【作者】 马建辉；

【导师】 喻树迅；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2013， 博士

【摘要】 陆地棉（Gossypium hirsutum L.）是我国重要的经济作物和纺织原料，具有明显的杂种优势。目前，棉花杂交种的生产主要通过人工去雄、授粉和细胞核雄性不育的“一系两用”法这两种方法，然而这两种方法制种程序复杂，需要消耗大量的人力和物力资源，造成棉花杂交种的成本过高，限制了杂种棉的推广应用。因此，选育出一个适宜棉花杂交种生产的雄性不育系，成为棉花育种家的一个关键任务。本研究通过航天诱变选育出一个具有芽黄标记的棉花光敏雄性不育突变体，对其性状特性和败育机理进行了全面的分析，并构建了一个棉花花药全生育期的均一化全长cDNA文库，与数字化表达谱相结合第一次对棉花花药发育的分子机制进行了系统的分析，所得结果如下：1.本研究第一次通过航天诱变育种在中040029的诱变后代中得到了一株具有芽黄标记的棉花光敏雄性不育突变体，经过在河南安阳和海南三亚两地、四年的回交和嫁接选育，于2010年获得了能稳定遗传的突变体材料，并将其命名为中9106，中9106的发现与成功选育为棉花杂种优势的利用提供了一个良好的种质材料；2.中9106的子叶和真叶期的顶叶表现芽黄，通过形态学和叶绿素含量分析发现，随着植株生长其叶色逐渐转绿，发育至第五真叶时，其叶绿素含量和叶色均与野生型相近，所以通过一定的栽培措施，芽黄性状对材料长势的影响可以消除，能满足大田应用的要求；3.以中9106为母本，H559和中040029为父本，构建了两个分离群体，在安阳分别调查两个群体F1和F2代材料的性状，并进行统计分析，结果表明中9106的芽黄和不育性状受一对共同的隐性基因控制；4.分析中9106在安阳和三亚两地的育性变化和光温条件，并与人工气候室的处理结果相结合可得：中9106在光照周期为13至14.5小时的长日照条件下生长时表现不育，在光照周期为11至12.5小时，日平均温度大于等于21.5℃的短日照条件下生长时表现可育，能满足短日高温地区自交繁殖不育系，长日照地区进行杂交种生产的要求，从而能在棉花杂交种的生产过程中极大的减少制种程序、降低制种成本，加快棉花杂种优势的应用进程；5.本研究通过对中9106和中040029不同发育时期花药的细胞学观察发现，中9106的花粉在其发育的单核期开始发生败育，至成熟期花粉完全失活。进一步分析两个材料四分体时期和单核期花药的表达谱发现，长日照激活了中9106单核期花药的泛素蛋白酶水解途径，从而引起花粉粒细胞质物质的降解，使花粉粒失活，最终导致中9106在长日照条件下表达不育；6.本研究以中棉所36为材料，第一次构建了棉花花药减数分裂至成熟期的全生育期均一化全长cDNA文库，在3′进行测序得到9,896条高质量的EST，拼接得到6,643条unigene，并经过生物信息学和荧光定量分析，筛选得到了花粉壁合成相关基因和其它一些在棉花花药发育过程中起重要作用的生物学途径，为深入开展棉花花药的研究提供了大量的参考序列；7.本研究以中040029的四分体时期、单核期、双核期、成熟期花药，以及根、茎、叶、胚为材料，构建了8个数字化表达谱文库，并以cDNA文库中的花药相关基因作为参考序列，第一次对不同发育时期的棉花花药进行了表达谱分析，得到10,178个棉花花药表达基因，其中包括1,165个花药优势表达基因，并通过分析发现黄酮类化合物和维生素C-谷胱甘肽循环在棉花花药的发育过程中起重要作用，同时还发现了一些与有丝分裂和激素响应相关的基因，这些研究为进一步开展棉花花粉、花药发育的研究和败育机理的分析，提供了一个良好的平台。更多还原

【Abstract】 Upland cotton (Gossypium hirsutum L.) is an important economic crop and textilematerials in China. It has strong heterosis. Currently, hybrid seeds are produced mainlythrough hand emasculation-pollination or genetic male sterile techniques. Both methods needa large labor force, making the hybrid seeds more expensive and restricting the utilization ofhybrid seeds in upland cotton. So, creating a novel sterile line of upland cotton, which issuitable for hybrid seeds production, becomes a primary mission of breeders.In this study, we got a novel photosensitive sterile cotton with virescent marker throughspace mutation and further analyzed the characters and sterile mechanism of this mutation.Meanwhile, we first constructed a normalized, full-length cDNA library from cotton anthersand systematically analyzed anther development in upland cotton by digital gene expressionprofiling (DGE). The results were shown as following.1. We fist got a novel photosensitive sterile cotton with virescent marker from CCRI040029through space mutation. After a four-year selection by backcrossing and grafting inAnyang and Sanya, the characters of this mutant had been stable and it was named as CCRI9106. It will perform an excellent germplasm resource for the utilization of heterosis inupland cotton.2. Cotyledon and the top leaf show virescent. By the morphology and chlorophyll cotentanalysis, we found that the virescent leaf turns to be green gradually and is similar to wildtypewhen the top leaf grows to the fifth leaf. So, the virescent does not have a negative effect onplants grown with good field management.3. We constructed two populations of CCRI9106×H559and CCRI9106×CCRI040029. And the F1plants and F2plants were sown and investigated in Anyang. It showedthat both characters, virescent and fertility, were controlled by a single recessive gene.4. We fully analyzed the fertility in Sanya, Anyang and phytotron considering the lightcycle and temperature and found that the CCRI9106was sterile with a13-to14.5-hphotoperiod and fertile with an11-to12.5-h photoperiod when the temperature was higherthan21.5°C. Thus, it could be utilized as a sterile line in long-day areas to produce hybridseeds and as a maintainer line in short-day areas to produce sterile line seeds.5. Through cytological observation about the anthers of CCRI9106and CCRI040029, we found that the pollen grains showed abnormal at uninucleate pollen period under long-dayconditions. And the transcriptome analysis of anthers, which were at tetrad pollen anduninucleate pollen periods, was performed and it indicated that the ubiquitin-proteasomesystem is induced in anthers of CCRI9106at uninucleate pollen period by long-dayconditions, which results in the degradation of cytoplasm protein. So, the pollen grainslost their activities and CCRI9106performed sterile.6. We first constructed a normalized, full-length cDNA library of anthers, which werefrom tetrad pollen period to mature pollen period of CCRI36. Ninety-eight hundred andninety-six high quality ESTs, which assembled into6,643unigenes, were obtained from their3′-ends and were further analyzed. Through bioinformatic analysis, we found many pollenwall related genes and many important biological processes during anther development. Itprovides a mass of reference sequences for anther research in upland cotton.7. We constructed eight digital gene expression profiling libraries of anthers at tetradpollen, uninucleate pollen, binucleate pollen and mature pollen periods along with roots,stems, leaves and embryos. From the analysis, we found10,178anther expressed genes and1,165stage-enriched in anthers. In additional, we found the flavonoid and ascorbate-glutathione cycle should be important during anther development in upland cotton. And manymitosis and hormone response genes were also identified. It would lay a good foundation forthe studying of pollen or anther development and the sterility mechanisms in upland cotton.更多还原