Study On The Genetic Characteristics And Expression Regulatory Network Of High Oil And Acidity In Peanut

Peanut(Arachis hypogaea L.)is one of the important oil and cash crops in China,which provides abundant vegetable oil and protein for people.With the continuous improvement of people's living standards and the diversified development of consumer demand,high oleic acid peanut has become a new trend of peanut industry development in the world.However,there is a shortage of high oleic acid peanut resources in China.Therefore,it is of great significance to study the genetic rule of oleic acidity in peanut grains and the molecular mechanism of oleic acid content regulation for breeding new high-oleic acid peanut varieties,vigorously developing high-oleic acid peanut industry,and promoting the adjustment of agricultural industry structure.In this study,two different types of hybrid combinations were combined(1701 combination: low oleic acid × high oleic acid;1702 combination: high oleic acid × high oleic acid).Genetic and correlation analysis was conducted on oleic acid and other quality traits in F2:3 population.Transcriptome sequencing of two materials with high oleic acid and two materials with low oleic acid was carried out to study the molecular regulation mechanism related to oleic acid metabolism.And a preliminary establishment of a peanut genetic transformation system based on Agrobacterium rhizogenes infection and transformation of hairy roots.The main results are as follows:1.Genotype detection of three peanut strains with different oleic acid content.In this study,two high-oleic acid materials(Yuhua 37,HY963)and one low-oleic acid material Baisha 1016 were tested for Ah FAD2 A and Ah FAD2 B genotypes,respectively.The results showed that the two high-oleic acid lines had a G>A mutation at Ah FAD2 A 448bp,and there was a base A(ins A)insertion in Ah FAD2 B 442bp,while the low-oleic acid line Baisha 1016 had no sequence changes.2.Construction of two hybrid combinations and genetic variation and correlation analysis of F2:3 population quality traits.In this study,three different peanut strains of oleic acid were combined with 2 hybrid combinations,which were respectively 1701(Baisha 1016×HY963)combination,low oleic acid × high oleic acid,and 1702(Yuhua 37×HY963)combination,high oleic acid × high oleic acid.It was found that the variation range of oleic acid content and other quality traits was relatively large in the two hybrid groups F2:3,and the variation coefficient of linoleic acid was the largest.The genetic analysis showed that all the quality traits showed continuous variation and expressed as quantitative traits,which may be controlled by micro effect polygenes or main genes plus micro effect polygenes.Further correlation analysis found that in the two combinations,the correlation between oleic acid and linoleic acid is the strongest in the two combinations,and both have a very significant negative correlation.The correlation coefficients are r =-0.996 or r =-0.987,respectively.There was a very significant negative correlation between oleic acid and palmitic acid,and the correlation coefficients were r =-0.983 or r =-0.816,respectively.In addition,it was found that there was a super-parent separation phenomenon in the quality traits of the two F2:3 populations.For example,in the 1702 F2:3 population of two high-oleic acid materials,individuals with common oleic acid phenotypes were selected,indicating that besides Ah FAD2 A and Ah FAD2 B,there may be other genes regulating the content of peanut oleic acid.The results of the study laid the foundation for mining other genes regulating oleic acid metabolism.3.Transcriptome sequencing analysis was performed on the seeds of two high oleic acid and two common oleic acid materials 50 days after flowering,and a total of 97.70 Gb Clean Data was obtained.The Clean Data of each sample reached 6.94 Gb and the percentage of Q30 bases was 93.85% and above.Clean Reads of each sample were compared with the reference genome of cultivated peanuts.Through further analysis,a total of 636 differentially expressed genes were obtained.The GO and KEGG enrichment analysis of all the differentially expressed genes showed that: in the GO function annotation,the differentially expressed genes were mainly enriched in the metabolic process in the biological process,indicating the grain metabolism between peanut strains with different oleic acid content There is a big difference.KEGG metabolic pathway analysis found that most genes are enriched in starch and sucrose metabolism,phenylpropane biosynthesis,and cyanoamino acid metabolism pathways.In addition,pathways directly related to fatty acid metabolism,such as unsaturated fatty acid biosynthesis,alpha-linolenic acid metabolism,fatty acid degradation and fatty acid elongation are also enriched in more genes,indicating that these differentially expressed genes are directly or indirectly involved in the formation and metabolism of peanut oleic acid.Further,13 genes were randomly selected for q RT-PCR verification,and their gene expression was consistent with the analysis results of transcriptome data.4.Construction of editing vector of arachidonic acid regulatory genes(Ah FAD2 A,Ah FAD2B).According to the high similarity of Ah FAD2 A and Ah FAD2 B gene sequences,this study designed three sg RNA primers that can simultaneously target Ah FAD2 A and Ah FAD2 B genes.Using enzyme digestion experiments and homologous recombination methods,the sg RNA expression cassette and Cas9 gene were accurately linked to the Ri plasmid p GWB405.We successfully constructed a CRISPR-Cas9 vector that can simultaneously target three sites of the Ah FAD2 A and Ah FAD2 B genes.In addition,the p GWB405 vector carries a GFP tag,which can be used to monitor Cas9 protein expression.