【作者】 曹景林；

【导师】 张献龙；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 体细胞胚胎发生是体细胞向胚胎发生途径转变的发育再建过程,也是植物全能性预言的最好说明。体细胞胚发生现象被看作是开展生物学理论研究的良好模型。研究棉花体细胞胚发生对于阐明植物发育的分子机理,促进作物的遗传工程改良,无论在基础理论还是在实际应用价值上都具有极其重要的意义。以陆地棉品种Coker201为材料,对体细胞胚发生过程中的同步控制技术、组织细胞学、基因表达、蛋白质表达和磷酸化、酪蛋白激酶活性以及蛋白质双向电泳技术等进行了研究。研究结果如下:1.棉花体细胞胚胎发生的频率低,且体细胞胚的发育存在着不同步性,给体细胞胚胎发生发育过程的生理学、生物化学和分子生物学的研究带来许多困难。以陆地棉品种Coker201为材料,首先筛选了适合体细胞胚胎发育的培养基配方,然后以此培养基为基本培养基,建立了一种简单有效的棉花体细胞胚高频率发生和同步发育的培养体系。经过多次继代获得的胚性愈伤首先在液体培养基中振荡培养2d使其分散,然后过30目筛去除大的颗粒,重新悬浮于同样的液体培养基中。悬浮培养14 d后,过50目筛,将筛上的胚性愈伤重悬浮于新鲜的培养基中,并用吸管吸取悬浮液,将其均匀地接种在表面垫有滤纸的同成分固体培养基(附加2.46μmol L^-1IBA和0.70μmol L^-1kinetin)上。培养21d后,用垫有滤纸的相同的固体培养基继代培养。利用这种培养体系,获得的体细胞胚数量分别是单纯悬浮培养和固体培养（不挚滤纸）的16.5倍和4倍。其中,球形胚、鱼雷形胚和子叶胚的同步发生率分别为70.2%、52.3%和73.0%。2.以陆地棉品种Coker 201下胚轴为外植体诱导愈伤组织,并以介于30-50目筛之间的胚性愈伤细胞团为材料进行单个细胞团培养,采用石蜡切片法观察棉花愈伤组织诱导和分化过程及体细胞胚发生和发育过程。结果显示棉花胚性愈伤组织有下胚轴两端发生、内发生、非胚性细胞转化、外发生和腺体处发生5种形成途径,其中两端发生和内发生是主要的形成方式,都是由下胚轴形成层细胞直接分化产生;棉花体细胞胚存在单细胞起源,多为表面发生,少量为内发生,并经历球形胚、心形胚、鱼雷形胚、子叶胚4个过程,与合子胚形成过程相似。棉花体细胞胚发育过程中最关键的事件似乎是极性的建立、顶端分生组织的形成以及前形成层的分化和生长轴变化。球形胚时期表皮原层的分化标志着体细胞胚结构分化的开始。初始前形成层的形成是体细胞胚形态转变的第一个信号。球形胚内部等径细胞轴向延长,接着前形成层生长轴发生变化,这些是心形胚或椭圆形体胚中的重要事件。鱼雷形胚基本分生组织中液泡的出现开始了组织分化的过程。三种主要的胚性组织系统包括芽顶端分生组织,根顶端分生组织和原形成层束在鱼雷形胚阶段清晰可见。随后鱼雷形胚上层分化形成芽原基和两个叶原基。子叶胚的维管组织从两片子叶伸向胚根,呈V形或Y形。对一些停滞发育的球形胚和鱼雷形胚的解剖检查表明,极性建立可能是决定体细胞胚能否萌发的关键因素。3.在体细胞胚胎发生和合子胚发育的过程中,无论是胚胎发生的启动还是胚状体的形态建成都受到多种因子的调控,体细胞胚发生是一个由多种基因参与的复杂时空调节的发育途径。本研究从基因表达水平上对体细胞胚胎发生过程中所涉及的基因转录、转录后与翻译后修饰以及糖类、脂类及氨基酸与蛋白质的合成转运与代谢等生化进程进行了一定的分析。在所研究的33个基因中,有25个来自一个棉花的体细胞胚胎发生的SSH文库,这些基因在棉花胚性愈伤和各时期胚状体中有差异表达,另外5个SERK基因和3个CKⅡ基因来源于相关Genebank库。根据qRT-PCR的实验结果,按目的基因的表达最高峰所对应的体细胞胚胎发生阶段可将这些基因分为3类:（1）与体细胞胚起始发生与形态建成相关。供试的基因中几乎所有涉及代谢与转运的基因均在这一阶段出现表达高峰,这与前人研究中体细胞胚发生过程中DNA含量明显增加,RNA合成速率明显提高,mRNA种类丰富,氨基酸含量升高,蛋白质合成活跃的特征是不谋而合的,高水平的脂质转运蛋白更是成为体细胞胚发生的分子标记物质;后转录因子在体细胞胚的形成与发育阶段的高表达预示着其生命过程的复杂性。（2）与体细胞胚发育与成熟相关。从供试的基因表达高峰所对应的胚胎发育阶段来看,体胚与合子胚的发育成熟过程中对后转录因子、CKⅡ、腺嘌呤、氨基酸与类脂物质代谢相关的基因需求量是不一致的。体细胞胚胎发育与成熟要求较高水平的细胞壁相关蛋白基因、腺嘌呤与氨基酸代谢相关基因与CKⅡ基因;以SERK为代表的翻译后修饰基因在体细胞胚发育阶段的高表达被认为是复杂的蛋白质相互作用的标志,证明了体细胞胚的发育和成熟与起始发生和形态建成一样是相当复杂的。（3）与合子胚发育相关。合子胚的发育和成熟要求更高水平的酰基转移酶;除ERT和SCL转录因子是体细胞胚胎发育所特有的,其余转录因子的表达水平在两种类型的胚胎发育过程中没有明显特异之处。这可能与转录因子的DNA结合域所能识别的顺势因子的差异有关。本研究结果首次证实了HMG转录因子和GRP蛋白在植物体细胞胚和合子胚发生发育中及CKⅡ在植物体细胞胚发生发育中的重要作用。APRT编码基因在体细胞胚发育过程中的活跃表达,意味着在高等植物胚胎发生或发育过程中存在核苷酸补救合成途径。本研究还发现一个编码氧化还原酶的基因在胚性愈伤组织中表达极度活跃,可能是该阶段的标记基因。4.蛋白激酶和磷酸酶主要参与通过蛋白磷酸化和去磷酸化调节的几个细胞事件。这些事件包括细胞生长、分化和细胞代谢。CKⅠ和CKⅡ参与了几种底物的磷酸化。在棉花体细胞胚发生过程中研究了蛋白质的磷酸化和酪蛋白激酶活性。在测试的体细胞胚发生的5个阶段（非胚性愈伤组织、胚性愈伤组织、球形胚、鱼雷胚和子叶成熟胚）观察到许多不同的多肽被磷酸化。外源ATP增强了体细胞胚发生各阶段的蛋白磷酸化作用。冈田酸和矾酸盐在磷酸化反应中的使用增加了蛋白磷酸化水平,这暗示除酪氨酸磷酸酶外,在体细胞胚发生过程各阶段还存在丝氨酸/苏氨酸磷酸酶。实验中采用CKⅡ抑制剂和胶内激酶分析所得结果表明,体细胞胚中存在CKⅡ。5.棉花愈伤组织和体细胞胚中富含多糖、多酚化合物、脂质、色素和其他干扰物质,因而采用常规方法难以制备高质量的蛋白质样品。对陆地棉品种Coker201的胚性培养物,通过添加PVPP研磨和丙酮、TCA/丙酮、甲醇/醋酸铵等有机溶剂的系列洗涤,然后再进行酚抽提,蛋白质质量得到显著改善,获得的2-DE图谱质量高、可分辩的蛋白质点数多。比较分析3种裂解液对蛋白质的溶解效果发现,在裂解液中混合采用7mol·L^-1尿素、2mol·L^-1硫脲以及4%CHAPS是有效的。对3种考马斯亮蓝染色方法和4种银染色方法分别进行比较分析,认为由Neuhoff法改进而来的Blue silver法敏感性比其他考染方法好,稳定性也好;银染色灵敏度高于考马斯亮蓝染色,但由于显色难以掌控因而稳定性差,相比较而言,Mechin银染法表现较好。本实验显示的结果为进一步的研究奠定了基础。更多还原

【Abstract】 Somatic embryogenesis is the developmental reprogramming of somatic cells toward the embryogenensis pathway and a remarkable illustration of the dictum of plant totipotency.It has been viewed as a potential model system for the study of the basic mechanisms of development reprogramming among the higher eukaryotic organism. Study on cotton somatic embryogenensis could provide a useful way to understand plant development such as embryogenesis and may have merit as a study presenting an application in plant biology due to its biological