【作者】 丁安明；

【导师】 孙玉合；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2014， 博士

【摘要】 PPR基因家族在植物生长发育过程中至关重要，参与质体基因转录后加工、调控细胞质雄性不育基因的表达、调控胚胎发育等。番茄与烟草基因组计划的完成，为其PPR基因家族的全基因组鉴定奠定了基础。细胞质雄性不育恢复基因（Rf）除个别外均编码PPR蛋白，是PPR基因家族的成员。在农业生产中，多利用雄性不育系生产烟草杂交种。但是，目前尚未在普通烟草中发现不育系的恢复系，也未见有烟草雄性不育/恢复作用机理的相关研究报道。本研究分析了番茄和绒毛状烟草PPR基因家族，克隆了绒毛状烟草中的Rf相关基因，不仅对其进行了在不同品种不育系中的过表达分析，还对K326进行了RNAi研究，探讨其与烟草雄性不育发生的关系，为烟草雄性不育/恢复机理的研究提供参考。获得的主要结果如下：（1）在番茄基因组鉴定了471个PPR编码基因。根据保守结构域将其分为P和PLS亚家族，各占序列数目的一半；PLS亚家族又分为PLS、E、E+和DYW四类。该基因家族分布在番茄12条染色体上，其中约60%的基因无内含子结构，平均每个基因含有1.03个内含子；约70%的PPR基因具有RNA结合活性。（2）在绒毛状烟草基因组鉴定了487个PPR编码基因，其中P和PLS亚家族各264和223条，PLS亚家族包含PLS、E、E+和DYW四个亚类型各22、76、56和69条序列；各个结构域高度保守，与番茄相比，S结构域的变异率最小，为12.9%，E+结构域最大，为29.0%。该基因家族较均匀的分布在绒毛状烟草12条染色体上，其中61%的基因无内含子结构，平均每个基因含有内含子个数为1.2个；74%的PPR基因具有RNA结合活性。（3）从绒毛状烟草基因组克隆了6条Rf基因相关序列，命名为NtomRf1~6，GenBank编号分别为KF770956~KF770961。其中，NtomRf2和NtomRf6存在碱基删除突变；其它4条为完整读码框，但NtomRf5包含3个PPR结构域缺失，且其在不育系MS K326中存在C/A颠换和T缺失突变，导致3’非编码区通读。NtomRf1、3、4、5均编码含有10~13个PPR结构域的PPR蛋白，定位于线粒体。6条Rf序列在检测的组织中均有表达，但存在表达偏好；其在MS K326中的表达量高于其保持系K326。（4）选择基因NtomRf4和NtomRf5构建了过表达载体，并分别转化不同烟草品种的不育系，其转基因植株阳性率为66.3%。NtomRf4和NtomRf5基因在过表达阳性植株不同组织中的表达量提高了1.9~3.7倍，但是却均不能使MS K326等不育系的雄蕊正常发育，也即不能使其恢复育性。说明MS K326及其他烟草不育系品种不育性发生的机制非常复杂，不能为单一Rf基因恢复。（5）构建了NtomRf4和NtomRf5基因特异序列的干扰载体，转化K326得到转基因植株的阳性率为100%。NtomRf4和NtomRf5基因在RNAi K326阳性植株中的表达量下降了57.1%~85.3%，但仍有可检测的表达量，可能是其花器官和雄蕊发育正常，且能产生花粉的原因之一。下一步收获T1代种子后，进行发芽实验，观察其发芽率是否降低或者T1代植株是否不育。更多还原

【Abstract】 PPR gene family plays essential roles in plant development, such as participating in RNAprocessing in plastid, manipulating CMS-related genes and regulating embryonic development, etc. Theaccomplishment of genome project of tomato and tobacco fixed foundation for identification of PPRgene family in these plants. All Rf genes encode PPR proteins besides several exceptions, which belongto the PPR gene family. In agriculture production, CMS lines were used to produce hybrid in tobacco.But until now, no restorer lines were found for CMS in tobacco, and no reports about the mechanism oftobacco CMS/Rf system were published. In this research, PPR gene family in tomato and N.tomentosiformis were identified, and Rf-like genes in tobacco were cloned. Overexpression of thesegenes was carried out in tobacco MS lines of different varieties, and RNAi in K326was also studied.The main results were as follows:(1). A total of471PPR genes were identified in the tomato genome, which were devided into P andPLS subfamilies, each accounting for half of the family. PLS could be classified to PLS, E, E+andDYW subclasses. The PPR genes distributed on the12tomato chromosomes,60%of which lack intronsand70%process RNA-binding activity. Each PPR gene harbors1.03introns averagely.(2). A totall of487PPR genes exist in the N. tomentosiformis genome. P and PLS subfamilycontain264and223sequences, respectively. And the223sequences harbor22PLS,76E,56E+and89DYW subclasses. The PPR motifs are highly conserved. Compared to tomato, S has the least variationrate of12.9%, while E+has the highest of29%. The PPR genes distributed on the12tobaccochromosomes more evenly than tomato,61%of which lack introns and74%process RNA-bindingactivity. Each tobacco PPR gene harbors1.2introns averagely.(3). Six Rf-like genes named NtomRf1~6were cloned from N. tomentosiformis, with genbank No.of KF770956~KF770961. Base-deletion mutations happened to NtomRf2and6, while the other fourORFs were intact. However, NtomRf5missed3PPR motifs, which harbors C/A transvertion and aT-deletion in MS K326, causing3’ UTR being translated. NtomRf1,3~5encode PPR proteins containing10~13motifs, which were located in mitochondria. All the Rf-like genes were expressed in tissuesexamined, whose expression levels were higher in MS K326than that in K326.(4). NtomRf4and NtomRf5were selected to construct overexpression vector, the transformationrate of which were66.3%averagely. The expression levels of NtomRf4and NtomRf5in positivetransgenic CMS plants increased1.9~3.7times, however, the stamens of the positive plants were stillabnormal and failed to produce functional pollen. The mechanism of CMS/Rf in tobacco was muchmore complicated than thought, which could not be restored by single Rf gene.(5). Gene specific fragments were amplified for NtomRf4and NtomRf5to construct gateway RNAivector, the percentage of positive transgenic K326plants of which was100%. Although the expressionlevels of the two genes in the positive plants were reduced57.1%~85.3%, the floral organ and stamenswere still normal, which could generate pollen. The F1seeds will be collected for germination tests.更多还原