【作者】 李水根；

【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， 2013， 博士

【摘要】 落叶松（Larix spp.）是我国北方和西部高山地区具有重要经济和生态价值的针叶树种，而体细胞胚再生体系是进行落叶松分子遗传改良的基础，也是研究裸子植物胚胎发生机制的理想实验材料。受基因型和培养条件的影响，落叶松体细胞胚的诱导经常出现发育畸形，阻碍了实验和生产。生长素及其极性运输在拟南芥等植物胚胎的形态建成中扮演重要角色。已经有研究表明，转录因子HD-ZIP III可能参与生长素的极性运输过程，且HD-ZIP III为miR166的目标基因。但是在裸子植物体细胞胚发育过程中，miR166调控HD-ZIP III，及其与生长素的关系没有相关研究。我们以落叶松体细胞胚为实验材料，研究了生长素极性运输抑制剂N-1-萘基酞氨酸（NPA）和过表达miR166a对体细胞胚发育的影响，分析了2个生长素受体和4个HD-ZIP III家族成员的基因结构和表达模式，并得到以下结果：（1）采用NPA处理，模拟生长素极性运输被抑制的突变体，研究生长素极性运输对落叶松体细胞胚发育的影响。结果表明，10mg/L的IAA和3mg/L的NPA处理导致原胚团生长缓慢，胚性胚柄结构消失。成熟培养基中加入3mg/L的NPA，体细胞胚发育出现畸形，子叶融合不展开，顶端呈杯状，胚柄程序化细胞凋亡被延缓，且胚头的生长受到抑制。利用树脂半薄切片观察体细胞胚的显微结构，结果表明NPA处理导致体细胞胚的顶-基胚轴极性无法正常建立，细胞排列紊乱，各组织和器官没有正确有序的进行分化，胚头和胚柄的生长平衡被打破。（2）通过同源克隆获得了两个生长素受体基因LaAFB1和LaAFB2，两者都具有典型的LRR和F-box结构域。LaAFB1的3’端具有miR393结合位点，其体内切割产物通过RLM-5’RACE技术得到验证，且切割位点发生在miR393的第10和13位碱基，表明落叶松LaAFB1受到miR393的转录后调控。通过检索体细胞胚和幼苗的小RNA测序文库，发现LaAFB1被切割后能产生次级siRNA，且只存在于幼苗小RNA文库中，而不存在于体细胞胚的小RNA文库中，提示miR393介导LaAFB1切割产生的次级siRNA只在落叶松的胚后发育中发挥功能。采用qRT-PCR检测LaAFB1和LaAFB2在落叶松根茎叶等器官和体细胞胚中的表达模式。发现两个基因的表达规律相似，且在成熟体细胞胚中的表达水平最低，表明削弱生长素的信号转导作用，有利于胚胎的发育和成熟。（3）利用同源克隆和RACE技术获得了4个落叶松HD-ZIP III同源基因的全长cDNA，分别命名为LaHDZ31、32、33和34。它们都具有保守的蛋白结构域：HD、START和MEKHLA。在LaHDZ31-34的START中存在miR165/166的核酸互补序列，进一步采用RLM-5’RACE验证了LaHDZ33和34的体内切割产物，且切割位点均位于miR166的第10位碱基，表明在落叶松体细胞胚发生过程中，LaHDZ31-34受到miR165/166的转录后调控。利用qRT-PCR检测了LaHDZ31-34在正常发生和NPA处理的体细胞胚中的表达规律，结果表明LaHDZ31-34在成熟体细胞胚中的表达量高于原胚团中，且表达最高峰发生在后期单胚阶段。在体细胞胚发育早期，NPA处理的体细胞胚中LaHDZ31-34的表达量比对照高；而在发育的后期阶段，LaHDZ31-33在NPA处理的体细胞胚中的表达量比对照低，但LaHDZ34的表达不受影响。表明LaHDZ31-34与落叶松体细胞胚极性的形成密切相关，LaHDZ34在胚胎发育后期及子叶形成中的功能与其他3个基因不同。（4）通过qRT-PCR检测了miR166的两个前体基因LaMIR166a和LaMIR166b在落叶松根茎叶、正常发生的体细胞胚和NPA处理的畸形胚中的表达规律，发现两个基因的表达模式明显不同。LaMIR166a在根茎叶中均表达，且在原胚团中的表达水平比成熟体细胞胚中略高；而LaMIR166b在原胚团和叶中不表达，在体细胞胚成熟过程中表达上调。在体细胞胚成熟过程中，miR166及其目标基因存在共表达现象，但是在体细胞胚发育后期，LaMIR166a和LaMIR166b的表达受NPA诱导，其目标基因却受NPA抑制。构建Super启动子驱动LaMIR166a超表达的转化载体，通过农杆菌介导的落叶松胚性细胞的遗传转化，获得了五个抗性细胞系。经PCR检测验证，五个抗性细胞系均为阳性转基因。对过表达LaMIR166a的转基因体细胞胚的表型和显微结构进行观察，结果表明过表达LaMIR166a使胚轴伸长受到抑制，影响子叶的发生和茎顶端分生组织的形态。过表达LaMIR166a与NPA处理对落叶松体细胞胚顶端形态发育的影响类似，但NPA处理的体细胞胚顶端比过表达LaMIR166a的表型缺陷严重。过表达LaMIR166a与NPA处理的体细胞胚中顶端分生组织标记基因WOX表达上调，提示miR166与生长素对落叶松体细胞胚的形态建成可能具有共同的调控作用。这些实验结果对于进一步研究针叶树体细胞胚发育的分子机制提供理论依据。更多还原

