【作者】 郑诚乐；

【导师】 潘东明；

【作者基本信息】 福建农林大学 ， 果树学， 2008， 博士

【摘要】 锥栗（Castanea henryi Rehd.&Wils.）是我国特色果树资源,其种质资源丰富,遗传背景复杂,同时普遍存在空苞现象,造成产量和品质下降,影响该资源的有效利用和产业发展。本试验在前人研究基础上,通过对锥栗开展胚胎学、营养学、蛋白质组学、分子生物学及孢粉学研究,旨为锥栗种质资源的合理利用和解决空苞问题提供依据。主要研究结果如下:1、以锥栗品种‘黄榛’为材料,采用石蜡切片技术对锥栗大孢子发生、雌配子体形成和胚、胚乳发育过程进行了研究。结果表明:锥栗为雌雄异花,多心皮复合雌蕊,中轴胎座,通常子房8～10室,每室倒生二枚胚珠。锥栗珠心组织中的一枚孢原细胞直接发育形成大孢子母细胞,经减数分裂形成大孢子四分体,其中靠近合点端的功能大孢子发育成为成熟胚囊。胚囊发育类型为单孢子蓼型胚囊。双受精后,原胚历经球胚、心形胚、鱼雷胚和子叶胚等阶段发育为成熟胚。核型胚乳,种子无胚乳。研究还发现锥栗胚胎发育过程存在多种异常现象,如发育初期形成胚珠时子房室内部全空,受精完成后出现子房室完全干瘪,球形胚后期胚囊萎缩有黑色圈形物质环绕在周围等,并已确定这些现象发生的时期。这些都可能是导致锥栗空苞现象发生的原因之一。2、通过对锥栗不同品种果实生长发育规律进行系统研究,结果表明:锥栗的果实分球苞和坚果两部分,大部分品种的球苞内,仅着生坚果1粒,个别品种球苞内坚果数1～3粒。坚果形状、大小、重量,因品种而有较大差异。锥栗大部分品种在5月下旬至6月上旬谢花后,幼果开始形成,并逐渐生长发育。发育全过程可划分为2个阶段:前期即8月上旬以前,球苞增长迅速,而坚果重量增加较少,此前各阶段果实解剖可见,在8～10个心室中只见乳白色绒毛,仅在果顶处见一胚珠渐渐发育,其余变褐;后期即8月中旬以后,球苞体积增长缓慢,而单果重增加迅速,直至果实成熟,经解剖可知,胚珠快速发育成种子,白色绒毛逐渐消退,随着果实的成熟,肥厚的子叶占据整个果实空间。此期一旦胚珠发育异常,就导致空苞发生。不同锥栗品种间单果重量早期差异不大,后期差异明显。除个别品种外,多数品种的果实采收期较为集中。锥栗果实纵横径发育与单果重大小变化呈正相关。果实的生长发育动态呈“慢—快—慢”的“S”型曲线。通过测定锥栗果实发育过程刺苞中10种矿质元素含量的变化,结果表明,随着刺苞增大,坚果逐步发育,刺苞内N、P、Cu、Fe、Zn都表现出总体下降趋势。K先降后升,Mg先升后降,Ca、Mn、B呈现波动现象。所测定的10种矿质元素含量中,只有Zn和Cu含量达到极显著正相关,其他矿质营养元素之间的相关性均不显著,说明各种矿质元素之间不存在显著或极显著相互拮抗的作用。锥栗刺苞与果实均富含多种矿质元素,但同时期大多数元素含量差异明显,坚果中K、Fe、Mn、Ca含量明显低于刺苞中的含量,前三者含量大约只有刺苞含量的一半,Ca更低,果实含量不及刺苞含量的1/4。而果实中的Mg、B、Cu含量略高于刺苞含量,尤其是果实中Zn的含量是刺苞含锌量的2.2倍。这一结果说明,锥栗刺苞作为果实的组成部分,至少吸收贮藏一半以上的矿质营养,这对坚果发育及产量将产生重大影响。3、以锥栗8月8日正常发育和空苞的‘处暑红’品种为样品,建立具有高分辨率和稳定性的锥栗胚胎蛋白质的双向电泳体系,提取总蛋白质,进行双向电泳,用Image MasterTM 2D Platinum software软件进行分析,挖取其中的12个差异蛋白质点进行肽质量指纹（MALDI-TOFMS）图谱分析,获得了完整的肽指纹图谱,用MASCOT搜索NCBInr绿色植物蛋白质数据库,有4个蛋白质被成功鉴定。这4个蛋白质参与了细胞壁代谢、糖类和能量代谢、细胞骨架代谢过程。首次报道了锥栗的差异蛋白质组学研究,为进一步研究锥栗空棚提供了重要的蛋白质水平信息。4、利用ISSR分子标记对闽北地区22份锥栗资源进行基因组多态性分析,从100条引物中筛选的16条具有扩增多态性的引物共扩增出201条带,其中多态性条带193条,占总扩增条带的96%,平均每个引物产生12条带。ISSR标记揭示的22个锥栗种质的遗传相似系数变化范围为0.366～0.981,平均值为0.612。聚类分析可将22个锥栗种质进行分类。从亲缘关系聚类树状图可以看出,在遗传距离D=24时我们可以把锥栗分为两大类型。其中‘黄榛’为一大类,其果大是其它品种无法比拟的。当D=14时可以把供试材料划分为6类,基本反映出各类果实和刺苞的相似关系。分析供试锥栗种质资源的遗传相似系数可知,‘乌壳长芒’和‘温洋红’,‘红子榛’和‘紫榛’,‘吴榛’与‘麦塞子’它们相互之间的亲缘关系很近。5、通过对锥栗5个主要品种的雄花序及其花粉粒的若干性状肉眼与光学显微镜观测,结果表明:不同品种雄花序长度,花粉粒大小、形状及花粉管长度都存在差异,但其雄花序的总花簇数较接近,即雄花序较长的品种,其花簇密度较小,反之,则大。30-35℃温度条件最适于锥栗花粉萌发和花粉管的伸长,但低温有利于锥栗花粉的贮藏;不同浓度的赤霉素（GA₃）和硼酸对锥栗花粉的萌发和花粉管的伸长均有促进作用,其中赤霉素以50mg/L效果最好;硼酸以0.1%～0.5%为佳。室温下贮藏花粉以干燥、遮光条件为佳;贮藏5d的花粉发芽率最高。花粉电镜扫描结果进一步表明:锥栗花粉为小花粉,长球形,极面观三裂圆形,赤道面观有长椭圆形、宽椭圆形和菱形三种。锥栗不同品种花粉间存在差异,主要表现在花粉的大小、形态和纹饰上,‘黄榛’花粉最大,花粉条脊宽度和条脊间距值基本一致;花粉大小接近的还有4组。花粉纹饰则没有完全相同的两种纹饰,但品种内性状较稳定。本研究还就锥栗空苞问题进行了探索,结果表明,空苞发生时期是在胚胎发育阶段。影响空苞的因子主要有品种、营养与授粉受精等因素。‘油榛’等品种在许多产地空苞率均较高。利用RAPD、ISSR标记技术表明空苞率较高的品种亲缘关系较近。营养问题主要体现在锥栗大量开花、结苞以及刺苞发育等对坚果发育存在激烈的竞争关系,减少树体贮藏营养。授粉受精不良可能引起胚早期败育,没有完成受精而使栗实干瘦。此外,还发现在空苞样品中某些特异蛋白的消失,使得细胞内的物质运输、细胞运动、细胞的分化发育以及细胞分裂繁殖等生命活动都无法完成。更多还原

【Abstract】 Chinquapin（Castanea henryi Rehd.