Rice T-dna Insertion Mutant W378 And L395 Mutant Gene Cloning And Functional Analysis

T-DNA insertional mutantion is one of important tools in constructing rice mutant lines and functional analyses of rice genes. In recent years, a series of T-DNA insertional lines were collected in our lab, and T-DNA integrational mechanism, mutant phenotypes and genes function were investigated. The mutants, with obvious phenotype variations and the variations being segragted with T-DNA (Ac/Ds), were selected to further analyses. Firstly, the mutant genes were cloned via T-DNA tagging method. In addition, the relationships between rice mutant phenotypes and mutant rice genes, and the putative function of mutant rice genes were analyzed by forward genetic (genetic and molecular analysis of mutants) and reverse genetic (overexpression and RNAi analysis) method. The mutants, with no obvious phenotype variations, were also collected to isolate the T-DNA flanking rice DNA sequences. T-DNA localizations in rice chromosomes of different mutants were determinated by blasting assay with those T-DNA flanking sequences. If the T-DNAs were integrated into (around) any interesting gene loci, the specific experiments based on the putative function of mutant genes, would be applied to analyze the physiological or biochemical variations (such as variations of metabolism product, sensitivity to plant hormones or heavy mentals) between mutants and wild plants. In this paper, parts of text were two representative examples of analyzing of mutants with or without obvious phenotype variations.T-DNA insertional mutant W378 was a late-flowering and dwarf rice mutant. Former experiment showed that the late-flowering phenotype of mutant W378 was segregated with T-DNA integration. The putative mutant gene OsLFL1 (Oryza sativa LEC2 and FUSCA3 Like 1) in W378 was isolated by T-DNA tagging. The OsLFL1 gene codes a putative B3 DNA binding domain transcription factor and is named as OsLFL1 (Oryza sativa LEC2 and FUSCA3 Like 1) because the deduced protein sequences from the full length cDNA is similar to those of Arabidopsis seeding development protein LEC2 and FUSCA3. Expression assays showed that OsLFL1 gene was only expressed in spikes and young embryos in Zhonghua 11, but high OsLFL1 gene expression was viewed in roots, shoots, leaves and other tissues of W378. Expression of rice flowering time gene Ehd1 and its putative downstream genes, such as Hd3a, RFT1 and OsMADSs, were all detected to be downregulated greatly in W378. Overexpressed OsLFL1 gene in Zhonghua 11 delayed flowering time of transgenic rice plants. Downregulated OsLFL1 gene in W378 by RNAi method repressed the late-flowering phenotype of W378. These results suggested that the late-flowering phenotype in mutant W378 was caused by overexpression of OsLFL1 gene. EMSA and ChIP assay suggested that the OsLFL1 protein could bind specifically to the DNA fragment containing RY repeats (CATGCATG) in Ehd1 gene promoter region. Our work showed the putative mechanism of late-flowering phenotype of mutant W378: OsLFL1 was expressed specifically in spikes of wild type rice, but overexpressed in leaves of mutant W378 because of T-DNA integration. The OsLFL1 protein, accumulated in leaves of mutant W378, could interact with RY repeats in promoter of Ehd1 gene (expressed specifically in leaves) and repress expression of Ehd1 gene, and then delay the flowering time of mutant. Other biological functions of OsLFL1 gene were also discussed in this paper.L395 was isolated from T-DNA insertion lines and shown none phenotype variation, but the T-DNA flanking sequence was highly similarly with gamma-glutamylcysteine synthetase (GCS) gene of Arabidopsis. In mutant L395, T-DNA was likely to be integrating into the rice homologue GCS gene. Because of the GCS gene not been reported in rice, we cloned the rice GCS gene by T-DNA tagging in mutant L395. Sequence analyses showed that the mutant GCS gene localized in chr.5 (OsGCS5) and another copy of OsGCS was existed in chr.7 (OsGCS7). In mutant L395, a single T-DNA copy was integrated between the second intron and the second exon of OsGCS5 gene, causing one nucleotide deletion in the second exon and two nucleotide deletions in the second intron. None significant difference was found under Cd2+stress tolerance, rice GCS gene expression level and GSH content between mutant L395 and Zhonghua 11, suggesting that the function of mutant OsGCS5 might be complemented by OsGCS7 gene. The sequences of the coding region of OsGCS5 and the OsGCS7 and also the deduced protein amino acid sequences were highly identical, but DNA sequences of their promoter were highly different. About 2kb promoter fragments for these two genes were constructed with GUS reporter and introduced to rice. GUS staining results for the transgenic plants showed that OsGCS5 and OsGCS7 were expressed in almost all tissues, but OsGCS7 gene had much higher expression than that of OsGCS5. OsGCS5 mainly expressed in thin wall cell around of abdominal vascular bundle and OsGCS7 in abdominal vascular bundle. RT-PCR-RFLP analyses also confirmed the expression results in GUS staining assay. The expressional differences for two copies of OsGCS suggested that functional variations might exist in them.