【作者】 张立峰；

【导师】 齐力旺；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2014， 博士

【摘要】 落叶松（Larixspp.）是我国北方重要的速生造林用材树种，具有非常诱人的育种前景和经济效益。落叶松干细胞繁育体系的建立为现代分子育种技术进行遗传改良奠定了基础，是研究植物胚胎发育及细胞全能性的理想实验材料，为林木优良品系大规模繁育提供了一套全新的技术体系。然而，目前针叶树体细胞胚发生体系中，胚性细胞系诱导困难、畸形胚发生率高、体细胞胚生根率低等问题，成为体细胞胚发生技术应用于生产的“瓶颈”。大量研究表明，脱落酸（ABA）在植物胚胎发育过程中起重要调控作用。胚胎发育早期，ABA可以促进胚胎早期形成及顶端-基部对称轴的建立；胚胎发育后期能够促进营养物质的合成与积累，诱导并维持胚胎进入休眠状态。本研究以落叶松胚性细胞系d202不同发育阶段的胚性培养物为试验材料，建立模拟外源ABA有无的体细胞胚发育模型，观察并分析了ABA对落叶松体细胞胚发育的影响；研究了LaTCTP和4个LaNFYAs家族成员在体细胞胚发育过程中的表达模式；进一步利用RNA-seq技术对关键时期的材料进行转录组测序和表达谱分析，结合差异基因表达与生理生化分析，初步探讨了ABA调控落叶松体细胞胚发育的分子机理。主要结果如下：（1）将继代培养14d的落叶松胚性胚柄团（Embryonal Suspensor Mass，ESMs），分别转接到ABA和No-ABA分化与成熟培养基上，建立了模拟外源ABA有无的体细胞胚发育模型。结果表明，ABA可以抑制细胞分裂，诱导早期胚胎形成，并促进胚胎的进一步发育与成熟。而当ABA不存在的情况下，H2O2含量逐渐增加，组织内部胁迫程度加重，ESMs由白色逐渐转变为褐色，体细胞胚发育受阻，最终没有正常的胚胎形成；偶尔会有几个畸形胚，但并不能萌发形成植株。（2）通过同源克隆技术，获得了LaTCTP的cDNA及基因序列。序列分析表明其具有TCTP蛋白家族的特征序列TCTP1和TCTP2。利用qRT-PCR检测了LaTCTP在落叶松体细胞胚发育过程中的表达模式。结果表明，LaTCTP可能受生长素诱导。体细胞胚发育早期，随着外源生长素含量的下降，LaTCTP表达下调；此时H2O2含量达到最大值，这有助于PCD发生，促进早期胚胎形成；早期胚胎进一步发育过程中，LaTCTP表达量维持在较高水平，表明细胞活动较为旺盛，对促进胚胎的进一步发育有重要作用；而在褐化组织中H2O2含量较高，LaTCTP表达上调，可能与胁迫诱导相关。综上所述，LaTCTP可能参与了ESMs的快速增殖、早期胚胎形成及胚胎的进一步发育。（3）通过对比TAIL-PCR、FPNI-PCR扩增LaTCTP5′侧翼序列的优略性，进一步优化了PCR反应条件和参数；在此基础上，通过缩短接头序列的长度，引入阻抑PCR原理，将其命名为Suppression and nested mediated PCR（SNM-PCR）；此方法增加了简并引物与基因组DNA的结合效率和特异性扩增，提高了LaTCTP5′侧翼序列扩增效率，并成功克隆了LaNFYAs5′侧翼序列。该技术为快速、高效克隆落叶松基因启动子序列，进一步研究并分析基因功能奠定了基础。（4）通过同源克隆和RACE技术，克隆并鉴定了4个落叶松NFYA同源基因，分别命名为LaNFYA1、LaNFYA2、LaNFYA3、LaNFYA4。序列分析表明它们均具有亚基结合结构域（SAD）和DNA结合结构域（HD）。进一步分析发现，3’UTR区域均存在miR169结合位点，表明其可能受miR169调控。5′侧翼序列分析表明LaNFYAs是一类与抗逆和激素应答相关基因。qRT-PCR结果表明，正常体细胞胚发生过程中，LaNFYAs的表达量均在第7d时达到最大值；而No-ABA处理的ESMs中，前14d，LaNFYA1、LaNFYA2、LaNFYA3表达均没有明显变化；暗示这三个基因可能受ABA诱导。另外，还发现LaNFYA4表达模式与其它三个基因不同，前7d表达趋势与正常体细胞胚一致；第14d时，正常体细胞胚发育中表达量急剧下降，而No-ABA处理ESMs中的表达量则继续上调，表明LaNFYA4不仅与ABA应答相关，还与ESMs细胞褐化后产生胁迫诱导相关。（5）通过RNA-seq分析发现：当ESMs转接到分化与成熟培养基后，由于生长素的撤除，导致376个与细胞生长相关基因下调表达；在ABA作用下，5～7d时形成黄色胚头，期间有302个基因上调表达，促进早期胚胎形成；而在No-ABA的情况下，细胞一直处于渗透胁迫状态，7～10d时胚性细胞逐渐褐化，在此期间有333个与胁迫及活性氧清除相关基因上调表达，以抵御或缓减胁迫对细胞造成的伤害。综上所述，通过以上研究结果表明，ESMs转接至ABA和No-ABA分化与成熟培养基2d时，由于生长素的缺失，维持细胞旺盛分裂的条件不复存在，诱使与细胞生长相关基因下调表达，导致细胞生长、分裂速度下降；此时两种ESMs生理状态相似，下调基因基本相同，表明此时ABA对落叶松ESMs作用不明显。在No-ABA情况下，细胞长期处于渗透胁迫状态，为抵御或缓减胁迫所造成的伤害，启动了大量与胁迫及活性氧清除相关基因，但仍无法阻止ESMs细胞由白色变为褐色，最终逐渐死亡。而在ABA作用下，2d后诱导了ABA应答基因（LaNFYAs）上调表达，可能促进了内源生长素合成；5d后诱导了生长素应答基因（LaTCTP）上调表达，促进早期胚胎形成及进一步发育。相关研究结果为深入研究ABA在落叶松体细胞胚发育的分子调控机理奠定了基础。更多还原

【Abstract】 Japanese larch (Larix leptolepis) is an important conifer species, which has significanteconomic and ecological value. Somatic embryogenesis provides a useful experimental systemto investigate the regulatory mechanisms of plant development, which can be applied toimprove genetic traits and large-scale breeding. However, a number of challenges are presentin conifer somatic embryogenesis, such as difficulties with embryonal-suspensor mass (ESM)induction, a high frequency of abnormal embryos developing during the maturation stage, anddifficulties with in vitro synchronization. These obstacles create a bottleneck in the productionof somatic embryos. Therefore, understanding the molecular mechanisms underlying thedifferent developmental stages of somatic embryogenesis is important to overcome theseproblems. Several studies have suggested that abscisic acid (ABA)plays