【作者】 吴国良；

【导师】 张大鹏；

【作者基本信息】 中国农业大学 ， 果树学， 2004， 博士

【摘要】 本研究以普通核桃(Juglans regia L.)为试材，利用组织切片技术观察了核桃果实的组织学构造。由结果可知，核桃果实结构上可分外、中、内果皮三层，中内果皮为果肉的大部分，其中散生有多束维管束。内果皮的细胞在前期小而透明，后期迅速木质化而形成硬壳，其外的维管束组织高度发达呈网络状。发现内果皮(果壳)在发育过程中自始至终不存在维管束发生和分布，成为果实组织学上的天然隔离层。 利用透射电镜技术对发育过程中核桃肉质果皮及种皮细胞的超微结构研究表明，核桃肉质果皮韧皮部及其周围薄壁细胞的SE-CC复合体中，筛分子体积大于伴胞，二者之间存在胞间连丝并呈分枝状，筛分子一侧为单通道，伴胞一侧为分枝通道。伴胞细胞质稠密，富含各种细胞器，SE-CC复合体与周围韧皮薄壁细胞间未发现胞间连丝，形成了共质体隔离。在种皮韧皮部的超微结构中发现，种皮SE-CC复合体的伴胞体积大于筛分子，二者间存在分枝型的胞间连丝，SE-CC复合体与周围韧皮薄壁细胞之间存在有大量的胞间连丝，种皮薄壁细胞含有浓厚的细胞质和大量细胞器。因此认为核桃肉质果皮中SE-CC复合体的同化物卸载可能通过质外体途径进行，种皮内SE-CC复合体中的同化物卸载则采取共质体途径进行。活细胞荧光示踪试验结果表明，果皮中CF荧光染料被限制在韧皮部内而无卸出，种皮中的荧光染料则卸出至周围薄壁细胞中，说明在核桃肉质果皮韧皮部组织中存在着共质体隔离，同化物卸载采取质外体途径而种皮内采取共质体途径。 利用western blotting技术、胶体金免疫定位技术结合透射电镜观察，对核桃果肉韧皮部及其周围薄壁细胞组织中酸性转化酶进行了定位研究。结果表明：以果肉提取的总蛋白进行了酸性转化酶的western blotting试验，检测到一条60kD的酸性转化酶，果实发育前期(花后30天)，酶表达量迅速增加，后期(花后80天)表达量较前期大为降低。在核桃肉质果皮韧皮部SE-CC复合体及其周围薄壁细胞的细胞壁上都特异性地定位到了酸性转化酶(金颗粒)。在发育早期(花后10天)金颗粒密度较高，随着核桃果实的迅速膨大，金颗粒密度迅速增加，随后在果实发育后期(花后80天)，金颗粒密度大幅度降低。可以认为，核桃肉质果皮韧皮部SE-CC复合体上的同化物卸载主要采取质外体途径，定位于细胞壁上的酸性转化酶随发育阶段不同，酶活性表达量不同，但整个生长期内的定位区域没有变化。 研究了核桃肉质果皮发育过程中糖含量及其相关酶活性的变化，发现各种可溶性糖因种类不同而含量各异，酸性转化酶在果实发育前期活性很强，后期降低。更多还原

【Abstract】 The pathway of phloem unloading was studied in developing walnut (Juglans regia L.) fruit using a combination of tissue slice, carboxyfluorescein transport, immunogold electron microscopy, and immunoblotting assay. The results showed that the tissue structure of the fruit pericarp may be divided into three parts: exocarp, mesocarp and endocarp. The developmental stage can be divided into the early stage and late stage. The mesocarp was the main part of pericarp, and the cell of endocarp was small and transparent in early stage, and it became into hard shell that was made of lignification stone cells. The tissue of vascular bundles was well developed and became ereticulate vein. There were no found vascular bundles in the endocarp, so it was a natural layer of isolation in walnut fruit.The ultrastructure of phloem and its surrounding parenchyma cells in developing walnut fruit was for the first systematically investigated via transmission electron microscope during whole developmental process of the fruit. The results showed that plasmodesmata exist at the thickened part of cell wall between sieve elements (SEs) and companion cells (CCs) both in pericarp of fruit and pericarp of seed, with the plasmodesmata furcating at the CC side and keeping a single oriface at the SE side .In pericarp of fruit the SEs are larger than the CCs, while in pericarp of seed the SEs are smaller than the CCs. The CCs contain an electron-dense protoplasm and nucleus, and are enriched with mitochondrion, endoplasmic reticulum and Golgi apparatus or dictyosomes in their cytoplasm. There was almost no plasmodesmata between the SE-CC complex and its surrounding phloem parenchyma cells in the pericarp of fruit, but it was opposite in the pericarp of seed, resulting in the symplasmic isolation between them in pericarp of fruit, which may be associated to apoplasmic transportation, of course there were symplasmic links between them in pericarp of seed. Plasmodesmata were found numerous both between phloem parenchyma cells and between flesh parenchyma cells (non-phloem parenchyma cells), as wells as between phloem parenchyma cell and flesh parenchyma cell. The relationships between the ultrastructure of the fruit phloem and its surrounding parenchyma cells and the photoassimilate unloading pathway in developing walnut fruit were discussed. It was considered that the apoplasmic unloading may be the main pathway of the photoassimilates unloaded from SE-CC complex of pericarp of fruit, and that the apoplasmic and symplasmic pathways may coexist during postphloem transport of photoassimilates in developing walnut fruit.The phloem-translocated carboxyfluorescein remained confined to the phloem in the pericarp of fruit and oppositional, it diffused into the surrounding parenchyma tissues in the pericarp of seed after application on the treated petiole during the fruit developing. This provided a supporting proof for assays of ultrastructure. The immunoblotting of acid invertase detected a 60-kD polypeptide for acidinvertase, and moreover, the apparent amount of acid invertase also increased at the early developmental stage (30DAFB) and decreased at the late developmental stage (80DAFB). The immunogold electron-microscopy technique was also used to determine the subcellular localization of cell wall invertase in developing walnut fruit. The antibody against apple fruit acid invertase was used. The results showed that the cell wall invertase mainly in the cell wall and the two antibodies can recognize each other. The gold particles representing acid invertase were found to reside predominantly in the cell walls of sieve element / companion cell (SE-CC) complex, phloem parenchyma cells and other parenchyma cells. There were almost no gold particles at other subcellular compartments (such as protoplasm and vacuole). The density of immunogold particles was low at the early developmental stage (10DAFB), but obviously high at the after developmental stage (30DAFB) and lately, was low at the late developmental stage (80DAFB). The distribution更多还原