| Peanut(Arachis hypogaea L.)originated in South America and is one of the most important oil crops in the world.It is grown in many regions of the world.Peanut is an important source of edible oil in China.With the improvement of economic level and people’s increasing demand for edible oil,the quality of edible oil is also more focused.The self-sufficiency rate of edible oil in our country is seriously insufficient and we rely on imports for a long time,which has led to lower prices of oil crops and reduced planting area in our country.However,increasing peanut production has long been the focus of research,whereas the research on peanut oil content and peanut oil quality is relatively lagged.Therefore,people pay more and more attention to how to use genetic engineering and molecular biotechnology to regulate the related genes of peanut oil synthesis,and then improve the quality and oil content of peanut oil.In higher plants,the synthesis of fatty acids(FAs)mainly occurs in plastids.Transshipment of FAs depends on the activity of acyl-ACP thioesterases(FAT)that hydrolyze acyl-acyl carrier protein(acyl-ACP)to release free FAs and ACP.Therefore,FAT plays an important role in controlling the content and composition of FAs in plants.In this study,two acyl carrier protein thioesterase genes(AhFATBs)were cloned from "Fenghua 1",a peanut cultivar,and their sequences were analyzed by bioinformatics.The expression pattern and subcellular localization were analyzed.The two genes were transformed into yeast and Arabidopsis for function identification.The promoter of the two genes was cloned and the vectors were constructed for function identification.Results are as follows:(1)In order to better understand the peanut FATs(Ah FATs),we carried out the bioinformatics analysis of Ah FAT gene family.There are 20 FATs in peanut genome,and unevenly distributed on 12 chromosomes in 2 genomes.Because the Araip.T3UA2 gene sequence is incomplete,and will influence the analysis result,so it is not used.Through phylogenetic tree analysis,it is found that FATs exist widely in plants and are the older genes.19 Ah FATs gene are divided into two subfamilies: Ah FATA and Ah FATB.There are 2 members in Ah FATA subfamily,17 members in Ah FATB subfamily.The Ah FATA genes have 6 or 8 exons,and the Ah FATB genes have 5 to 8 exons respectively.None of the Ah FATs have transmembrane domains,but they all have the conserved Acyl-ACP＿TE domain.Results of alternative splicing analysis of Ah FATs showed that only a few of Ah FATs had alternative splicing events,and it accounted for about 25%,and the splicing isoforms had tissue-specific expression patterns.(2)According to the sequence information obtained by transcriptome sequencing,two AhFATBs named Ah FATB2-1 and Ah FATB2-2 were cloned from the peanut cultivar "Fenghua 1".Sequence analysis showed that the nucleotide sequence similarity was 99.36%,encoding 414 amino acids,and amino acid sequence similarity was 100%.Same as the other plant FATBs,AhFATBs have a typical conserved Acyl-ACP＿TE domain.Phylogenetic tree analysis showed that Ah FATB2-1 and wild peanut FATBclustered in the same branch,indicating their closest relationship;and they were clustered together with Glycine max,Medicago sativa and Vigna radiata,indicating their closer phylogenetic relationship.(3)The results of AhFATBs expression pattern analysis showed that the two genes were expressed in all tissues,but there were significant differences between them.In the four tissues(root,stem,leaf,flower),AhFATBs expressed at the higher level in flower,then in root;whereas in stem and leaf,it was lower.In the four different seed development stages,AhFATBs expressesd at the higher level at 15 d and 30 d,whereas at a low level at 45 d and 60 d.(4)Using the transient expression system of Arabidopsis protoplast,the subcellular localization of AhFATBs was analyzed.Results showed that they were located in chloroplasts.(5)The yeast expression vectors were constructed for genetic transformation of yeast strains.Gas chromatographic method was used for analysis of the content and composition of the FAs of each transformed yeast strains.Results showed that the composition of the FAs did not change between the transformed yeast strains and the control,but the total FAs content increased,whereas the content of C16:1,C18:0,C18:1 was significantly increased by 24.7% 、 36.5% 、 56.1% and 24.7% 、 22.6% 、 50.6%,respectively.(6)The plant over-expression vectors were constructed for genetic transformation of Arabidopsis.Gas chromatographic method was used for analysis of the FAs content and the composition.Results showed that the total FAs content and the main FAs content of the transgenic Arabidopsis seeds were significantly higher than that of wild Arabidopsis seeds.The total content of FA increased by 69.0% and67.8% respectively,the content of C16:0 and C18:0 were 87.5 mg/g dry weight,19.3 mg/g dry weigh and88.0 mg/g dry weight,19.5 mg/g dry weight,respectively,increased by 5.4 and 3.5 times with the control.(7)The promoter sequences of the two genes were cloned,and they were 1316 bp and 1489 bp,respectively.Sequence analysis showed that there were typical CAAT-box and TATA-box elements within the promoters.In addition,there were some photoresponse elements and cis-acting elements related to defense and stress.To verify the promoter function,we successfully constructed the GUS-plant expression vector. |