& Wils.）is a special fruit resource in China,with abundant germplasm resources and complex genetic background.The phenomenon of ’empty-seeded ness’ is common in chinquapin productions,causing the declines of yield and quality and affecting the efficient application and industry development of the resources.Based on former studies,the experiment was conducted to study the embryology,nutriology, proteomics,molecular biology and palynology of chinquapin,with a view to providing the basis for rational use of chinquapin germplasm rescources and overcoming problems of ’empty-seededness’.The results of major studies were as follows:1.Using ’huangzhen’ as the material and taking advantage of paraffin section technology, studies were carried out on the megasporogenesis,female gametophyte formation,the development of embryos and endosperm.The results showed that chinquapin was diclinous with polycarpous compound pistils,axile placentas,commonly 8 to 10 loculament and 2 anatropous ovules in each loculament.An archesporial cell in the nucellus tissue would directly develop into the megaspore mother cell that subsequently formed linear megaspore tetrads.The functional megaspore near the chalazal end developed into mature embryo sac,which was monosporic polygonum-type.After double fertilization,the proembryo would develop into a mature embryo going through globular,heart,torpedo and cotyledon stages.Endosperm is nuclear endosperm and it didn’t exist in seeds.The study also found that there were several anomalies in the development of chinquapin embryos.For example,the insides of loculi were fully empty in the early stage when ovules were forming;the loculament would completely shrivel after the fertilization and the embryo sac would wither and have black circular materials around in the later globular stage.The timings when these phenomena happened were also determined.All these may lead to ’empty-seeded hess’ phenomenon in chinquapin.2.The fruit growth and development of different varieties of chinquapin were systematically studied.The results indicated that chinquapin fruits could be divided into two parts as the bur and the nut.The burs of most varieties only bore one nut while some few bore one to three nuts. The shapes,sizes and weights varied from varieties to varieties.Most varieties of chinquapin shed flowers between late May and early June and then formed young fruits that would gradually grow and develop.The whole development process could be divided into two stages.In the early stage before early August,the bur rapidly grew while the increase of nuts’ weights declined.The anatomies of fruits in different early stages showed that only milky white hairs were seen in 8 to 10 loculi and only an ovule under development was seen on the top of fruits while the others turned brown;during the later stage after middle August,the increase of the volumes of prickly burs slowed down while the weight of single fruit increased rapidly before finally matured.The anatomies showed that ovules quickly developed into seeds with disappearing white hairs.As fruits matured,fleshy cotyledons would occupy the whole fruit space.During this stage,once ovules developed abnormally,the phenomenon of’dmpty-seeded hess’ would happen.The single fruit weights of different varieties varied little in the early stage but significantly during the later stage.Other than a few varieties,most varieties had concentrated harvest periods.The transverse and vertical development of Chinquapin fruits were positively related with the changes of single fruit