【作者】 崔彬彬；

【导师】 续九如； 李云；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2009， 博士

【摘要】 细胞质遗传是指核外遗传,即质体和线粒体的遗传。国内外关于白杨派树种细胞质遗传至今未见很全面、系统的报道,开展细胞质遗传研究对杨树系统进化、遗传多样性、抗性研究及遗传改良和杂交育种工作具有重要的理论和实践意义。本研究以白杨派毛白杨、银白杨、中国山杨、响叶杨、新疆杨、毛新杨、银腺杨、银毛杨16个种及其三倍体杂种为材料,通过不同发育时期花粉细胞学观察、成熟花粉核酸酶检测、PCR-RFLP分子标记技术,围绕质体和线粒体及其DNA在花粉发育过程中的存在状况、变化规律与遗传机理进行了初步研究。同时,对不同亲本来源的三倍体毛白杨杂种叶绿体性状变异进行了分析,检测是否有细胞质遗传效应发生,取得一定的进展。主要研究结果如下:1)应用电镜结合DAPI-DiOC荧光法,发现小孢子第一次有丝分裂时质体由于极性分布导致初形成的生殖细胞已不含质体;线粒体DNA在雄配子发育和成熟过程中发生特异性的降解,但是一定数目的线粒体一直存在。研究结果为白杨派树种质体、线粒体母系遗传提供了确切的细胞学证据。同时,细胞学观察显示银腺杨少数质体及其DNA被排除和降解时期相对迟缓。2)利用DNA-SDS-PAGE方法在毛白杨、三倍体毛白杨、中国山杨和毛新杨成熟花粉中检测到了高活性的特异性核酸酶。其中,毛白杨、三倍体毛白杨花粉中检测到3个Ca²⁺依赖性花粉核酸酶（17KD,21KD,33KD）和1个Zn²⁺依赖性花粉核酸酶（26KD）;中国山杨和毛新杨花粉中检测到2个Ca²⁺依赖性花粉核酸酶（17KD、21KD）和1个Zn²⁺依赖性花粉核酸酶（26KD）。上述细胞器DNA的选择性降解很可能是由花粉特异性核酸酶引起的。3)利用PCR-RFLP技术对白杨派16个种及其F₁代的叶绿体和线粒体遗传进行了分析,结果表明:包括毛新杨×毛白杨、毛新杨×银腺杨等在内的22个白杨派杂交组合F₁代的叶绿体DNA为母性遗传,支持质体细胞学研究的结论;全部杂交组合线粒体基因片段的PCR扩增与酶切图谱完全一致,未发现多态性,表明白杨派线粒体基因序列高度保守。首次为白杨派树种毛白杨、新疆杨、中国山杨、响叶杨、银毛杨、毛新杨和银腺杨及其杂种的叶绿体母性遗传提供了直接证据。4)利用7个不同亲本起源的三倍体毛白杨杂种无性系和3个二倍体无性系对叶绿体大小、数目、单位体积叶绿体DNA含量和单个细胞叶绿体DNA含量等性状变异进行了研究。结果表明加倍前后毛白杨叶绿体性状的变化呈现多样性,并不完全因倍性的增加而增加,与亲本的关系密切相关。具体表现为大部分三倍体毛白杨杂种与二倍体亲本在叶绿体大小上差异不大;叶绿体数目则因倍数的增加而增加,其中卵细胞加倍获得的三倍体达到极显著水平;在单位体积、单个细胞叶绿体DNA含量方面,花粉加倍的三倍体比卵细胞加倍的三倍体表现突出;其中叶绿体大小与单位体积的个数呈负相关变化,单位体积叶绿体DNA量的增加与叶绿体大小和单位体积叶绿体数目关系不大。更多还原

