【作者】 黄晋玲；

【导师】 李炳林；

【作者基本信息】 山西农业大学 ， 作物栽培学与耕作学， 2003， 博士

【摘要】 植物细胞质雄性不育(cytoplasmic male sterility，CMS)是一种普遍的生物学现象。研究其发生的机理有助于促进细胞质雄性不育在作物杂种优势上的利用、了解线粒体和叶绿体遗传系统的特点和核质两套遗传系统的相互作用规律。本研究对晋A细胞质雄性不育系进行了小孢子发育的超微结构观察、生理生化分析、线粒体和叶绿体DNA分子标记和育性恢复的研究，主要结果如下： 1．我们已对晋A细胞质雄性不育系进行了细胞生物学基础方面的研究，研究结果表明：晋A不育系的败育时期主要在造孢细胞增殖——小孢子母细胞形成时期。小孢子发育的超微结构观察发现，晋A不育系在造孢细胞增殖时期即表现出败育迹象。绒毡层细胞质膜断裂破碎，细胞质解体，线粒体、质体等细胞器解体，绒毡层细胞提前解体退化。并可看到明显的细胞质和细胞核浓缩现象，因此我们认为，小孢子败育过程中的绒毡层细胞解体是一种与PCD有关的有序过程。 2．酶活性的变化与育性有着密切的关系。我们对与线粒体有关的过氧化物酶(peroxide)、超氧化物歧化酶(superoxide dismutase)，细胞色素氧化酶(cytochrome oxidase)和琥珀酸脱氢酶(succinic dehydrogenase)进行了酶活性的分析，结果表明：在花粉发育的各个时期不育系的酶活性比保持系明显要低。这表明了不育系与其保持系之间体内代谢强度的差异，也说明酶活性的变化与雄性不育现象有着直接的关系。 3．线粒体基因组的变异与细胞质雄性不育密切相关。而叶绿体同线粒体一样，具有独立于核基因组以外的遗传系统，因此在研究细胞质雄性不育机理时，也应将其作为核质互作导致植物雄性不育的原因之一。本研究采用5对叶绿体和3对线粒体通用引物对(universal primer pairs)对晋A不育系及其保持系、104-7A和哈克尼西棉不育系进行了基因组DNA的PCR扩增，都没有检测到多态性，但扩增产物用18种限制性内切酶酶切后，发现8个有多态性的叶绿体CAPS(Cleaved Amplified Polymorphic Sequences)标记，而线粒体之间无多态性。用线粒体中常见的基因DNA作探针对上述材料的基因组DNA进行了RFLP(Restriction Fragment Length Polymorphism，限制性片段长度多态性)研究，在晋A不育系与其保持系之间和不同的不育系之间都检测到了多态性。RFLP方法可用于棉花胞质的快速鉴定分类。带型差异反映了DNA水平的差异，这为寻找晋A不育系的相关基因提供了线索。同时，从分子水平上揭示了晋A不育系是一种具有新的胞质的细胞质雄性不育系。 4.细胞质雄性不育系及其保持系、恢复系的选育是杂种优势利用的基拙性研究工作。经过5年8代选育，我们已经转育出巧个稳定的不育系及同型保持系。研究表明，晋A不育胞质对F，的育性有一定程度的负效应。然而，这种负效应可以通过优良不育系和强恢复系的选配得以克服或减轻到不显著水平。通过采用测交筛选和系统选育相结合的方法，已选育出具有明显的产量优势的杂交组合，产量超过推广良种“晋棉12”。为晋A不育系的高效利用提供了育种基础。更多还原

【Abstract】 Plant cytoplsmic male sterility (CMS) is a common phenomenon in higher plants and is manifested as the inability of a produce viable pollen. CMS is a trait of economic importance in the production of hybrid seed from crop plants as it eliminates the need for emasculation. A correlation between CMS and unique arrangements of the mitochondrial (chloroplast) genome has been found. CMS systems make excellent models for studying nuclear-cytoplasmic interactions because fertility restoration relies upon nuclear-encoded gene products that suppress cytoplasmic dysfunction. In the study reported here. Ultra-structural observation on the microsporgenesis, analysis of physiological and biochemical bases, CAPS and RFLP analysis for mitochondrial DNA and chloroplast DNA, as well as in Jin A CMS line. The main results are summarized as blow.1. we had found that the auxocyte abortion of Jin A took place at the stage of sporogenous cell division-microspore mother cells formed. Through ultra-structure observation, it is found that the microspore of Jin A began to abort at the stage of sporogenous cell division. During the abortion period of the CMS line, the ceil membrane of microspore and tapetum dissolved, cytoplasm degenerated, the cell organelles such as mitochondria and plastid and the tapetal cells became degenerated and disorganized. In addition , it is found that nuclear and cytoplasm began to concentrated in tapetal cells, these are morphological features of PCD. So, we think that tapetal cells disorganization may be one type of PCD that occurs in plants. Certainly, these must be proved on molecular level.2. A correlation between CMS and varies of activity of enzymes. The activity of four enzymes with mitochondrial function in cotyledon, leaf and anther of Jin A cytoplasmic male sterile and in those of its maintainer line. The results showed that activity of peroxide (POD), cytochrome oxidase (COD), superoxide dismutase (SOD) and succinic dehydrogenase in the anther of the sterile line were lower than those in the fertile line; but such differences were not found between the leaves of the sterile line and the fertile line, and only the activity of succinic dehydrogenase was lower in the cotyledon of the sterile line than that of fertile line.3. CMS is maternally inherited trait. Both the chloroplast and the mitochondrial genomes can be maternally inherited in higher plants; however, to date, only mitochondrial genome mutations have been correlated with cytoplasmic male sterility (CMS). A correlation between CMS and unique rearrangements of the mitochondrial genome has been found. Cleaved Amplified Polymorphic Sequence (CAPS) wassuccessfully applied to analyze the organelle composition among Jin A its maintainer 104-7A and sterile with cytoplasm of Gossypium harknessii. Five chloroplast and three mitochondrial universal primer pairs were used. All mitochondrial primer pairs (nad I exon B-nad 1 exon C,18SrRNA-5SrRNA, nod exon 1-nad 4 exon 2) and four chloroplast primer pairs ( rbcL-rbcL, rbcL-PSA I, TrnH-TrnK, TrnD-TrlT) of the five ehloroplast primer pairs, were efficiently amplified, but no polymorphism was detected, when the PCR products were digested by eighteen restriction endonucleases, including, Alu I , Bshl236 I. BsuR I, Csp6 I, Hin6 I, HpaII. Rsa I, Taq I Trul1 . Msp I, EcoR.I. EcoRV. Pst I, HindIII BamH I . Beg I . Beg II Xba I and Sal I respectively, eight polymorphic cpDNA-CAPS markers (rbcL-PSA I / Bshl236 I, rbcL-PSA I / BsuR I, rbcL-PSA I / Hin6 I, rbcL-PSA I / Rsa I, TrnH-TrnK/ Bshl236 I, TrnH-TrnK/ Csp6 I, TrnH-TrnK/ Rsa I, TrnH-TrnK/ Taq I ) were found and no mtDNA-CAPS marker was found. In this research, four lines of cotton were used for mitochondrial RFLP, including JinA,104-7A,the cytoplasm of G. harknessii Brandegee three types of cytoplasms. The plentiful polymorphism has been detected in some probe/enzyme combinations (atp9/HindIII,cox II/BamH, cox II /EcoR I, coxII/EcoR V). The RFLP results can be used for classification of CMS lines quickly and also gives更多还原