theory and economical importance.To explore the mechanism of somatic embryogenesis in cotton（Gossypium hirsutum L.）, synchronization control technology,histo-cytology,gene expression,protein expression and phosphorylation and casein kinase activity during somatic embryogenesis,and two-dimensional electrophoresis technology were studied by using cotton cultivar Coker 201 as materials.The results are as follows:1.Low efficiency of somatic embryogenesis and asynchronous embryo development results in a lot of difficulties to physiological,biochemical,and molecular biological studies of embryogenesis processes in cotton.A simple and efficient method was developed to improve somatic embryogenesis frequency and synchronous development of mass somatic embryos from cultured cells of the cotton cultivar Coker 201 on the basis of the developed medium which was more suitable for somatic embryo development compared with other tested media.The embryonic calli obtained after several rounds of subculture were scattered in a liquid medium by shaking for 2 d and then resuspended in the same liquid medium after discarding the larger callus aggregates over 30 mesh-size-sieve.The suspensions cultured for 14 d were filtered through 50 mesh-size-sieve and the aggregates over the sieve were incubated for 21 d onto the surface of Whatman filter paper that were placed on the solid medium containing 2.46μmol L^-1 indole-3-butyric acid（IBA） and 0.70μmol L^-1 kinetin.The amount of somatic embryos obtained by this system was 15.5-fold and 3-fold higher than that of suspension culture and solid culture without filter papers,respectively.About 70.2%for globular, 52.3%for torpedo-shaped,and 73.0%for cotyledonary embryos were obtained during the culture.The method combining suspension culture and solid culture（with filter paper） proved to be efficient for synchrony of somatic embryogenesis and mass embryo development.2.Calli were induced from hypocotyl explants of cotton cultivar Coker 201,and individual embryogenic cell clusters with 30-50 mesh sieve,which isolated from embryogenic suspension cultures,were cultured.The development of the calli and single few-celled aggregates was observed through paraffin sections.The results revealed 5 pathways of embryogenic calli origination including origination from both ends of hypocotyls,origination in inner,differentiation from non-embryonic cells,origination from outer cells of calli,and origination in the surrounding of gland.Among these,the origination from both ends of hypocotyls and in inner were the most important pathways that embryogenic calli were differentiated directly from cambium cells of hypocotyls. Cotton somatic embryos originated from individual embryogenic cells,which were in exterior of embryogenic calli in most cases and in inner of embryogenic calli in a few cases.Somatic embryogenesis that the emhryoids were sequentially differentiated through globular,heart-shaped,torpedo embryo and cotyledonary embryo stages was similar to that of the zygotic embryo.The most critical events appeared to be the establishment of apical-basal patterns of symmetry,formation of apical meristems,and differentiation and axial change of procambium during the development of cotton somatic embryos. Differentiation of the protoderm layer marked the beginning of the structural differentiation in globular stage.Incipient procambium formation was the first sign of somatic embryo transition.Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the procambium was an important event in heart-shaped or oblong-stage somatic embryo.Vacuolation in the ground meristem of torpedo-stage embryo began the process of histodifferentiation.Three major embryonic tissue systems,shoot apical meristem,root apical meristem,and the differentiation of procambial strands,were visible in torpedo-stage somatic embryo. Subsequently,partitioning of cotyledon and shoot primordia in the upper layer of torpedo-stage somatic embryos took place.Vascular tissue of the cotyledonary-stage somatic embryos from the two cotyledons to radicle presented V or Y shape.Histological observations of some globular and torpedo-shaped embryos which faired to advance showed that establishment of polarity was the key factor which determined germination of somatic embryos.3.During somatic embryogenesis and zygotic embryos development,the start-up and morphogenesis of embryos are regulated by a variety of factors.In particular,somatic embryogenesis is a temporal and special regulated developmental process.This study attempted to analyze the process of somatic embryogenesis on the expression level of related genes,which involved in the process of transcription,post-transcription and post-translation modification as well as the synthesis,transport,and metabolic biochemical processes of carbohydrate,lipid,amino acids and protein.Among 33 genes in this study,25 were selected from a cotton somatic embryogenesis SSH array, considered differential expressed at different development stages such as the formation of embryogenic callus and somatic embryogenesis,the others including 5 SERK genes and 3 CKⅡgenes were acquired from the Genebank.From the qRT-PCR analysis,some genes were preponderant expressed in special stage of somatic embryogenesis,and these genes could divide into three categories:（1） associating with the start-up and morphogenesis of embryos.Almost all of the genes involving in metabolism and transporter presented a peak expression in this stage.This coincided with the previous studies including remarkable increase of DNA content,evident improving of RNA synthesis rate,mRNA variety,hoist of the levels of amino acids,and stirring synthesis of protein during somatic embryogenesis.Lipid-transporter proteins with the high level of expression were considered as the molecular marker substances of somatic embryogenesis. Post-transcription factors expressed actively at the formative and developmental stage of embryo indicating the complexity of this process.（2） Associating with the development and maturity of somatic embryo.During the development and mature of embryos, somatic and zygotic embryogenesis were different with the genes relating with post-transcription factor,CKⅡ,adenine,amino acid and lipid metabolism-related.Cell wall-related protein genes,adenine and amino acid metabolism-related genes and CKⅡgenes were behaved high expression level during the development and mature of somatic embryos.The high expression level of the post-translation modification genes,such as SERK gene,was considered as a sign that proteins interacted complexly.It was proved that the development and maturity of somatic embryos was complex as the start-up and morphogenesis of somatic embryos.