Ginkgo Chloroplast Ndhf Gene Rna Editing Sites Measured And Editing Efficiency Analysis

RNA editing is the phenomenon of nucleotide insertion, deletion or replacement during RNA maturation, which is a post-transcriptional modification process, including complete editing, partial editing and silence editing. RNA editing is an important way of chloroplast gene expression, which is common in terrestrial plants.Basing on its chloroplast genome sequencing, annotation and location, the gymnosperm Ginkgo(Ginkgo biloba)was studied. RT-PCR technique was used to study RNA editing of Ginkgo chloroplast ndhF gene, bioinformatics were used to analysis ndhF gene before and after editing. Meanwhile, the critical editing sites which affected protein structure was determainted. In order to investigate the possible function of chloroplast ndhF gene, we further studyed the changes of its editing efficiency under different environmental factors and during 24h period. The editing efficiency of Ginkgo chloroplast ndhF C290 site under different environment conditions was further determined using monoclonal enzyme digestation method.The studies and results were as follows:1. The RNA editing sites of ndhF gene in Ginkgo chloroplast was determined using RT-PCR method and its relevant characteristics were analyzed. In the 2214bp open reading frame of ndhF gene, there were totally 21 editing sites detected, which were all C to U conversation and partial editing, of which severn sites occurred in the first codon, thirteen in the second, one in the third place. RNA editing of ndhF gene was codon bias and increased the proportion of hydrophobic amino acids and the protein conservation.2. The bioinformatics analysis of ndhF gene before and after editing were performed in Ginkgo chloroplast. The results showed that the editing of C290 site affected ndhF gene structure, which might affect the protein folding correctly. There commonnly existed C to U conversations in ndhF C290 site of dicotyledons by comparativing different species, while they were Ts in genome level among the monocot speices.RNA editing of ndhF C290 site leaded to amino acids conservation increase between different species, which totally transformed to Leu at last.Basing on these, it could be inferred that C290 was the critical editing site of ndhF gene in Ginkgo chloroplast, whose editing might influence the function of ndhF gene.3. The editing efficiency of 21 editing sites were determined in Ginkgo chloroplast ndhF gene by using direct sequencing meothd, including different developmental stages and 24h growth period respectively. The results showed that editing efficiency of 21 editing sites changed in different developmental stages, and it first increased then decreased during 24h growth period. 4. An effective method of determining editing efficiency, named monoclonal enzyme digestion method, was established,which improved the existing methods greatly. It provided the possibality for RNA editing function study by determining RNA editing efficiency.5. The editing efficiency of Ginkgo chloroplast ndhF C290 was determined using monoclonal enzyme digestation method, under different environment conditions, including low temperature, high temperature, drought, darkness and salt stress.Meanwhile, the editing efficiency of ndhF C290 was also deteminated during 24h growth period in the same way. The results showed that the editing efficiency responsed to environment stress, for example, after 6h cold treatment, editing efficiency had a downward trend, yet under dark treatmentwere a rising trend was observed on editing efficiency. During the 24h growth period, its editing efficiency was first down then up.