【Abstract】 Cytoplasmic inheritance is a form of extranuclear inheritance,which includes plastid and mitochondria inheritance.So far there are no comprehensive and systematic reports on cytoplasmic inheritance of section Leuce in Populus at home and abroad.The research on cytoplasmic inheritance has theoretical and practical significance in the fields of phylogenetic evolution,genetic diversity,resistance research, genetic improvement and cross breeding of Populus.Sixteen species and clones in section Leuce, including P.tomentosa,P.bolleana,P.alba,P.davidiana,P.adenopoda,P.tomentosa×P.bolleana, P.alba×P.glandulosa,P.alba×P.tomentosa and their triploid hybrid clones were selected as samples.The plastids,mitochondria and their DNAs in the pollen development of section Leuce were studied by a combination of cytological detection of pollen development,pollen nuclease detection and PCR-RFLP analysis.Moreover,the chloroplast traits of triploid clones of Populus tomentosa from different hybrid parents were analyzed to measure genetic effects of the cytoplasm.By these experiments,the obtained major results and conclusions are described as follows:1) The observations of electron microscopic and DAPI-DiOC fluorescence microscopic have demonstrated that the formed generative cells were free of plastids because of extremely unequal distribution of plastids in the first microspore mitosis.The fluorescence observation of DNA showed that cytoplasmic（plastid and mitochondria） nucleoids gradually degenerated and disappeared during pollen development,but a certain amount of mitochondrion with complete structure still were present. These results provide precise cytological evidences of plastid and mitochondria maternal inheritance in section Leuce.At the meantime,relatively later eliminattion or degeneration of some plastid and their DNAs was founded in P.alba×P.glandulosa.2) High activity of pollen nuclease was found in P.tomentosa,triploid clone of P.tomentosa,P. davidiana and P.tomentosa×P.bolleana by DNA-SDS-PAGE method.Three Ca²⁺ dependent pollen nucleases（17,21,33 KD） and one Zn²⁺ dependent pollen nuclease（26 KD） were detected in P. tomentosa and triploid clone of P.tomentosa.Two Ca²⁺ dependent pollen nucleases（17,21 KD） and one Zn²⁺ dependent pollen nuclease（26 KD） were found in P.davidiana and P.tomentosa×P. bolleana.These results show that the activity of pollen nuclease may induce the selective degradation of organelle DNA.3) The inheritance of chloroplast and mitochondria DNA of 16 species and clones belonging to the section Leuce and their F₁ progenies was analyzed by PCR-RFLP analysis.Restriction fragment length polymorphism（RFLP） analysis has revealed that the chloroplast DNA in 22 F₁ hybrid individuals including（P.tomentosa×P.bolleana）×P.tomentosa),（P.tomentosa×P.bolleana）×（P.alba×P. glandulosa） is maternal inheritance.This supports the result of cytological analysis in plastid.Moreover, there was no variation among these 22 hybrids in the PCR amplification pattern and the restriction pattern,indicating that mitochondrial genes are highly conserved in section Leuce.These results provide direct evidences of maternal chloroplast in P.tomentosa,P.bolleana,P.davidiana,P.adenopoda,P. tomentosa×P.bolleana,P.alba×P.glandulosa and P.alba×P.tomentosa.4) The size,number,DNA content of chloroplast were measured in seven triploid clones of Populus tomentosa clones from different hybrid parents and three diploid Populus tomentosa clones.Results show that variations of chloroplast traits before and after the chromosome doubling incompletely increase with polyploidy increasing,are diversity and closely related with their parents’ characteristics.more detail as follows:there is an unobvious difference in chloroplast size between most triploid clones of Populus tomentosa and their diploid parents,while the chloroplast number increased with chromosome doubling.Especially the triploid Populus tomentosa clones from female gamete doubling reached the very significant level.Variations in chloroplast DNA content per unit volume or per cell in triploid clones of Populus tomentosa from male gamete doubling were more significant than those from female one.Chloroplast size was negatively related to chloroplast number per unit volume, Chloroplast DNA content per unit volume was not closely correlated with size and number per unit volume of chloroplast.更多还原