weights.The growth and development of chinquapin fruits featured a S-shape curve representing ’Quick-Slow-Quick’ trend.By measuring the changes of the contents of 10 mineral elements in prickly burs during the course of development,the results indicated that as the prickly burs grew larger and nuts gradually developed,the contents of N,P,Cu,Fe,and Zn in prickly burs declined in a whole. Amongst 10 measured mineral elements,only the contents of Zn and Cu were extremely positively related to each other while there were no significant correlations between other mineral elements,demonstrating that there are no significant or extremely significant mutual antagonisms between various mineral elements.Both chinquapin prickly burs and fruits were rich in a variety of mineral elements,most of whose contents varied significantly in the same period.The contents of K,Fe,Mn and Ca in nuts were obviously lower than those in prickly burs,with the contents of the former three minerals in nuts half of those in prickly burs and the content of Ca in nuts one fourth of that in prickly burs.Fruits had a bit higher contents of Mg,B and Cu than prickly burs,especially the content of Zn in nuts 2.2 times than that in prickly burs. The results indicated that as a part of fruit,chinquapin prickly burs at least absorbed half of mineral nutrients,having a significant influence on the development and yield of nuts.3.By using ’chushuhong’ under normal development and dmpty-seeded ness ’chushuhong’ on August 8 as the samples,a two-dimensional electrophoresis system of high resolution and stability for chinquapin embryonic proteins was established to extract the total protein and conduct two-dimensional electrophoresis.Image Master TM 2D Platinum software was adopted in the analysis.Image Master TM 2D Platinum software was adopted in the analysis.12 differential protein spots were taken out for peptide mass fingerprinting（MALDI-TOF MS） analyses and a complete peptide fingerprinting was obtained.MASCOT was adopted to search the protein database of green plants on NCBInr with 4 proteins successfully identified.These 4 proteins involved in cell wall metabolism,carbohydrate and energy metabolism and cytoskeleton metabolism.The article has reported for the first time the differential proteomic study,providing important information of protein levels for further studies of dmpty-seeded ness phenomena in chinquapin.4.DNA polymorphism analysis was carried out on 22 chinquapin resources in northern Fujian by using ISSR molecular markers.A total of 201 bands were amplified by 16 primers chosen between 100 primers,including 193 polymorphic bands,accounting 96%of the total.On the average,each primer amplified 12 bands.Genetic similarity coefficients of 22 varieties of chinquapin germplasm revealed by ISSR markers ranged from 0.336 to 0.981,with an average of 0.612.These chinquapin germplasm could be classified by polymorphism analysis.The phylogenetic tree showed that chinquapin could be divided into two groups when the genetic distance D equaled 24,with ’huangzhen’ as a major class,which was second to none in fruit