| Leaf is an important organ for photosynthesis and the major source for carbohydrates. Photosynthetic efficiency is closely related to chlorophyll content, the shape and structure of chloroplast, the place for plant photosynthesis, which will affect the photosynthesis, growth and development of the plant. Leaf color-related mutants are important materials to study the synthesis, regulation and degradation of the chlorophyll, the growth and development of the chloroplast and the signal transmission between chloroplasts and the nucleus. Rice is the cereal crops which has smallest genome size, and as a model plant, it is used to research the genetic and development of the cereal crops. Following the completion of the rice genome sequencing project and the continuous development of correlation technology, and the constructing of all kinds of rice mutant library, it is an essential task in post-genome era to identify the important genes in specific biological process and learn to know the functions by using forward genetics and reverse geneticsIn this study, there were three leaf color-related mutants which were selected from restorer lines and derived from Ethylmethyl Sulfone (EMS) mutagenesis, respectively, they were natural mutant Chy (chlorophyll) with the semi-dominant yellow-leaf trait, natural mutant stll (stripe11) with the temperature-sensitive white-stripe leaf trait and the pgl4(pale green leaf4). We studied the morphological identification of the mutants, the electron microscopy of the chloroplast development, genetic analysis, the measurement of photosynthetic and physiological traits, the mapping and cloning of genes, the constructions of each vector, the complementation test and the regulation of the genes express. And the main results were summarized as follows:1. Genetic analysis indicated that Chy belongs to a semi-dominant genetic model which was controlled by one gene locus. Chy lacked chlorophyll a and b, and the development of chloroplast obstructed, then the shape of chloroplasts were abnormal and into vacuole shape. There were not complete thylakoids in Chy which was lethal at three-leaf stage. Heterozygote Chy/+showed yellow-leaf, and the content of chlorophyll a and b were both lower than the wild-type at two-leaf stage, seedling stage, tillering stageand heading stage, respectively, but the ratio of chlorophyll a and b of Chy/+was higher than the wild-type at the stage above and stayed consistent, the development of the chloroplast disordered partially. The plant height, growth stages and the grain number per panicle showed significant difference between Chy/+and the wild-type. The CHY was located on chromosome3between the markers RM1164and RM8208, which was near the centromere, then the candidate region was delimited to a2.1Mb physical distance on either side of centromere by using the newly developed11pairs of molecular markers.2. We found that the CHLI gene (LOC_Os03g36540) which encoded Mg-chelatase I subunit on the BAC AC084767may be related to the mutants Chy. The result of sequence analysis showed that there was a single substitution (G to C) in coding region of CHLI, which resulted in the amino acid changed from glutamate into glutamine, the substitution was in the conserved AAA+domain, Chy and the mutant chl9were allelic, then CHLI considered as the candidate gene for CHY. We connected the6.9Kb DNA fragment from CHY including the promoter region,5’non-translated region and3’ non-translated region to the complementary vector, and transferred to Nipponbare by agrobacterium-mediated approach, and we got5positive plants. Compared with the control, in To generation the transgenic plants did not show significant difference in leaf colour, while in T1generation, there were a quarter of (pale) yellow leaf individuals, then we crossed the (pale) yellow leaf individuals with chl9, and the analysis on the F1showed that the segregated (pale) yellow leaf individuals were transgenic complementary plants rather than the leaf color-related mutants which were generated from the tissue culture. The complementation test further identified that the CHY was the CHLI. CHY showed dose effect. Semi-qRT-PCR analysis indicated that CHY down-regulated the CHLH, PORA and CAO-1expression, and CHY affected indirectly the development of chloroplast by down-regulating the CHLH expression. Meanwhile, CHY also down-regulated expression of CablR, Cab2R, PsbA and RbcL encoded the proteins in chloroplast and up-regulated PsaA expression.3. Genetic analysis indicated that pgl4was a mutant with a recessive gene controlled, the leaf showed virescent at three-leaf stage, after which only the newly growing leaf showed virescent, and the leaf color gradually become green with the leaf completely unfolded. At heading, the chlorophyll content was not significant difference between pgl4and the wild-type, while the photosynthetic rate and the stomatal conductance of pgl4were obviously lower than that of the wild-type. At three-leaf stage, the development of thylakoids of pgl4disordered and vacuole appeared, while there was not difference at heading compared with the wild-type. PGL4was mapped to a21Kb physical distance on the chromosome, the result of sequence analysis indicated that there was a single substitution (G to A) at the926th base in PGL4which encoded CLP proteolytic enzymes, and the substitution was at the splice site between the second intron and the third exon, which resulted in the splice site moved backward for nine bases. PGL4in T-DNA insertion line lost the function and the mutant showed albino and lethal. The transgenic plants which transferred with the RNAi vector showed pale yellow, and the leaf turned to green when completely unfolded. Both the insertion and the RNAi results further identified that PGL4encoded a CLP proteolytic enzymes. PGL4expressed in the root, shoot, leaf, sheath and panicle organs, but it expressed highest in the leaf and the sheath, while lowest in the root. When inserted by T-DNA, it down-regulated the nuclear genes OsGLK expression, and then affected the PsaA,,PsbA and RbcL expression, which encoded the protein in chloroplast. In addition, we also analyzed that leaf color-related mutants were put into use for hybrid breeding as marker traits.4. stll showed white stripe during the seedling, and the glume turned white. stll that cultivated in24℃was normal, while in28℃, it showed white stripe, and in32℃, it almost showed albino, which indicated that stll was temperature-sensitive white-stripe leaf mutant and the critical temperature was28℃. Genetic analysis indicated that stll was a recessive mutant and controlled by a single gene. But when the mutant selfing, there were albinos in the descendant, and the plant showed cytoplasmic effect..To cross stll as the maternal parent, the individuals of the F1of all cross showed half albino, and the genetic model are still being worked out. The ST11was located on the short arm of the chromosome6between the markers RM225and RM217, and the genetic distance was3.9cM. Then the candidate region was delimited to a143Kb physical distance between the RM584and WP-4by using the developed5pairs molecular markers. There were12open reading frames in the candidate region, among which there was a gene (LOC_Os06g07210) encoded the large subunit nucleoside diphosphate reducase, the sequencing analysis showed that there was a missense mutation in the fifteen exon (G-A), which was the3859th base, this missense mutation resulted in the amino acid changed from methionine into isoleucine., There was no difference in the expression of ST11when stll subject to24℃,28℃and32℃. And, ST11down-regulated the expression of Cab1R and RbcL at32℃... |
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