| Perilla frutescens(L.)Britt.,as a new type of oil crop,has attracted more and more attention because of its rich α-linolenic acid.It is one of the first plants of medicinal and edible homologous crops announced by National Ministry of Health.Alpha-linolenic acid in perilla seed oil is as high as 60%.Long-term edible is beneficial to human health,especially in promoting brain development and preventing cardiovascular diseases.Lysophosphatidic acid acyltransferase(LPAT)is a key enzyme in the process of plant seed oil synthesis and accumulation.It can catalyze the transfer of acyl-Co A fatty acids to the sn-2 position of triacylglycerol(TAG)to form phosphatidic acid(PA).Moreover,different types of LPAT have preferences for different substrates,and are often used to improve the fatty acid composition of vegetable oils in genetic engineering.In this study,the LPAT enzyme genes were identified and analyzed from perilla transcriptome databases,and the temporal and spatial expression profile of PfLPATs genes were detected.Those highly expressed genes in seeds were screened for further molecular cloning,and their biological functions were identified through heterologous expression yeast(Saccharomyces cerevisiae)and two kinds of tobacco(Nicotiana tobaccum and N.benthamiana).It provided a theoretical basis for in-depth exploration of the mechanism of LPAT genes in the process of perilla oil synthesis and accumulation,and at the same time provided excellent genetic elements for the specific accumulation of C18:1 in oil crops.The main results are as follows:1.Using the Arabidopsis AtLPATs protein sequence as a probe,BLAST search was carried out in the perilla transcriptome databases.A total of 11 PfLPATs gene sequences were obtained and renamed.The results of bioinformatics analysis showed that these 11 PfLPATs sequences all contained a complete coding region,and they all had a conserved region similar to lysophosphatidic acid acyltransferase.The coding amino acid lengths were between 250 and 384 aa.Except for PfLPAT1 which was located in the chloroplast,other proteins were located in the endoplasmic reticulum.The results of cluster analysis showed that the 11 PfLPATs gene sequences of perilla belonged to three subtypes.Among them,type 1 LPAT had one gene,type2/3 LPAT contained four genes,and type 4/5 LPAT contained six genes.But no B-type LPAT sequence was found.2.The expression profiles of 11 PfLPATs genes in different tissues and different development stages of’Jinzisu 1’ seed were analyzed by RT-PCR and q RT-PCR techniques.The results showed that these 11 genes were expressed in different tissues,but the expression patterns were different.PfLPAT1 was relatively high in flowers and early seeds,the overall expressions of type 4/5 LPAT genes were low,and the expressions of type 2/3 LPAT were high.Among them,the expressions of PfLPAT2-1 and PfLPAT2-3 in seeds were significantly higher than others genes.It was speculated that these two genes may be involved in the accumulation of perilla seed oil.Therefore,using the c DNA of seeds on 20 days after flowering as a template,the coding sequences of PfLPAT2-1 and PfLPAT2-3 were successfully amplified with high-fidelity RT-PCR,and their sizes were 1155 bp and 1149 bp,respectively.3.In order to verify the function of the PfLPAT2 gene of perilla,we constructed yeast recombinant expression vector p YES2.0-PfLPAT2-1 and p YES2.0-PfLPAT2-3,heterologously expressed PfLPAT2-1 and PfLPAT2-3 in INVSc1 Saccharomyces cerevisiae,and analyzed the total fatty acid content and fatty acid composition of the recombinant yeast.The results showed that the total lipid content of the two transgenic yeasts increased by 1.4% and 2.5%,respectively,and the proportion of C16:1 and C18:1 in the transgenic yeast increased significantly,especially C18:1 increased by 6.36% and 10.27%,respectively.The substrate selectivity of PfLPAT2-1 and PfLPAT2-3 were further analyzed by adding exogenous fatty acids(C18:0,C18:1,C18:2 and C18:3),and the yeast growth viability and fatty acid composition were detected.The analysis results showed that these two genes have substrate specificity for C18:1.4.The plant overexpression vector p CAMBIA1303-PfLPAT2-1 and p CAMBIA1303-PfLPAT2-3 were successfully constructed,and consequently transformed into Nicotiana benthamiana leaves by Agrobacterium-mediated infiltration for transiently express the target gene.The results showed that the total fatty acid content increased by 2.1% and 3.2%,respectively,and the proportions of each fatty acid component were changed.Among them,C18:1 increased by 6.21% and 8.37%,and C18:3 increased by 3.47% and 2.46%,respectively.C16:0 reduced significantly.Furthermore,the two target genes were separately introduced into common tobacco by Agrobacterium-mediated leaf disc transformation.The total lipid content and fatty acid composition of transgenic tobacco leaves were analyzed.It showed that the heterologous expression of PfLPAT2-1 and PfLPAT2-3 genes could increase the total fatty acid content of tobacco leaves and change the proportion of fatty acid components.The C18:1 content increased by 10.15% and 11.84%,respectively.The C18:3 content also increased.These results further showed that PfLPAT2-1 and PfLPAT2-3 have more preferences for oleic acid. |
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