The Gene Identification Of An Arabidopsis Thaliana Yellow Leaf Mutant And Its Preliminary Fuction Study

Photorespiration is the metabolic process by which green plants absorb oxygen and release carbon dioxide under light conditions.The metabolic process is very complex and involves chloroplasts,peroxisomes,mitochondria and cytoplasm.The overall result of its metabolism is the production of one molecule of carbon dioxide and one molecule of phosphoglycerate.In the whole photorespiration metabolic pathway,glycolic acid produces glyoxylic acid under the action of glycolate oxidase?GOX?,and also produces H₂O₂.However,excessive accumulation of H₂O₂ will cause some cell death.Especially under conditions of enhanced photorespiration such as high light and drought,plant cells produce a large amount of reactive oxygen species?ROS?.Once the level of reactive oxygen is increased,it will not be conducive to the growth and development of plants.It is not entirely clear what mechanism the plant uses to reduce the level of oxidation and protect it from harm.Superoxide dismutase?SOD?is known to be a metal-containing antioxidant enzyme,which is ubiquitous in living organisms.According to the different metal ions binding to SOD enzyme,it can be divided into MnSOD,FeSOD,Cu/ZnSOD,and NiSOD.SOD is one of the important antioxidant enzyme systems,but it has not been reported whether it plays a role in clearing ROS in photosynthesis or photorespiration.In this study,a mutant with stable genetic inheritance from Arabidopsis thaliana Col-0 mutant library generated by EMS was obtained.This mutant has yellow phenotype and dwarf plant phenotype in the atmosphere.The phenotype at 3000 ppm CO₂ is consistent with the wild-type Col-0,and map cloning indicates that the mutant was caused by a point mutation in AtFSD3?At5g23310?.This thesis uses this mutant as a research material and obtained the following research results:1.Obtaining yellow leaf mutant 27-24We screened the mutant library 27?EMS mutagenized Col-0?and finally got a stable.The genetically inherited yellow leaf mutant,We named the mutant 27-24.Some of the mutants that grow in the atmosphere can flower on the platform,but the branches of the plants are very slender and dwarf.The leaves of the whole plant are in the state of yellowing,the candied fruit is short and the number of seeds is small,while the mutants growing at 3000ppm CO₂ are in the form of Is consistent with wild-type Col-0.2.Measurement of physiological indices of 27-24 mutantExtracts 27-24 and wild-type Col-0 grown under atmospheric and high CO₂ were used to extract chlorophyll using acetone and absolute ethanol and measured at wavelengths of 663nm and 645 nm,and also with a SPAD instrument.The relative content of chlorophyll in leaves was determined.The results showed that the chlorophyll content of the mutants grown in the atmosphere was significantly different from that of the wild type,but the chlorophyll content of the mutants grown under high CO₂ had no significant difference from the wild type.At the same time,the maximum photochemical efficiency Fv/Fm of the leaf was measured with chlorophyll fluorescence imager,and the results were consistent with the results of chlorophyll content determination.We then used wild-type and mutants grown in the atmosphere and under high CO₂ for 5 weeks as a material,and we performed statistics on rosette leaf number and mean leaf weight.The results showed that the number of rosette leaves and leaves of mutants growing under the atmosphere The average weights were all significantly lower than those of the wild type,while the mutant rosettes grown under high CO₂ had almost the same number of rosette leaves and average leaf weight as the wild type.At the same time,wild type and mutants grown under atmospheric and high CO₂ were stained by NBT and DAB to detect ROS level in plants.Finally,it was found that the ROS accumulation in mutant leaves grown under the atmosphere was significantly higher than that in the wild type,whereas the ROS accumulation in mutant leaves grown under high CO₂was close to the wild type.3.Location of mutant genesThe 27-24 mutant and wild-type Ler were crossed to construct a population of map-based populations,and then 24 pairs of Marker primers uniformly distributed on the chromosome of Arabidopsis thaliana were used for the primary mapping.The mutant gene was initially mapped between 5.44M and 13.58M on chromosome 5,and then new primers were designed for fine mapping.Finally,the mutant genes were mapped between 6.61M and 8.60M on chromosome 5.The mutant was sent to the company for genome re-sequencing,combined with SNP mutations and mutant phenotype analysis between 6.61M⁸.60M:the mutation of At5g23310 was thought to be the cause of this mutant,that is,the 748th base in the cDNA sequence of the gene changes from G to A,resulting in the conversion of Glu250 to Lys250in the FSD3 protein.4.Complementary experiments to verify the mutated geneIn combination with genome re-sequencing and the phenotype of the mutant with yellow leaves and short stature under ambient air,the phenotype of the mutant was found to be identical to that of the superoxide dismutase mutation by searching the literature.The gene sequence of At5g23310 was downloaded from TAIR?https://www.arabidopsis.org/?,the full-length sequence of AtFSD3 with native promoter was cloned to construct its complementary vector,and then use the flower-dipping method to transform mutants.After screening on gentamicin plates,complementary plants that were similar to Col-0 were finally obtained.According to the candidate gene AtFSD3,the specific primers were redesigned,an overexpression vector was constructed,and the mutant was infested with the dip flower method.After screening with gentamicin,several positive strains were finally obtained.Subsequently,the chlorophyll content and the maximum photochemical efficiency Fv/Fm of complementary plants and over-expressed plants were measured.The results showed that both the chlorophyll content and Fv/Fm value were close to the wild-type Col-0.It was finally determined that the phenotypes of the mutants 27-24,such as yellowing and dwarfism,were caused by mutations in the AtFSD3 gene.5.Expression analysis of AtFSD3 in Arabidopsis thalianaOverexpression of the protoplasts of the plants and fluorescence microscopy were used for fluorescence observation.The results indicated that the AtFSD3 gene was located on the chloroplast.At the same time,the GUS expression vector was constructed and GUS staining was performed on the T1 generation positive seedlings that had grown for 5 weeks.Finally,the leaves,fruits,and petals of the T1 generation positive seedlings were not stained,and only the pollen grains were stained blue.After another generation,GUS staining was performed on seedlings of the T2 generation,and it was found that the cotyledons of the T2seedlings were stained blue.