山核桃成花机理研究

【作者】 黄有军；

【导师】 王明庥；

【作者基本信息】 南京林业大学 ， 林木遗传育种， 2010， 博士

【摘要】 成花是植物发育过程中的重要时期,是植物生活史中从营养生长向生殖生长、孢子体向配子体转变的重要转折点,处于形态更替和世代交替的关键时期。100多年来科学家们孜孜不倦地探索着成花的机理。山核桃(Carya cathayensis Sarg.)属胡桃科山核桃属(Carya Nutt.),是浙江区域优势明显的特色经济干果,是高档的干果和木本油料植物。山核桃已经成为浙西北山区农民实现富裕的重要经济支柱。山核桃营养生长期长,始果期需要十年以上。雌雄同株,雄花为下垂的葇荑花序,雌花为短穗状花序。雌雄花的形成存在时空上的差异。山核桃雌花形成直接决定当年的产量,无论从成花理论还是为解决实际问题都有必要研究山核桃的成花问题。本文利用石蜡制片技术结合扫描电镜观察,研究山核桃雌雄花发育的形态发生;利用cDNA-AFLP技术、基因芯片技术结合454深度测序,探讨山核桃成花过程的基因代谢网络调控体系;利用PCR法从山核桃中同源克隆FT、AG、AP1等成花重要基因,从形态解剖学和分子生物学角度探讨山核桃成花机理,并利用拉枝、喷施生长调节剂等园艺措施研发成花控制技术,主要结果如下:(1)从形态解剖学角度掌握了山核桃雌雄花发育过程。3月下旬在雄花原基顶部形成雄蕊原基,4月中旬形成雄蕊并进一步分化。4月下旬,花药内形成花粉囊,花粉母细胞减数分裂形成四分体,之后四分体解体产生单胞花粉粒,5月上旬形成2-胞花粉粒。雌花发育过程从3月下旬开始到5月中旬基本完成,整个过程可分为未分化期、分化初期、花序分化期、总苞形成期和雌蕊形成期共五个时期。分化初期生长点趋于半圆形;花序分化期三个小花原基形成;总苞形成期每一小花周围形成1个总苞片和3个小苞片;雌蕊形成期小花继续发育,胚囊形成。山核桃胚珠为直生胚珠,单珠被,厚珠心型。雌配子体为八核七细胞蓼形胚囊。(2)利用cDNA-AFLP技术克隆获得山核桃成花过程的278个TDFs,其中65个TDFs为未知基因序列,213个TDFs具有同源序列。已知功能的同源基因可分为11类,分别是:细胞信号转导、细胞生长、衰老与死亡、细胞分化与植物成熟、激素合成、胁迫与抗病、物质运输、光合作用、物质和能量代谢、矿物质同化与有机物合成、光形态建成,在山核桃成花过程中起着直接或间接的作用。利用Real-time RT PCR技术对cDNA-AFLP分析进行了验证,两者基本吻合,从而证实了cDNA-AFLP结果的可靠性。对278个TDFs进行了功能注释和代谢分析,共65(23.4%)个无功能注释,213个有功能注释。有功能注释的TDFs中,158(54.7%)个属于生物进程,211(73.0%)个属于细胞组分,分子功能包含173(59.9%)个TDFs。生物进程中占比例最大的是氧化还原作用,其次是翻译功能,然后是蛋白水解、光合作用、对紫外线B的反应、负调控、运输,等等;细胞组分中占比例最大的是叶绿体,其次是类囊体、线粒体、质膜,等等;分子功能中,占比例最大的前两个是ATP绑定和蛋白绑定,其次是丝氨酸型肽链内切酶,等等。(3)通过基因芯片杂交分析,在山核桃成花过程各时间点总共有232个基因表达发生变化,占总基因数的1.0%左右,有196个基因表达至少发生2倍及以上的变化,占总基因数的0.9%左右。其中,总共有106个上调基因和90个下调基因。山核桃成花过程中响应的基因主要涉及到细胞壁合成,细胞周期调控,细胞信号转导,胁迫响应,光合代谢,脂类代谢,蛋白代谢,碳水化合物代谢,转录调控,细胞凋亡,激素合成与运输,核酸代谢,物质运输,氨基酸代谢等等,从分子水平上证实了现代成花的细胞生物学理论。(4)根据已知FT同源基因的保守区设计特异引物和简并引物,对山核桃DNA进行PCR扩增克隆到4个FT基因片段,长度分别为382bp、82bp、80bp和152bp,其中第四片段已在GenBank注册(注册号FJ858260.1),命名为CcFT。CcFT经4次步移共获得1526bp片段,该片段含FT基因的第三外显子和第四外显子。其次克隆到2个AG同源基因片段,长度分别为131bp、122bp,在GenBank上注册(注册号FJ858261.1)命名为CcAG。还克隆到486bp的AP1同源基因片段。该片段包含2个内含子,长度分别为86bp和291bp,在GenBank上注册(注册号EU155118)命名为CcAP1。(5)山核桃CcLFY基因在山核桃不同部位的表达量有差异,花芽中表达最高,其次是幼茎,再次是幼叶,根中表达量最低。成花各时期CcLFY的表达情况大致是,3月上旬表达量较低,在3月18日表达量达到高峰,随后表达量下降。(6)用潮霉素初选获得转CcLFY基因烟草并经PCR检测鉴定确认。形态学观察表明,转基因植株较对照高大,开花提前,种粒大。(7)454测序共获得876,664个reads,样A和样B读数分别为431,759和444,905。平均长度为332bp。序列拼接后样A和样B分别有25339和26935个contigs。从样A和样B的contigs中分别获得15,085和16,387个ORFs。样A与样B特异序列(B与A)分别为6,138和7,346个;两者共有序列(AI B)分别为19,201和19,589个;两者共含不重复的contig序列(AU B)最小为32,685个。对样A与样B共七个集合(A、B、A+B、AU B、AI B、B、A )进行功能注释。在集合AU B共32,685个contigs中,无功能注释的有1,506(4.6%)个,有功能注释的有31,179(95.4%)个。把这些有功能注释的contigs分为生物进程(Biological Process)、细胞组分(Cellular Component)和分子功能(Molecular Function)3种类型,其数量(比例)分别是22,515(68.9%)、21,788(66.7%)、24,037(73.5%)个。其它六个集合的相似性序列的功能注释和分类与上述情况基本一致。对四个集合(B、A、AU B、AI B)的contigs进行了数据库比较的代谢分析。与拟南芥KEGG数据库相比,约有5~8%的contigs有代谢途径;与杨树KEGG数据库相比,约有8~13%的contigs有代谢途径。获得的已知成花候选基因有:GI、AP1、AP2、EMF1、TFL2、EMF2、FLC、PAF1、DET1、FRI、PLE、PI、HD1、AGL24、VRN1、SOB3、CRY1、FCA、FKF1、PIE、PIE1、AGL75,等等。山核桃雌花成花可能存在多条途径。从样A共25339个contigs中,共检出792个含有SSR的序列,占3.1%;从样B共26935个contigs中,共检出783个含有SSR的序列,占2.9%。更多还原

