| Perilla(Perilla frutescens) as a new oil crop with a unique medicine value for human health, is beginning to take notice. The perilla seed oil contains higher level of a-linolenic acid, a omega-3 fatty acid essentially required for human nutrition and healthy. In this study, PfPDAT gene was cloned, related to regulation of perilla seed a-linolenic acid synthesis and accumulation. Gene expression in different tissues and single nucleotide polymorphism analysis of different varieties was performed to investigate the relationship between PfPDAT gene expression and the synthesis of a-linolenic acid accumulation. The present study will generate new knowledge for the further application of transgenic technology and metabolic engineering to cultivate new varieties of perilla enriched a-linolenic acid, and provide genetic element and technology system for other ordinary oil crop new lines. The main findings obtained in this study were described as the followings:(1) The full-length cDNA sequence of the target gene 2097 bp was obtained, containing 1554 bp open reading frame (ORF) which encoded a pepitide of 517 amanio acides, and two non-coding regions,44 bp 5’UTR and 499 bp 3’UTR.(2) According to the PfPDAT gene cDNA squence, bioinformatics analysis was performed. The results showed that:perilla PDAT protein molecular weight was 56923.5 KDa, theoretical isoelectricpoint pI was 5.45, the protein was a stable hydrophilic protein, and there were 3 transmembrane helix models. Multiple sequence alignment and phylogenetic tree analysis showed that PfPDAT protein had the closer relationship with sesame PDAT1. PfPDAT protein was highly conservative. By the prediction of protein secondary structure, PfPDAT protein accounted for 32.30% α-helix,39.46% random coil,17.99% extension chain and 10.25%β-corner. Protein domains encoded by PfPDAT gene belonged to the PLN02733 super family.(3) The expression characteristics of PfPDAT at transcription level were investigated by real-time qPCR. The results showed that the expression of PfPDAT gene in roots, stems and flowers of perilla was quite low level, but the expression leave of PfPDAT was higher in leaves and seeds. With the continuous development of the seed, Jin Sul, Zhong Bei, Bai Su and Y-4 at 10 days after flowering achieved the highest expression levels and then decreased; while PfPDAT gene expression in Y-7, Y-2 and Y-Zao was first increased and then decreased, the seed of 20 days after flowering had highest expression levels.(4) In this study,7 perilla varieties with different oil content as materials, single nucleotide polymorphisms of the full-length cDNA sequence of PfPDAT gene were analyzed by direct sequence. The results showed that:the total length of the nucleotide sequence was 14679 bp, including 9 SNP and 2 InDel. The frequencies of SNP and InDel were 1/1631 and 1/7340 respectively, which 2 SNP sites from the coding region,7 sites from non-coding region. The SNP frequencies in coding region and in non-coding region were 1/5439 bp and 1/543 bp respectively. The π of the coding region was less than the non-coding region. Therefore, the variation frequency of PfPDAT gene in coding region was lower than in non-coding region. PfPDAT gene sequence Ka/Ks was less than 1, showed that the nucleotide sequence of the gene was relatively conservative. Different haplotypes were detected in the tested accessions according to DNA sequence polymorphism of PfPDAT. Haplotype H1 and H2 both included the higher oil content materals and low content materials, synchronously. It seemed that PfPDAT gene haplotype classification was incompletely consistent with perilla oil content, meanwhile it also showed further that plant seed oil synthesis was very complicated. |
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