Construction And Analysis Of The Light Independent Anthocyanin Accumulation T-DNA Mutant In ’Tsuda’ Turnip

The construction of large-scale mutant library is an important content of functional genomics research. Analysis of the mutant phenotype and identification of the gene function are the most direct and effective method of functional genomics research. In recent years, using T-DNA as an insert element to build mutant library, screen mutant and research gene function has been found in many plants. In the early studies, the results showed that anthocyanin synthesis of ’Tsuda’ turnip was specifically induced by UV-A, which suggested that there may be a UV-A specific receptor which is different from the UV-A/blue receptor in plant. Creating the light independent anthocyanin accumulation T-DNA mutant in ’Tsuda’ turnip will build a platform for the research of genes and signal transduction factors in anthocyanin biosynthesis pathway specifically induced by UV-A.In this study, a T-DNA mutant library about anthocyanin synthesis of’Tsuda’turnip was built and then the mutant population was analyzed. We obtained the following main results:The full-length of GUS from pBI121plasmid was successfully cloned. Sequencing analysis showed that the sequence of full-length is1812bp, and the result of blast defined that we obtained the correct sequence.The expression vectors-pH7WG2D,1-GUS was successfully constructed using the Gateway technology, which contained the GFP gene, the GUS gene, hygromycin as selectable marker and the CaMV35s promoter.The tissue culture genetic transformation with’Tsuda’turnip rachis was explorated. We setted tirty-five different mediums with different6-BA and NAA combination in accordance with the characteristics of the turnip. Yet the results showed that those mediums could not get regeneration buds. The non-tissue culture genetic transformation of infiltration germinating seeds with agrobacterium vacuum was a successfully attemp. Four experimental conditions was setted which were dry seeds without ultrasound, dry seed with ultrasound, soaked seeds without ultrasound, soaked seeds with ultrasound. Green fluorescent protein screening and the GUS histochemical preliminary identification showed that conversion rate of soaked seeds with ultrasound was highest and the positive results reached60%.After screened the T-DNA mutant library, light independent anthocyanin accumulation-red type and no anthocyanins accumulation regardless of light condition-white type were obtained. We screened about10000T-DNA mutants of generation T1, and obtained200target mutant strains. We obtained17red and60white T-DNA mutants in generation T2. The PCR identification of four marker genes:gfp、gus、hyg and35s prmoter showed that number3and4in red mutant, number7and9in white mutant had all four genes.The result showed they were the positive transformants.The TAIL-PCR results showed a T-DNA insertion site was in the chromosome07between20,002,828and20,003,156, which was may be between two genes or downstream of the gene promoter zone. After blasted Arabidopsis genome database to analysis the gene of upstream and downstream, we found that the upstream gene was pentatricopeptide (PPR), the downstream gene encoding the protein which belonged to the family of Multidrug and Toxic Compound Extrusion (MATE).