size. When D equaled 14,the experimental materials could be classified into 6 groups,basically reflecting the similarity relationship between various fruits and pricey burs.The analyses on genetic similarity coefficients of experimental chinquapin showed that there were close relationship between ’wukechangrnang’ and ’wenyanghong’,’hongzizhen’ and ’zizhen’ as well as ’wuzhen’ and ’maisaizi’.5.Naked-eye and microscopic observations were conducted on some properties of male inflorescences and pollens of five major varieties of chinquapin.The results indicated that the lengths of male inflorescences,the sizes and shapes of pollens as well as the lengths of pollen tubes varied in different varieties,while the total numbers of blossom clusters of male inflorescences were close.The longer male inflorescences,the lower densities of clusters,and vise versa.The temperature between 30 to 35℃was most suitable for the germination of chinquapin pollens and the extension of chinquapin pollen tubes while low temperature was favorable for the storage of chinquapin pollens.Different concentrations of gibberellin（GA₃） and boric acid could promote the germination of chinquapin pollens and the extension of chinquapin pollen tubes,with 50 mg/L of gibberellin the most effective and 0.1%to 0.5%of boric acid having better effects.Dry and shading conditions were better for the storage of pollens in arnbient temperatures.The pollens that had been stored for 5 days had the highest germination rate.The results of pollens scanning by electron microscopes further demonstrated that chinquapin pollens were small pollens in prolate shape,with 6-lobate-circular in polar view and oblong-elliptic,broad-elliptic,rhombic shapes in equatorial view.Pollens of different chinquapin varieties were different,principally manifested in the pollen sizes,shapes and ornamentations."huangzhen" pollens were the largest ones with basically the same ridge width and space.There were four groups of pollens with similar sizes.There were no ornamentations exactly the same.However,the properties of different varieties were stable.The reason of ’empty seeded ness’ on chinquapin also discussed in this study.The result showed that the ’empty seededness’ on chinquapin occurred at the stage of embryo’s growth.The factors of variety,nutrition,pollination and impregnation can be regared as some reasons of ’empty seededness’.Variety of ’youzheng’ had relative high ’empty seededness’ at some producing areas.Varieties with high ’empty seededness’ had close relationship by using the technologies of RAPD and ISSR.Mass a blooming,and the development of prickly burs had drastic competition on nutrition,as a result,the storing nutrition of tree reduced.Bad pollination and impregnation may caused embryo’s abortion at the forepart,those nonimpregnation had ’empty seededness’ in the end.Besides,we also discovered that the disappearing of certain differential protein in ’empty seededness’ resulted in several life activities stopped,for example,the matters transportation in cell,cell moving,cell differentiation and growth,cell division and propagating.更多还原