【Abstract】 Process of flowering is very vital in lifetime of plants, it is also the turning point that plants development from vegetation to generation, and from sporophyte to gametophyte. Many researchers have worked on the mechanism of flowering for around a century.Hickory(Carya cathayensis Sarg., Carya Nutt., Juglandaceae) is one of important economic trees, which is also one of very important oil tree plants and has become one of the resource of income for indigene in Zhejiang Province of China.Hickory has a long vegetative period, and it needs nearly 10 years for the first fruit set. The flowers of hickory are monoecism. Its staminate flowers are catkin, and female flowers spica. Flowering (staminate flowers and female flowers) of hickory is different in space and time. Because of the importance of flowering for female flowers, which decides to the yield, more attention should be paid to the flowering mechanism.Olefin slices about different period of time of flowering were observed through scanning electron microscope for the scale of studying the development of staminate flowers and female flowers. The technique of cDNA-AFLP and genechip were exerted to study the network control system of flowering. The important homologous genes on flowering, FT, AG, AP1 and so on, were cloned through the technique of PCR; The mechanism of flowering was studied from the point of view of anatomy and molecular biology. And also flowering control was studied with the use of the means of pulling branches and spraying plant growth regulators. The results were as follows:(1) The formation and development of female and male flower of hickory was mastered in anatomy. Androecium anlages form in the top of male flower anlages in the last ten-day of March. Stamens come into being and farther differentiate in the middle ten-day of April. In the last ten-day of April, pollen sacs forms from anther and microspore mother cells forms into tetrad spores with miosis. Then, tetrad spores disaggregate and produce one-cell microspores. In the first ten-day of May, two-cell microspores come into being. The development of female flower mainly accomplish from the last ten-day of March to the middle ten-day of May. The course can divided into five periods: the period of undifferentiation, pre-differentiation, inflorescence differentiation, involucrum formation, and gynoecium formation. In the early days of differentiation, the growing point goes semi-circle. In the period of inflorescence differentiation, three flowerets anlages come into being. In the period of involucrum formation, one involucrum and three bracts forms around the floweret. In the period of gynoecium formation, the flowerets continue to develop, and the embryo-sac comes into being in this period. The ovule type of hickory is orthogropous with single integument and thick nucellus. The female gametophyte is an embryo-sac with eight-nucleolus or seven-cell.(2) 278 TDFs during flowering in hickory were obtained by cDNA-AFLP technology. The 278 genes were composed of 65 genes of unknown function and 213 genes of known function. The 213 genes of known functions were involved in cell signal transduction, cell growth, senescence and death, cell differentiation and plant mature, plant hormone synthensis, stress and resistance, material transport, photosynthesis, material and energy metabolism, mineral assimilation and organic synthesis, photomorphogenesis, which play a direct or indirect role in hickory flowering process. The expression patterns of these genes were confirmed by Real-time RT PCR analysis. Both are basically consistent, which confirms the reliability of the results of cDNA-AFLP. There are 213(76.6%) annotated TDFs and 65(23.4%) non-annotated ones. Of annotated TDFs, the number or percentage of Biological Process, Cellular Component ,and Molecular Function are 158(54.7%), 211(73.0%), 173(59.9%), respectively. Of biological process, the turn of percentage in descending order is oxidation reduction, translation, proteolysis, photosynthesis, response to UV-B, negative regulation, transport, etc. Of cellular component, the turn of percentage in descending order is chloroplast, integral to membrane, thylakoid, mitochondrion, plasma membrane, etc. Of molecular function, the turn of percentage in descending order is ATP binding, protein binding, serine-type endopeptidase, etc.