【Abstract】 Mainly distributed in northern China and western mountains, Larix spp. is an importantconifer species with highly economic and ecological values. Somatic embryogenesis is anuseful tool to improve genetic traits of larch by molecular methods. It is also an idealexperimental material to study the mechanism behind gymnosperms embryogenesis. However,stable and efficient induction of somatic embryos is affected by various genotypes and cultureconditions, and abnormal embryos were initiated frequently in our experiments. Auxin and itspolar transport play important roles in embryogenic morphogenesis of Arabidopsis and otherplants, and it had been suggested that the HD-ZIP III transcription factors may participate inpolar auxin transport, but it is still largely unknown the functions of auxin and miR166regulation of HD-ZIP III in gymnosperm somatic embryogenesis. Based on the somaticembryogenesis of larch, we had studied the effects of polar auxin transport inhibitor1-N-naphthylphthalamic acid (NPA) and over-expression of miR166a on somatic embryodevelopment of larch, and analyzed the sequence structures and expression patterns of twoauxin receptors and four HD-ZIP III members. The following results were obtained:(1) To study polar auxin transport in somatic embryogenesis of larch, somatic embryostreated with NPA was used to mock the larch mutant in which auxin polar transport wasinhibited. As a result, after treated with10mg/L IAA and3mg/L NPA, the pro-embryogenicmass (PEM) grow more slowly, with the polarity destructed.3mg/L NPA-treatment had noeffect on the frequency of induction of somatic embryos, but the NPA-treated somatic embryosformed a cup-sharp apical embryo, cotyledons fused and programmed cell death of suspensorsdelayed. The microstructure of NPA-treated somatic embryos were observed by semithinsection, and the cells in NPA-treated embryos were not organized normally and patternformation was disrupted, indicating that the growth balance between embryo and suspensorwas changed by NPA-treatment. (2) Two homologues of auxin receptors from larch were identified and designatedLaAFB1and LaAFB2, both of which contained LRR and F-box domains. Identification of thecleavage products of LaAFB1in vivo confirmed that it was regulated by miRNA, and thecleavage sites occurred at10th or13th base of miR393. Secondary siRNAs that were derivedfrom miR393-guided cleavage of LaAFB1were predicted by bioinformatics, and they wereonly found in small RNA transcriptome of seedling, but not in somatic embryos, indicating thatmiR393-guided cleavage of LaAFB1occurs in post-embryogenesis. The expression patterns ofLaAFB1and LaAFB2in PEM, mature somatic embryos, root, stem and leaf were analyzed byqRT-PCR. The results showed that they shared similar expression pattern and both showedlower transcript levels in mature somatic embryos, which suggested that it is required todepress the auxin sensitivity for maturation and development of somatic embryos in larch.(3) Four HD-ZIP III homologues from larch were identified and designated LaHDZ31,32,33and34. They all contained HD、START and MEKHLA domains. The occurrence of amiR165/166target sequence in all four cDNA sequences indicated they might be targets ofmiR165/166. Identification of the cleavage products of LaHDZ31and LaHDZ32in vivoconfirmed that they were regulated by miRNA. Their expression patterns during somaticembryogenesis and the effects of NPA on their expressions were investigated. The resultsshowed that the four genes had higher expression levels at mature stages than at theproliferation stage, and that NPA treatment down-regulated the expression of LaHDZ31,32and33at cotyledonary embryo stages, but had no effect on the expression of LaHDZ34. Weconcluded that these four members of Larix HD-ZIP III family might participate in polar auxintransport and the development of somatic embryos.(4) Two miR166precursor genes were obtained and designated LaMIR166a andLaMIR166b. Their expression patterns in root, stem, leaf, normal somatic embryos andNPA-treated embryos were studied, and both genes showed very differential expressionpatterns. The transcripts of LaMIR166a could be detected in all organs and developmentalstages of somatic embryos, while the LaMIR166b showed undetectable transcript levels inPEM and leaf. Taken together, miR166and their targets were co-expressed at late stages of somatic embryogenesis, but showed inverse NPA-responses. LaMIR166a was cloned into thepSuper1300+binary vector allowing over-expression under a Super promoter. Subsequently,five hygromycin-resistant embryonic cell lines were obtained by Agrobacteriumtumefaciens-mediated transformation, and they were identified to be positively transgenic byPCR amplification. Study on the phenotypes and microscopic structures of transgenic somaticembryos indicated that over-expression of LaMIR166a inhibited hypocotyl elongation, andaffected the initiation of cotyledons and morphology of stem apical meristem.From the research of auxin and miR166in somatic embryogenesis, we found that theeffects of LaMIR166a over-expressing on apical morphogenesis of somatic embryo weresimilar with that of NPA-treatment, although the developmental defects of LaMIR166aover-expressor was slighter than that of NPA-treated somatic embryos. The expression level ofWOX was up-regulated in LaMIR166a over-expressing and NPA-treated somatic embryos,indicating that the miR166and auxin might share common regulatory pathways during somaticembryogenesis of larch. These results provided a theoretical basis for further study of themolecular mechanisms behind conifer somatic embryogenesis.更多还原