| Tomato is an important economic crop in the world. The quality and yield of tomato are influenced by photosynthetic efficiency, directly related to the contents of chlorophyll in leaves. Leaf coloration mutants are prone to occur and exhibit obvious phenotypes. The study of leaf coloration mutants has great significance to clarify the pathways of chlorophyll biosynthesis and degradation, chloroplast differentiation and development, and the mechanism of photosynthesis regulation in higher plants. Furthermore, it is also propitious to use genetic engineering techniques to improve the photosynthetic efficiency of plants and increase the yields. In this research, we studied a white virescent(wv) mutant LA1526 of tomato. To explore the molecular mechanism of mutant phenotype, the phenotypic identification, genetic analysis and gene mapping were studied at the levels of plant physiology, heredity and molecule biology, which would provide theoretical basis for photosynthetic physiology, function genomics and seed breeding in tomato. The results of the study are summarized as follows:1. wv mutant LA1526 is an environment sensitive mutant, the phenotype may be influenced by both of temperature and illumination. The mutants grown in the field of seedling in winter, showed albino phenotype before 3th leaf stage. Then the leaves became to turn green, and the mature leaves eventually appeared normal green, while the apical leaves showed yellow phenotype until death. However, the apical leaves of mutants grown in the field of seedling in summer or in a room with constant temperature(25℃), didn?t show albino phenotype, but still keep yellow.2. The results of photosynthetic physiological indexes in wv mutant LA1526 indicated that, the contents of chlorophyll a in the apical leaves of LA1526 had no significant difference compared with the control Ailsa Craig, but the contents of chlorophyll b and total chlorophyll were both significantly lower than those in the control, and the contents of β-carotene was significantly higher than the control; The Y(II) and electron transfer rate(ETR) in the apical leaves of LA1526 were drastically lower than those in the control; The ultrastructure of chloroplast was characterized by transmission electron microscopy(TEM). The results showed that the ultrastructure of chloroplast in the apical leaves of LA1526 was basically complete. While the chloroplasts in the apical leaves of LA1526 had less grana lamella, starch grains and more osmiophilic bodies. And the arrangement of grana lamella showed loose and irregular.3. By comparing the published IL map with the genetic map of tomato, we conjectured that wv was located in the middle region of the long arm of chromosome 2, linked with Prx-2,3, and in the upstream of Wo, corresponding to the region of IL2-3. F2 genetic segregation population was constructed by crossing IL2-3 with LA1526. Genetic analysis indicated that the mutant phenotype of LA1526 was controlled by recessive nuclear gene.4. To localize the target gene, we exploited newly In Del markers according to the tomato genome data and finally got 12 pairs of In Del markers with polymorphism; By using 5 pairs of In Del markers and 186 progenies showing the wv mutant phenotype separated from the F2 mapping population, wv was located in a region between the In Del markers wv-c13 and wv-c24, which is 0.27 c M apart with wv-c13 and 1.09 c M apart with wv-c24; Then we further expanded the F2 mapping population of the cross IL2-3 × LA 1526 and carried out fine mapping. Based on 2368 progenies showing the wv mutant phenotype separated from the F2 mapping population using newly exploited markers between wv-c13 and wv-c24, we finally delimited wv in a 94 kb DNA region between the In Del markers wv-c53 and wv-c75, which were both closely linked to wv with respectively one and five recombination event identified.5. Gene prediction of this region showed that there were 19 opening reading frames(ORFs) in this region. To define the molecular lesions of wv mutant, we designed amplification primers of all the genes and promoters of Solyc02g079720, Solyc02g079730, Solyc02g079740, Solyc02g079750, Solyc02g079760, Solyc02g079770, and amplified the fragements in LA1526 and Ailsa Craig. Blast analysis showed that all the fragement could be amplified successfully in LA1526, but all the fragements didn?t have the mutations of base sequence and the changes of fragment length. Therefore, we inferred that there were two possible reasons for this result: Mutation occurs in an un-sequenced promoter; Or the mutant phenotype is caused by the changes of gene expression, which may be related to the epigenetic variation.6. The expressions of Solyc02g079720 、 Solyc02g079730 、 Solyc02g079740 、Solyc02g079750、Solyc02g079760、Solyc02g079770, which may be related to plant photosynthesis, chloroplast development and signal transduction, were determined by Q-Real-time PCR in LA1526 and Ailsa Craig. The result showed that the expression of Solyc02g079730 in LA1526 is less than that in Ailsa Craig. To verify the functions of candidate genes, we used VIGS technique to further study. The p TRV2 virus expression vectors of Solyc02g079730, Solyc02g079740, Solyc02g079750, Solyc02g079760 were constructed and transiently expressed in the leaves of Ailsa Craig by agroinfiltration. The results showed that the inhibition of these four genes couldn?t make Ailsa Craig show the mutant phenotype. |
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