Expression Analysis Of FAD2 In Peanut And Preliminary Screening Of FAD2 Upstream Regulatory Factors

Low temperature is an important factor limiting the distribution of plants and affecting plant growth and development.When plants are in a low temperature environment,they sense the cold signal,and the organism changes in levels of Ca²⁺,hormones（such as ABA）,and various osmotic adjustment substances.These changes,through a series of signal transduction,eventually induce the initiation of cold-regulated gene regulatory networks,thereby regulating plant resistance to cold.In the cold stress response not only includes these physiological index which play an important role,but also the participation of various transcription factors.The FAD2 gene is a gene encoding a fatty acid dehydrogenase.In most higher plants,their genomes have two copies or more of the FAD2 genes,and the expression patterns and functions of different copies of the FAD2 gene are also different.The FAD2 genes in plants are divided into two categories,one of which is constitutively expressed.It is involved in the synthesis of unsaturated fatty acids in membrane lipids;the other is seed-specific expression,which mainly plays a role in the process of desaturation of storage lipids.In the previous study,our laboratory had obtained two copies of the FAD2 gene（AhFAD2A and AhFAD2B）.There are alternative splicings in these two genes,of which the A2 and B2 splice isoforms expressed in seeds have been identified to participate in the synthesis of unsaturated fatty acids of storage lipids.The A4B4splice isoforms express as a constitutive expression pattern and it is speculated that to be involved in the synthesis of membrane lipids.In addition,there may be other copies of FAD2 in the peanut genome.Based on these problems,the EST comparison analysis was conducted on the peanut database;new FAD2 genes were discovered through homology analysis of the splicing contigs sequences;the expression pattern of FAD2 gene was analyzed by qRT-PCR in different tissue and analyzed under cold stress.The expression pattern was further analyzed by the transgenic Arabidopsis GUS expression system;the yeast expression library of leaf cDNA under cold stress was constructed,and the regulatory factors interacting with FAD2 were through yeast one-hybrid.Through the above studies,the following major results have been achieved:（1）Two novel copies of the FAD2 gene,AhFAD2-2.1 and AhFAD2-2.2,were cloned from the peanut ESTs database.The cDNA of the two genes are 1152bp and encode 383 amino acids.There are 12 nucleotide difference in the cDNA level,among which only the differences located at 483bp and 720bp lead to the differences at amino acid level;the gDNA size of these two genes are 5089bp and 5090bp,respectively;The introns of both genes are located in the 5′UTR region upstreaming of the start codon ATG and are 3821 bp and 3822 bp in length,respectively.By comparing the amino acid sequence of AhFAD2-2 with the FAD2 amino acid sequences from other species,it was found that AhFAD2-2 is clustered in housekeeping-type FAD2.Among them,phylogenetic relationship with wild-type peanut FAD2-2 is the closest,followed by closely related with soybean GmFAD2-2,cotton GhFAD2-2 and Caragana korshinskii CkFAD2,but the genetic relationship Arabidopsis thaliana AtFAD2,rape BnFAD2,sunflower HaFAD2 is relatively faraway.Peanut AhFAD2-2 is clustered with AhFAD2A and AhFAD2B in different branches.This shows that these are two types of FAD2 genes,the latter belonging to seed-type FAD2.Besides,there is a highly conserved structure in the amino acid sequence of FAD2-2 consistent with the homologous sequences from other species.For example,there are five transmembrane domains,three highly conserved histidine clusters（HECGHH,HRRHH,HVAHH）and the aromatic amino acid-rich sequence（-YNNKL）at C-terminal.In addition,the regulatory elements in introns located at the 5′UTR region of AhFAD2-2 was analyzed.The results showed that there are some elements that control high level transcription of gene,some related to light regulation,and some participate in stress response.（2）Through the expression pattern analysis of AhFAD2-2,it was found that AhFAD2-2 was constitutively expressed,and significantly responded to low temperature stress.It was expressed in all organs,among which the highest expression level in leaves,the lowest in seed,and the the expression level at the early stage of the developmental seeds is much higher than that at the later period.In the cold-treated leaves,the expression level increased significantly within 12 h after treatment,and the expression level decreased significantly after 12 h.Within 24 hours after recovery to room temperature,the expression of the sample group was always lower than that of the control group.For the expression analysis of AhFAD2 A4B4 splice isoforms,the result showed that expression of A4B4 splice isoforms in peanut leaves was induced by low temperature stress.The expression level reached the highest level after cold treatment for 2 hours,and then the expression level decreased as the duration of treatment time.When recovery to room temperature,the expression level increased slightly within 24hours.Using the GUS expression system of Arabidopsis,the expression patterns of different structures were analyzed,the results showed that in the cotyledon at the seedling stage,GUS genes of the P2 structure and the P3 structure were slightly expressed,and the P4 structure strongly expressed;in the anther and stigma of the flower organ,GUS expression level of the P2,P3,and P4 structures were similar;at the tip and stem-leaf junction of the cauline leaves,GUS of all structures detected were slightly expressed.In embryos,the P2,P3,and P4 structures are strongly expressed.Under the cold stress,GUS gene expression didn’t change significantly in seedlings of each transformed structure.（3）A yeast cDNA library of peanut leaves under cold stress was constructed,which has library capacity of 7.8×10⁷/m L.Promoter analysis revealed that low temperature-related regulatory elements such as ACGT ABRE MOTIF and CGCG BOX AT were present in the promoter region of the AhFAD2 A4B4 splice isoform.The bait sequences and their corresponding mutant sequences were designed for exploring the regulatory factors by yeast one-hybrid method,and bait vectors and bait yeast strains were constructed.These studies will provide the basis for discovering regulatory factors that interact with FAD2.The results obtained in this study are still relatively preliminary.Furthermore,for the AhFAD2-2 gene,we will further study its expression pattern and cold stress response pattern through mRNA in-situ method.Again,combined with the result of transgenic function analysis,we will gain insight into the effect of AhFAD2-2expression on the composition of unsaturated fatty acids in membrane lipids.Whether the intron in the AhFAD2-2 has regulatory functions such as enhancing gene expression,we will also verify by the function analysis of regulatory elements.For the AhFAD2A and AhFAD2B,we expect that the transcription factors interacting with the motifs metioned will be found out as soon as possible by the yeast one-hybrid.Then,the selected transcription factor genes will be analyzed in different aspects including expression pattern analysis,and the regulatory regions that are bound to the AhFAD2.Finally,functional verification that the transcription factors interacting with AhFAD2 regulate the accumulation of unsaturated fatty acids in membrane lipids will be performed.