an importantregulatory role in plant embryogenesis. In somatic embryogenesis in conifers, exogenouslyapplied ABA not only can block the proliferation of ESMs and induce further development ofearly proembryos, it can also stimulate the deposition of storage compounds within the somaticembryo, induce and maintain embryo dormancy. Based on the somatic embryogenesis of larch,we had studied the effects of exogenously applied ABA, established somatic embryogenesismodel of mock exogenous ABA presence or absence and analyzed the sequence characteristicsand expression patterns of TCTP and four NFYA members. Furthermore, RNA-seq was appliedto the analysis of expression profiles between morphological changes’ materials, together withthe analysis of physiological and biochemical, we initially explored the ABA regulatorymechanism during somatic embryogenesis in larch. The following results were obtained:(1) To study the effects of ABA during somatic embryogenesis, ABA was removed fromthe differentiation and maturation medium. ESMs were cultured on subculture medium for14days, transferred to differentiation and maturation medium either with ABA (normal somaticembryogenesis) or without ABA, established somatic embryogenesis model of mockexogenous ABA presence or absence. As a results, ABA can inhibit cell division and induce the early embryo formation, and promote further development and maturation of embryo. WhenABA was removed from the medium, H2O2levels will increased, ESMs underwent browningon the8day and gradually died. Eventually, only a few partially malformed embryosdeveloped at42days and did not have the ability to germinate.(2) cDNA and genomic sequences of a TCTP gene were cloned from L. leptolepis,designated LaTCTP, which contained two signature sequences TCTP1and TCTP2. Theexpression profile of LaTCTP during somatic embryogenesis was determined by qRT-PCR. Asa results, the expression of LaTCTP decreased gradually during the first5days after transfer ofESMs to differentiation and maturation medium, which may be caused by the absence of auxin.At this point, H2O2level reaches the maximum, which can induce the occurrence of PCD topromote early embryos formation; In somatic embryos rapid development stage, LaTCTPexpression remained high expression levels, which have an important role to promote thefurther development of somatic embryos; while upregulation of LaTCTP in browning,whichhas higher H2O2levels, may be associated with stress-induced. In summary, LaTCTP probablyparticipates in the proliferation of ESMs, formation and further development of early embryos.(3) By comparing the amplification of LaTCTP’s5’flanking sequence using TAIL-PCRand FPNI-PCR, the PCR conditions and parameters were optimization. On the basis of this,new method has created by reducing the length of the linker sequenceand integrated into theprinciple of suppression PCR, designatedsuppression and nested mediated PCR (SNM-PCR);This method increases the binding efficiency of random primers with genomic DNA andamplification specificity, which improved the amplification efficiency of LaTCTP5’flankingsequences and successfully obtained LaNFYAs5’flanking sequences. SNM-PCR has laid thefoundationfor fast and effectively cloning promoter sequences and further analysis of genefunction.