| Linseed flax is a kind of ancient oil-bearing crop,its grains contain a variety of vitamins,amino acids.And the linseed is rich in α-linolenic acid,which is essential for human body.α-linolenic acid is a precursor of the EPA(EPA,eicosapntemacnioc acid)and DHA(DHA,docosahexaenioc acid).All these three fatty acids help lower blood sugar,blood fat,prevent the happening of the disease of heart head blood-vessel.Therefore,linseed has the high nutrition function and edible value.Previously,the capsules of Macbeth with high-oil content and Heiya No.14 with low-oil content were collected at 5 d,10 d,20 d and 30 d after pollination and performed transcriptome sequencing.Meanwhile,the four stages of seed oil content were measured respectively.It is found that the oil contnet of Macbeth accumulated heavily.Some of the LEA genes expressed a lot at the late seed development stages as well.Thus,it is deduced that LEA genes play an important role in the seed development process.To explore the mechanism of LEA genes in the seed development,studies conducted are presented as follows:1.A total of 50 LuLEA genes were identified in flax genome by BLAST and HMM,divided into 8subfamilies: LuLEA＿1～6,Ludehydrin and Lu SMP.LEA phylogenetic tree was constructed by MEGA software using maximum likelihood method.With a variety of online tools,the prediction of the physical and chemical properties,subcellular localization,chromosome distribution and sequence characteristics of LuLEA were analyzed.The results showed that the length of most LuLEA genes is shorter,and the proteins’ molecular weight is small.Most LuLEA proteins are hydrophilic,and located in the nucleus.Whereas,several LuLEA also exsist in the mitochondria and chloroplasts.LuLEA genes were distributed on all 15 chromosomes,in which Lu1 chromosome was the most widely distributed.The number of introns of LuLEA genes was less than 2,and the protein motifs of each subfamily differed greatly.2.The heat maps of each LuLEA subfamily were ploted and analyzed.It was found that 36 LuLEA genes expressed throughout the four stages of seed development,among which LuLEA＿1,LuLEA＿4～6and Lu SMP genes were accumulated abundantly at 20 DAP and 30 DAP,while the others did not show consistent regularity.3.Three LuLEA genes of LuLEA1,LuLEA2,LuLEA42 were screened from the transcriptome database with high expression level,and overexpressed in Arabidopsis thaliana.The transgenic seeds of T3 generation were obtained,whose characteristics and fatty acid content were analyzed.It is found that the values of the 1000-seed weight,seed area means,seed circumatances and fatty acid content of the LuLEA1 and LuLEA2 transgenic seeds have dropped significantly compared to that of WT,while LuLEA42 transgenic seed weight also declined significantly,and the mean area and circumference also decreased,but the total fatty acid content increased slightly.All these seed traits results suggested that LEA＿1 genes may play a negative role in regulating the seed size and fatty acid content by participating in the seed development process.4.The homologous genes of LuLEA1 and LuLEA2 were knocked out with CRISPR technology in Arabidopsis thaliana,and 9 T1 generation genome-edited plants were obtained.The 1000-seed weight of most genome-edited seeds was significantly increased,and the seed area and circumference mean were significantly increased,demonstrated that LEA genes may negatively regulate the seed size further.5.Seven LuLEA1 flaxseed mutants were screened by TILLING technique,and the changes of fatty acids in the mutants C18:1 and C18:3 were the most obvious,showing a roughly negative correlation,indicating that LuLEA1 gene may affect the changes of the fatty acid proportion.To sum up,LEA gene family has many members,and most LEA proteins are hydrophilic proteins.LEA＿1 subfamily genes may play a negative regulatory role in seed development,as well as affecting the fatty acid content of seeds.And its specific function mechanism remains to be studied further. |
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