| Part I Gene cloning and expressional analysis of rice long glume mutantsThree long sterile lemma mutants, Oslg-1, Oslg-2 and Oslg-3 were screened from YixianglB (Oryza sativa L. subsp. indica) treated by ethyl methanesulfonate (EMS). In order to explore molecular mechanism and genetic basis of long sterile lemma development, mutants and wild type were analyzed in phenotypes of floral organs, allelism of three mutants, gene-related mapping, bioinformatics analysis and expression analysis by quantitative PCR. The main results were as follows.1. Oslg-1 mutant showed there were no significant difference involved in glume during the early development of panicle compared with wild type. However, the epidermal cells of glume presented significant difference at the mature stage. In mutant, epidermal cells of abaxial glume presented bumps and rough, odules formed axially alignment, more hairs appeared, more grooves formed on the surface, quite imilar to the structure of epidermal cells in lemma.2. Genetic analysis by F2 suggested that the mutant phenotype of Oslg-1 was controlled by one recessive nuclear gene. The Oslg-1 mutant gene was mapped between SSR markers RM5344 and RM20934, on the short arm of rice chromosome 7, with genetic distances of 1.11 cM and 0.82 cM, respectively, and with a physical interval of 246.3 kb.3. By analyzing and sequencing for the candidate genes in this genomic region, it was found that there was a single nucleotide change (T61A) in exon of LOC_Os07g04670 gene, which caused a missense mutation (Leu to His) in the encoded product. Detecting the gene OsLG in Oslg-2 and Oslg-3 mutants, and found that there was a single nucleotide change (T316A and T1 19C) in exon of OsLG gene, which caused a missense mutation (Trp to Arg and Leu to Pro) in the encoded product, respectively.4. The results suggested that candidate gene may regulate elongation of rice glume by the homologous gene sequence alignment and phylogenetic analysis. On the gene sequence alignment and homologous evolution analysis, g1 is similar and grandiglumis phenotype, So we support the sterile lemma floret degradation hypothesis, the gene mutations can lead to glume elongation.5. This study also analyzed expression levels involved in candidate gene and another glume gene PAP2 controlled glume trait by real-time PCR (qRT-PCR). The results showed that OsLG gene was expressed in roots, leaf sheath and panicle, and presented rather higher expression in panicle than that of in root and leaf sheath. By contrast, PAP2 gene just only was checked in panicle. Therefore, OsLG and PAP2 genes both possess tissue-specific, and PAP2 gene was stronger than that of OsLG gene. Besides, two gene expression is declining, which indicates that it has coordinated expression characteristics.Part II Rice stripel-2 and stripel-3 mutants encoding the small subunit of ribo nucleotide reductase are temperature sensitive and are required for chlorophyll biosynthesisTwo mutants stripel-2 (stl-2) and stripel-3 (stl-3) from 9311 (Oryza sativa L. subsp indica) treated by Ethyl methanesulfonate (EMS) were used in study. The mutant phenotype present retarded growth rate, and stably inherited. The main results were as follows.1. St1-2 and stl-3 plants exhibit a normal green leaf as wild-type before L2, and next they both presented chlorotic leaf form L2 until tillering stage. And then visible white-striped green leaves started to be expressed from the tillering stage until mature. Stl-2 presented more apparent white-striped leaf than stl-3 from the maximum tillering stage. Although, white-stripe leaves of st1-2 do not still disappear until mature, green slowly recovered with temperature increased. We investigated agronomic traits among mutants and wild type, such as plant height, number of tillers, numbers of spikelet, panicle length,1000-grain weight, setting rate. St1-2 and st1-3 significantly decreased than that of wild type. Besides, St1-2 was significantly lower for 1000-grain weight and Setting rate than that of stl-3.2. Photosynthesis for WT, stl-2 and stl-3 showed that WT is the highest than mutants as results expected, and ordered as follows:WT> st1-3> stl-2, in the whole development stage. WT for chla content is significant higher than that of mutants during the whole development stage. Chlb content for WT is just only slightly high than that of mutants in heading stage, and not differentiation in other stage. Stl-3 mutant for Chla and b contents is the higher than that of stl-2 in tillering and heading stage.3. We compared to the ultrastructures and number of chloroplast among blades of WT, st1-2 and st1-3 plants growth in jointing and heading stage, respectively. WT presented a considerable number of starch granules than that of st1-2 and st1-3 in jointing stage. But structure of chloroplast in mutants still displayed well developed lamellar structures equipped with normally stacked grana and thylakoid membranes, and there was no significant difference in the number of chloroplasts per cell between wild type and mutants. However, thylakoid structure of stl-2 appeared visibly loosely arranged compared to wild type in heading stage. Besides, st1-2 and st1-3 all presented a considerable number of osmiophilic acid particles than WT in jointing and heading stage.4. Using PCR-based markers, the st1-2 locus was initially mapped two simple sequence repeat (SSR) markers, between RM3438 and RM3431, on the short arm of chromosome 6. Stl-2 locus was finally narrowed to a 64-kb interval between Ch6-819-1 and Ch6-825-l. Four of 13 candidate genes possess function in this region in the Rice Genome Research Program. In the st1-2 mutant revealed that in the RNA small submit gene (designed RNRS1; LOC_ Os06g 14620) comprising only one exon, a signal-base change (G511T) occurred in the st1-2 allele, which caused a missense mutation, Val to Phe compared to wild. Genetic mapping of the stl-3 gene was performed using F2, were generated from a cross st1-3 (indica) mutant and 02428 (Japonica). And st1-3 also was a allele gene with stl-2, but locus of mutant was different from stl-2 compared to wild, which caused C685T, Leu to Phe.5. Protein structure prediction analysis shows that there are 15 alpha helix, no beta fold area. Conservative Val 171 mutations in alpha helix area. Leu229 loci in saccharomyces cerevisiae, mouse, people such as creatures are proline, sequences of the gene in rice and Plasmodium yoelii have the same loci. Although there are some differences between these sequences, but the secondary structure are consistent. Mutations cause phenotypic differences show that both the change of the amino acids caused changes in the structure of space.6. We studied genes expression directly involved in Chls biosynthesis pathway using real-time PCR in different development stages. The results showed that RNRS1 mainly affected Porphobilinogen synthase, Coproporphyrinogen oxidative decarboxylase, Protoporphyrinogen oxidase, Chlorophyll synthase, Chlorophyllide a oxygenase of chloroplast biosynthesis pathway, and pheophorbide a oxygenase of degradation pathway.7. In different growth phase with different temperature to deal with stl-2 and st1-3, the results said RNRS1 only regulates partial genes involved in photosynthesis, in L20/D16, L26/D22 and L30/D26 conditions, mutants RNRS expression in quantity lower than RNRL is more serious.But under the condition of L30 RNRL and RNRS expression quantity are low. Suggest that RNRS1 can control partial genes involved in photosynthesis. |
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