（3） Associating with the development of zygotic embryos.The development and maturity of zygotic embryos required a higher level of acyltransferase.Our results showed that ERT and SCL were unique during somatic embryo development,the rest of transcription factors were not remarkable difference with the expression level between two types of embryo development process.It was related to the difference of the relevant factors identified by the DNA-binding domain of transcription factors.The results of this study first pointed out that HMG transcription factor and GRP protein played a very important role during the development of somatic and zygotic embryo and CKⅡwas very important for somatic embryo development in plant.APRT coding gene was active during the development of somatic embryogenesis, this indicated a way of nucleotide remedial synthesis existed during embryogenesis or embryo development in higher plant.In this study we also found that an oxidoreductase gene exhibited an extremely high expression level in embryogenic callus.It might be a marker gene in this stage.4.Protein kinases and phosphatases are responsible for several cellular events mediated by protein phosphorylation and dephosphorylation.Among these events are cell growth and differentiation and cellular metabolism.CKⅠand CKⅡare involved in the phosphorylation of several substrates.Endogenous protein phosphorylation and casein kinase activity were investigated during cotton somatic embryogenesis.It was observed that a number of different polypeptides are phosphorylated in the five tested development stages including non-embryogenic callus,embryogenic callus,globular embryos, torpedo-shaped embryos,and cotyledonary and mature embryos.Exogenous ATP enhanced protein phosphorylation in these stages.The use of okadaic acid and vanadate in the phosphorylation reactions increased phosphate incorporation in several polypeptides suggesting the presence of serine/threonine as well as tyrosine phosphatases in the five stages of somatic embryogenesis.Also,the results obtained in experiments with CKⅡinhibitor and in-gel kinase assays indicated the presence of CKⅡin somatic embryos. 5.Preparation of high-quality proteins from cotton calli and somatic embryoids is difficult due to the high endogenous contents of polysaccharides,polyphenols,lipin, pigments,and other interfering compounds.To establish a routine procedure of protein extraction for proteomic analysis to cotton embryogenic cultures,a new protocol was developed by optimizing a phenol extraction method combined with methanol/ammonium acetate precipitation.The protein extraction for 2-DE was remarkably improved by the combination of chemically and physically modified processes including polyvinylpolypyrrolidone（PVPP） addition,acetone cleaning and TCA/acetone washes followed by aqueous methanol/ammonium acetate and acetone lavation.The protocol gave a higher protein yield and higher resolution and spot intensity in 2-DE analysis. Furthermore,three lysis buffers for 2-DE were compared on cotton embryogenic calli proteins isolation.Incorporation of chaotrope mixture(7 mol L^-1 urea,2 mol L^-1 thiourea) with 4%CHAPS was found to be the most effective step.In addition,three CBB staining and four silver staining protocols were also compared respectively.A modified Neuhoff’s CBB G-250 stain,dubbed "blue silver",exhibited a much higher sensitivity than all other CBB staining recipes and had a good stability.The silver stains were found to increase the sensitivity of protein detection over CBB staining,but had a poor stability due to difficulty in control of developing color.Of all silver stains,one protocol described by Mechin et al.was better on account of its sensitivity and stability.The results in this study laid the foundation for further research.更多还原