Cloning And Identification Of T-DNA Insertional Yellow-leaf Mutant In Tomato

Mutants are one of the important tools for plant functional genomics. Leaf coloration mutants exhibit obvious phenotypes, in which chlorophyll synthesis and degradation, chloroplast development are directyl or indirectyl restrained, leading to altered content of chlorophyll and leaf coloration. Leaf coloration mutants play vital roles in studying the structures, functions, and mechanisms of chloroplast and photosystem. In the study, the genetic behavior, mutant mechanism, photosynthetic characterization, cloning and function of mutated gene of tomato mutant yl (yellow-leaf) were studied at the levels of Plant physiol genetics and molecule biology, which should provide theoretical basis for tomato photosynthetic physiology, function genomics and agricultural productions. The results are summarized as following:1. The yl mutant displays yellow leaf phenotype from seedling stages with cotyledons. This yellowish leaf phenotype of yl mutant is kept throughout the whole plant growth stages. The yl mutant is much shorter than wild-type plants.2. Genetic anaylsis of yl mutant indicated that yl mutant is a deficient in a single recessive nuclear gene as a result of T-DNA knock-out with a single site T-DNA insertion. Expression anaylsis showed that the mutation phenotypeis not attributive to to exogenous expression of the target gene in the T-DNA.3. The result of photosynthetic characterization indicated that contents of chlorophyll a, chlorophyll b and carotene in the top third leaf of yl mutant were significantyl lower than those of wild-type plants. Chloroplast of wild-type plants possesses fully developed thylakoid membrane system composed of grana connected by stromal lamella. However, the chloroplast int the top third leaf of yl mutant had less thylakoid membrane stacks, indicating chloroplast degradation in yl mutant. Net photosynthetic rate of yl mutants was also significantyl lower than that of the wild-type plants.4. The right-border sequence of T-DNA of yl mutant was amplified by hiTAIL-PCR method and its candidate gene in the insertion locus was identified based on the blast anaylsis and co-segregation between yl phenotype and T-DNA insertion. T-DNA was intergated on tomato chromosome 11 and inserted at eighth exon of SGN-U579512 gene (1901bp), which encodes RuBisCO large subunit-binding protein subunit alpha according to the functional annotation. The candidate gene SGN-U579512 was named as RLBPa. As the cataylst for CO2 entrance to organism as well as inorganic carbon transformation to organic carbon, RuBisCO plays a decisive role in the net photosynthetic rate. Rubisco is located the chloroplast stroma in higher plants, accounting for 50% of the chloroplast soluble protein. Rubisco carboxylase activity and soluble protein content of yl mutants were determined, and the results showed that Rubisco carboxylase activity and soluble protein content of yl mutant were significantyl lower than those of wild-type control. The soluble protein in yl mutant was reduced by 44% as compared to control.5. We further used VIGS approach to study whether yellow leaf phenotype of yl mutant is caused by lack of RLBPa gene. pTRV2 virus expression vector was constructed and transientyl expressed in tomato leaves by agroinfiltration. The infected leaves of wild-type plants and yl mutants, turned yellow and white, respectiveyl,2 weeks after agroinfiltration. RT-PCR anaysis showed that the expression level of RLBPa gene decreased in the VIGS treated plants, and the content of chlorophyll and soluble protein were significantyl reduced as compared to the untreated control.