Creation Of A New Germplasm By Transgenic PfFAD To Increase The Content Of ?-linolenic Acid In Arabidopsis Thaliana

Perilla [Perilla frutescens(L.)],a traditional medicinal and oilseed crop,contain extremely high levels of polyunsaturated ?-linolenic acid(ALA)in its seeds.It is of great significance to dig and analyze the key genes and functions of ALA biosynthesis in Perilla for the creation of new germplasm of high-quality oil plants.In this study,based on the complete genome sequencing,we screened and analyzed the PfFAD of Perilla M40 with high oil content,cloned the key enzyme gene for ALA synthesis,and genetically transformed Arabidopsis thaliana,thus creating a new Arabidopsis germplasm with high ALA content.The main research contents and results are as follows:1.Screening and characterization of PfFAD.35 PfFAD candidate genes were screened from P.frutescens for subsequent analysis and belonged to 4 subfamilies.The same subfamily gene has similar intron-exon structure,conserved domain characteristics,subcellular localization,and contains functional conserved histidine boxes.Functional element analysis showed that PfFAD may be involved in plant growth and development and the regulation of defense response.2.Expression analysis of PfFAD in Spatiotemporal.The transcriptome data of tissues and seeds at different developmental stages in Perilla were analyzed.It was found that the expression of Pf SAD ware up-regulated in the middle and late stages of seed development mainly.However,the genes expression of Pf SLD and Pf DES were increased significantly in the early stage of seed development.There are two main gene expression patterns in ?12FAD and ?-3 FAD.The plastid PfFAD6 and PfFAD7/8 are mainly expressed in leaves,stems,flowers and other tissues,while the micro-type PfFAD2 and PfFAD3 are expressed Increased in the middle and late stages of seed development.Select some ?12FAD and ?-3 FAD subfamily genes for q RT-PCR verification,which is consistent with the trend of transcriptome gene expression.3.Cloning of ?-3FADs in Perilla.Five ?-3FAD were cloned in Perilla and constructed into plant over expression vector,which encodes an amino acid range of390 to 568.Amount them,Except for PfFAD7/8.5-Clone,which does not contain TMs and partially lacks histidine box,there are four ?-3FAD genes contained 3 hisboxes and 4 TMs.Functional analysis showed that two PfFAD genes have high catalytic activity for ala synthesis.the PfFAD3.1-clone was constructed into p CAMBIA2301 vector,and other genes were constructed into p CAMBIA1301 vector.4.Genetic transformation and functional analysis of A.thaliana.The PfFAD3.1was transformed into Arabidopsis thaliana by flower soaking method,and 8homozygous transgenic plants of T3 generation were obtained.Compared with the wild type,there was no significant difference in seed size,but the bolting time of transgenic plants was 2-3 days earlier,and the stem turned purple.The fatty acid composition of seeds was analyzed by GC-MS.It was found that the content of ALA increased by19.53-43.29%,and the content of other polyunsaturated fatty acids also increased.PfFAD3.1 expression can promote the growth and development of recipient plants and the accumulation of polyunsaturated fatty acids.5.Gene expression of ALA synthesis pathway.in order to explore the effect of PfFAD3.1.on plant growth and fatty acid synthesis,the expression characteristics of genes related to ALA synthesis pathway were analyzed.It was found that the key enzyme genes of fatty acid synthesis and accumulation,such as ACCase,FAT,FAD2,DGAT and the regulatory factors of seed development,such as WRI1,ABI3 and FUS3 were up-regulated in seeds.PfFAD3.1 may be involved in the regulation of seed growth and development by regulating the metabolic flux of fatty acids.