Identification Of PPR Gene Family In N. Tomentosiformis And Tomato And Function Analysis Of Rf-related Genes In Tobacco

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.