Construction And Genetic Transformation Of Peanut Leaf Color Mutant And AhPDS Gene CRISPR-Cas9 System

Peanut is one of the most important oil crops in the world.The annual output of peanut in China ranks first in the world,and the peanut industry is developing rapidly,but the basic theory research process of peanut lags far behind other crops.Chloroplast is an important place for photosynthesis,and as a plant energy supply station,it plays an important role in plant growth and development.Leaf color mutants can cause the decrease of photosynthetic efficiency,which is applied to breeding and molecular marker assisted selection due to its apparent phenotypic characteristics and easy identification.Leaf color mutants have been studied and applied in a variety of plants such as rice,but have not been reported in peanuts.In this study,yellow-green and white-green leaf color mutants were screened from the peanut mutant library created by EMS mutagenesis,and the changes of physiological indexes,cell microstructure and yield difference were analyzed at different developmental stages;At the same time,Ah PDS gene was analyzed,and its physicochemical properties and function were predicted.The expression vector of CRISPR-Cas9 was constructed by using Agrobacterium tumefaciens-mediated method and transferred into peanut seedling.It is necessary to achieve the knockout editing of peanut Ah PDS gene by this technique,which lays the foundation for further study of gene function and molecular breeding of leaf color phenotype.The main results of this study are as follows:(1)The chlorophyll a,chlorophyll b and carotenoids of peanut yellow-green mutants and white-green mutants were lower than those of wild-type at different developmental stages,and the chlorophyll content was the most significant at seedling stage and flowering stage.Chlorophyll fluorescence kinetics parameters showed that the main parameters Fv / Fm,?(PS?)and q P were significantly lower than wild type,Indicating that the mutant PS? is inhibited by light and the ability to replenish the original photon is also weakened,which is consistent with the result of the decrease of chlorophyll content affecting photosynthetic capacity;The subcellular structure of the chloroplast was observed by transmission electron microscopy(TEM).It was found that the chloroplast membrane structure and shape of the mutant were intact,Indicating that the mutant chloroplast membrane structure is not damaged,and the internal structure damage is more serious,the number of thylakoid membrane decreased,And the thylakoid membrane did not form the stacked granules,which were linear or scattered in the chloroplast.The incomplete structure of the chloroplast affected the photosynthetic performance.The agronomic traits of the mutants showed that the plant height,fruit number and grain weight of the mutant were significantly decreased,and the comprehensive analysis of the white-green mutant was slightly worse than that of the yellow-green mutant.(2)Cloning and Molecular Characterization of Ah PDS in Peanut.According to the PDS gene in the peanut transcriptome database,the sequence of peanut Ah PDS gene was searched and analyzed by bioinformatics in Peanut Base peanut website.The results show: The gene was located on the chromosomes of A04 and B04,and its open reading frame was 6385 bp.The coding region was 1752 bp,which could encode 583 amino acid residues with molecular weight of 64.66 k D,and its theoretical isoelectric point was 6.81;The protein contains 52 potential phosphorylation sites,no signal peptide structure,belonging to the chloroplast more stable hydrophilic protein;The protein has a longer amine oxidase domain and a shorter NAD binding protein domain;Phylogenetic tree shows that peanut Ah PDS is close to other species and retains the more conservative structure and function of PDS.(3)According to the characteristics of conserved NGG sequences in the first exon region of peanut Ah PDS gene,the expression vector of CRISPR-Cas9 gene was constructed and transferred into peanut plant by Agrobacterium tumefaciens,The results showed that the color of the four foliage in the leaves of the peanut was lighter or the macula appeared about 15 days after the genetic transformation.The sequence of Cas9 gene was amplified by PCR,The results showed that the target sequence was transferred to the peanut plant,but the analysis of the base mutation site of the Ah PDS gene was needed for further study.