(3) The expression patterns of total 232 genes during hickory flowering process were obtained by microarry, which accounted for about 1% of total genes. 196 genes with at least 2-fold changes were obtained, which accounted for about 0.9% of total genes. Among them, there were 106 upregulated genes and 90 downregulated genes. The genes during flowering in hickory were invloved in cell wall synthesis, cell cycle regulation, cell signal transduction, stress response, photosynthetic metabolism, lipid metabolism, protein metabolism, carbohydrate metabolism, transcription regulation, apoptosis, hormone synthesis and transport, nucleic acid metabolism, material transport, amino acid metabolism, etc., which confirmed in molecular level the modern flowering theory of cell biology.(4) We designed the special primers according to the conserved sequence of the known FT, AG and AP1 homologues and cloned four FT, two AG and one AP1 fragments, respectively. The length of the four FT sequences are 382bp, 82bp, 80bp and 152bp respectively. The 152bp sequence (GenBank accession number: FJ858260.1) named CcFT, which included the third and fourth exons. The length of two AG homologues fragments are 131bp and 122bp (GenBank accession number: FJ858261.1) named CcAG. One AP1 homologue fragments (GenBank accession number: EU155118) were cloned and named CcAP1, which included two introns (86bp and 291bp).(5) The gene expression of CcLFY in different position in hichory are different. The highest expression was noticed in blossom buds, followed by young stems and young leaves, and the lowest expression was in roots. In the flowering process, CcLFY expression generally was a low expression in early March, a peak in March 18, followed by a decline as time progressed.(6) Both hygromycin assays and PCR amplification demonstrated that CcLFY was integrated into T0 genomes. The Morphological observation showed that the transgenic tobacco plants that survived under natural condition were tall, early flowering and seed grain larger than the wild-type controls. (7) 876,664 reads are derived by 454 sequencing from two samples labeled as A and B in hickory. There are 431,759 and 444,905 reads in sample A and B, respectively. After sequence aligning, there are 25,339 and 26,935 contigs in sample A and B. And 15,085 and 16,387 contigs are derived fromsample A and B, respectively. The number of specific contigs in sample A and B are 6,138 and 7,346. The number of common contigs in the two samples are 19,201 and 19,589, respectively. And all contigs number in both samples is 32,685. Seven sets, A, B, A+B, AU B, AI B, B , A , are annotated. For example, there are 31,179(95.4%) annotated contigs and 1,506(4.6%) non-annotated ones in set AU B. For annotated contigs, there are 3 type named Biological Process, Cellular Component ,and Molecular Function which contig number or percentage are 22,515(68.9%), 21,788(66.7%), 24,037(73.5%), respectively. The rest 6 sets are similar to AU B. Pathway analysis are implemented for B , A , AU B, AI B sets. There are 5~8% contigs have pathways, in which Ncotinate and nicotinamide metabolism, oxidative phosphorylation, steroid biosynthesis have higher percentages than others, comparing to KEGG Arabidopsis thaliana database. And there are around 8~13% contigs have pathways, in which purine and pyrimidine metabolism have higher percentages than others, comparing to KEGG Populus trichocarpadatabase. Several known flowering candidate genes are derived, such as GI, AP1, AP2, EMF1, TFL2, EMF2, FLC, PAF1, DET1, FRI, PLE, PI, HD1, AGL24, VRN1, SOB3, CRY1, FCA, FKF1, PIE, PIE1, AGL75. It’s suggested that there are several flowering routes in hickory. Eventually, 792 (3.1%) and 783 SSRs (2.9%) are derived from sample A and B, respectively.更多还原