Study On The Mechanism Of FAD2 Gene Family Regulating Oleic Acid Accumulation In Peanut Seeds With Different Maturity

Peanut（Arachis hypogaea L.）is one of the most important edible oil crops worldwide.The oleic acid（18:1）and linoleic acid（18:2）account for more than 80%of the total fatty acids in peanut.The oxidation stability of oleic acid is 10 times higher than that of linoleic acid,therefore,the peanut with high oleic acid and its products have longer storage life.In common peanut or high oleic acid peanut varieties,the ratio of oleic acid and linoleic acid（O/L）varies at different developmental stages or maturity even in the same variety.AhFAD2,a key enzyme catalyzes the transformation of linoleic acid from oleic acid,and determines the fatty acid composition in plants.To understand the role of AhFAD2 in regulation of fatty acid composition is of great significance.In the process of high oleic acid peanut breeding,four stable genotypes AABB,aaBB,AAbb and aabb of FAD2A and FAD2B were obtained.In consideration of the possible influence of AhFAD2A and AhFAD2B on the expression of other FAD2 members,these four peanut genotypes were used to study the mechanism of the oleic acid accumulation regulated by different FAD2 members at different developmental stages.The main results of this study were as follows:1.According to the color and thickness of mesocarp,peanut pod development was divided into four stages in this study:stage with white and thick mesocarp（S1）,stage with white and thin mesocarp（S2）,stage with brown spotted and thin mesocarp（S3）,and stage with black spotted and thin mesocarp（S4）.The fatty acid content of peanut seeds at differently developmental stages in four genotypes was determined by gas chromatography.In all genotypes,the change trend of fatty acid composition was basically similar,that is,with the increase of maturity,the content of oleic acid increased,while the content of linoleic acid decreased,leading to the increased O/L ratio.Therefore,it is speculated that in addition to AhFAD2A and AhFAD2B,other FAD2 family members or transcription factors could be involved in linoleic acid synthesis.2.Three new AhFAD2 members,named as AhFAD2-7,AhFAD2-8 and AhFAD2-9,were obtained by analyzing the whole genome sequences of wild and cultivated peanuts.The length of AhFAD2-7,AhFAD2-8 and AhFAD2-9 genes were 1152 bp,1164 bp and 1164 bp,respectively.Among them,AhFAD2-7 was located on chromosome B06,while AhFAD2-8 and AhFAD2-9 were located on chromosome A09 and B09,respectively.The sequence identity of these two genes was 98.71%,with only 15 bp differences.3.AhFAD2-7,AhFAD2-8 and AhFAD2-9 proteins were verified to locate on the endoplasmic reticulum through scanning the Arabidopsis roots of T₁ seedlings with fluorescence confocal microscopy.4.Functional verification of AhFAD2-7,AhFAD2-8 and AhFAD2-9 proteins were performed in the yeast INVSc1 and the fad2 mutant of Arabidopsis.The results showed that linoleic acid could be detected after transforming yeast and Arabidopsis mutant fad2 with AhFAD2-7,AhFAD2-8 and AhFAD2-9 construction vectors,indicating that AhFAD2-7,AhFAD2-8 and AhFAD2-9 had the activity ofΔ12-fatty acid dehydrogenase and could transform oleic acid into linoleic acid.5.To analyze the roles of different FAD2 family members at different developmental stages of seed and in different tissues,the temporal and spatial expression patterns of FAD2family members in four peanut genotypes were analyzed by qRT-PCR.The results showed that AhFAD2A/B was expressed in seeds of different developmental stages and different tissues,with the lowest expression level in stem.With the increase of seed maturity,expression level of AhFAD2A/B increased firstly and then decreased.Unlike AhFAD2A/B,AhFAD2-3 was mainly expressed in seeds.In seeds,the trend of expression of AhFAD2-3 was the same as AhFAD2A/B.AhFAD2-7 was expressed in different developmental stages and tissues with the same expression patterns of AhFAD2A/B.with the increase of seed maturity,the expression of AhFAD2-7 in AABB and aabb plants decreased,which coincided with the changing trend of linoleic acid.While in aaBB and AAbb seeds,the expression levels of AhFAD2-7 decreased first and then increased when the seeds were fully mature.This may be caused by the decreased activity or inactivation of AhFAD2A or AhFAD2B.The expression levels of AhFAD2-8 and AhFAD2-9 increased with seed maturity.These results showed that the expression levels of all FAD2 members（AhFAD2A/B,AhFAD2-3,AhFAD2-7,AhFAD2-8and AhFAD2-9）were increased in roots of aabb plants to compare with other genotype.It was speculated that the declined activity ofΔ12-fatty acid dehydrogenase due to AhFAD2A and AhFAD2B mutation could lead to transcriptional change of FAD2 genes to meet the demands of FAD2 function involved in the fundamental biological processes in roots.The expression levels of AhFAD2A/B and AhFAD2-7 were relatively higher in four genotypes and four seed stages than other AhFAD2 genes.The expression level of AhFAD2-7 was the highest in S1 in AABB,AAbb and aabb genotypes,while AhFAD2A/B was the highest among the four genotypes in S2,S3 and S4.The trend of FAD2A/B expression is inconsistent with that of seed maturity.However,the expression level of AhFAD2-8 was increasing.The expression level of AhFAD2-8 was the lowest,which may be not enough to reverse the increase of oleic acid content change with the increase of maturity.The expression of AhFAD2-7 was relatively high in four genotypes and four seed stages,and the variation trend was consistent with that of linoleic acid content.Therefore,we speculate that AhFAD2-7 could be the key member leading to decreased linoleic acid with seed maturity.In addition,some members of the WRI1family may be involved in the process.