sinoamericamum SE,we overexpressed LhMIR397a,LhLAC11 and LhLAC11 mutant respectively.After 30 days of callus induction,a lower callus proliferation rate was identifi ed when LhMIR397a was overexpressed.While a higher callus proliferation rate was identified when LhLAC11 or LhLAC11 mutant was overexpressed.And then we conformed qRT-PCR,the result showed that CYCA、CYCB had a low expression when LhMIR39 7a was overexpressed,while a high expression of CYCA、CYCB was identified when LhLAC11 or LhLAC11 mutant was overexpressed.The results showed that overexpression of LhMIR397a decreased the cell division rate and the callus proliferation rate.After 23 days of somatic embryo induction,early cotyledonous embryos were observed when LhMIR397a was overexpressed,while there was no obvious early cotyledonous embryos in other group.Further observation showed that overexpression of LhMIR397a induced somatic embryos with high incidence.Subsequently,we examined the physiological and biochemical status of transgenic callus,and found that the content of soluble sugar,starch and soluble protein were higher in the callus that overexpred LhMIR397a,while the content were lower in the callus that overexpressed LhLAC 11 or LhLAC11 mutant.And then we observed the content of starch granule of the callus that overexpressed LhMIR397a,LhLAC 11,LhLAC11 mutantor without transgenic treatment.A higher content of starch granule was observed when LhMIR397a was overexpressed,and a lower content of starch granule was observed when LhLAC11 or LhLAC11 mutant was overexpressed as well as the callus without transgenic treatment.These results indicated that overexpression of LhMIR397a promoted the accumulation of cellular nutrients and thus promoted somatic embryo induction.更多还原