(4) Four NFYA homologs were isolated and characterized from L. leptolepis, designatedLaNFYA1, LaNFYA2, LaNFYA3and LaNFYA4. Multiple-sequence alignments showed that thefour putative proteins harbored two functional domains: a subunit association domain (SAD)and a DNA-binding homeo domain (HD). Further analysis showed that miR169target sequences were present in the mRNAsequences of all four genes, which suggested thatLaNFYAs may be regulated by miR169. Promoter analyses of the LaNFYAs indicated that theymay be stress-and hormone-responsive genes. The expression profile of LaNFYAs duringsomatic embryogenesis was determined by qRT-PCR. The level of LaNFYAs transcriptsincreased gradually during the first7days and reached their highest levels at7days.However,the removal of ABA resulted in no significant changes in the levels of LaNFYA1,LaNFYA2and LaNFYA3transcripts in the first14days, which was suggested that theexpressions of LaNFYA1, LaNFYA2and LaNFYA3were induced by ABA. In addition, it wasfound that the mRNA transcript levels of LaNFYA4increased significantly and had almost thesame mRNA accumulation pattern as in normal embryos during the first7days of culturing,and the mRNA transcript abundance of LaNFYA4increased continuously between7and14days on No-ABA treatment, which suggested that LaNFYA4may notonly be closely associatedwith the ABA response, but also involved in the stress response.(5)The RNA-seq was applied to the analysis of expression profiles betweenmorphological changes’ materials. As a results, When ESMstransferred todifferentiationandmaturation medium, the removal of auxin andcytokininresulted in376genesassociated withcell growth downregulated; In theinductionofABAandPEG4000, yellow embryo “head”appeared at5to7days,there are302genesupregulated to promoting the formationofearlyembryos; while the removal of ABA, PEG4000makeESMscells in astate ofosmotic stress,embryoniccellsgradually underwentbrowning at7to10days, in the meantimethereare333stress-related genesupregulatedin order to resistormitigate thedamage caused bystress.In conclusion, thisstudy showedthat during the first two daysafterESMstransferredtodifferentiation andmaturation medium(ABA and No-ABA), due to the absenceofauxinandcytokinin, the conditions of maintainvigorouscelldivision does not exist, whichinducea number ofgenes associatedwithcell growthdownregulated, leading to the rate ofcellgrowth anddivision decreased;At this stage, the ESMstreated by ABA and No-ABA, havesimilarphysiological status and substantially identicaldown-regulated genes, which indicatingthat the role ofABA is not obvious.while in the caseofonlyPEG4000, ESMs was in astate ofosmotic stress for long time, thecellsstarteda lot ofstress-relatedgenes to resistormitigatethedamage caused bystress. However, it still can notstop thecellsunderwentbrowningand diegradually; In theinductionofABAandPEG4000, ABA-responsive genes (LaNFYAs) wereupregulated after twodays, which might promotethe synthesis ofendogenousauxin;Auxin-responsive genes (LaTCTP) were induced after fivedays to promoteformationandfurtherdevelopment ofearly embryo. This study will benefit future studies on ABAregulatory mechanisms during somatic embryogenesis in Larix spp.更多还原