毛白杨雌配子核染色体加倍过程中质体和线粒体变异机理研究

A series of studies have demonstrated that the triploid Populus tomentosa were obtained from hybridization of 2n female gamete and 1n pollen has special traits than that from hybridization of 2n pollen and 1n female gamete, such as more chloroplast number, thick leaves and higher chlorophyll content. The reason for these phenomena is probably due to the doubling of cytoplasmic genetic material together with the doubling of nuclear chromosome induced by artificial mutation,furthermore the former could be inherited by the triploid hybrid produced from 2n female gamete and 1n pollen because of maternal heredity. To explore the mechanism, the 2n female gametes will be induced by colchicine and high-temperature. The materials are obtained from diploid Populus tomentosa. The 2n female gametes were isolated and selected by enzymatic digestion and mechanical dissection . Transmission electron microscopy ,LSCM and single-cell nested PCR technology are also being used to observe and determine quantitative cytoplasmic plastids and mitochondria morphology, number, distribution and the changes of DNA copy number before and after 2n female gamete chromosome doubling of the Populus tomentosa. In addition, immunofluorescence location method will be also used to detect the variation period of plastid and mitochondrial and indentify the function mechanism of cytoplasmic genetic material effected by microtubule system during mutation of Populus tomentosa 2n female gamete. These results will be helpfule to reveal variation mechanism of plastid and mitochondria during doubling of Populus tomentosa female gamete nuclear chromosome, and improve theories and strategies of cytoplasmic inheritance in Populus tomentosa ploid breeding.

多年研究发现由2n雌配子与1n花粉杂交比由2n花粉与1n雌配子杂交获得的三倍体毛白杨具有叶绿体数目多、叶片较厚和叶绿素含量高等性状，推测原因很可能是雌配子核染色体加倍时，引起了细胞质遗传物质加倍等变化，在杂交中这种变化随母系遗传进入到由2n雌配子与1n花粉杂交的三倍体中。为探究起因，本项目以二倍体毛白杨为材料，用秋水仙碱与高温诱导2n雌配子；采用酶解-解剖及镜检方法分离并筛选2n雌配子；应用透射电镜、激光共聚焦电镜和单细胞嵌套式PCR技术观察并精确定量毛白杨2n雌配子染色体加倍前后，其细胞质内质体和线粒体形态结构、数量、分布及其DNA拷贝数的变化；利用免疫荧光技术定位检测毛白杨2n雌配子诱导过程中质体和线粒体发生变异的时期以及微管系统对细胞质遗传物质变化的作用机制。以期揭示毛白杨雌配子核染色体加倍过程中质体和线粒体的变异机理，丰富毛白杨倍性育种中细胞质遗传的理论和方法。

在国家自然科学基金项目“毛白杨雌配子核染色体加倍过程中质体和线粒体变异机理的研究”资助下取得新进展。明确了毛白杨大小孢子母细胞减数分裂的进程以及与雌雄花芽外部形态的变化之间均存在一定的相关性；同时发现银腺杨大孢子母细胞减数分裂及胚囊发育进程与花芽的外部形态变化也存在一定的相关性。以新鲜的毛白杨雌花为材料，采用酶解-解剖法结合吸吹的方法分离出胚囊。筛选出分离毛白杨胚囊最佳的混合酶液的成分和浓度为：0.5%半纤维素酶、0.5%纤维素酶、0.1%果胶酶、1%BSA、10%甘露醇。在最佳酶液浓度条件下酶解胚珠2-2.5h，完整胚囊获得率高达8.7%左右。观察发现完整的毛白杨胚囊轮廓呈长椭圆形，大小为2.52µm左右，经透明处理后还可以在合点端观察到类似反足细胞的结构。以秋水仙碱和高温加倍处理的毛白杨和银腺杨雌花芽为材料，利用透射电镜技术观察雌配子加倍前后，其细胞质内质体和线粒体的变化情况。结果发现，毛白杨和银腺杨经过人工加倍处理以后，在雌配子细胞质内的质体、线粒体和高尔基体等细胞器的大小、数量和形态结构发生了变异，主要表现为个大、数量增多、形态结构多样化并且发生显著变化。在核染色体加倍成功获得的2n雌配子中，质体和线粒体等细胞器的变异现象更为明显和突出。确定在秋水仙碱人工诱导过程中，引起质体和线粒体变异时期发生在大孢子母细胞减数分裂时期，与秋水仙碱作用于细胞的最佳时期一致。建立了适合毛白杨胚囊微管免疫荧光的观察体系，即采用PEG包埋切片法，结合蛋清粘合剂粘合制片，不需要酶解处理，经过预处理后经过FITC免疫标记和PI复染的方法，可以得到毛白杨胚囊微管结构的清晰图像。为杨树雌配子核染色体加倍时会引起细胞质遗传物质加倍的假说提供了实验依据，揭示了毛白杨等白杨派树种雌配子核染色体加倍过程中质体和线粒体的变化规律。综上所述，本研究已经完成了原计划中的任务指标。