Investigation Of Regualation Of The Oil Biosynthesis By Nucleotide Triphosphate Transporter In Brassica Napus L.

Seed oil content is one of the important agronomic traits in rapeseed.Increasing the oil content of rapeseed can increase the oil production and improve economic benefit of rapeseed.The biosynthesis of fatty acids in plants is a very complex regulatory network,which is not only regulated by various enzymes,substrates,energy and transcription factors in plants,but also influenced by external environmental factors such as light,temperature and oxygen concentration.The nucleotide triphosphate transporter(NTT)can transport ATP from cytosolic into the plastid to meet the needs of fatty acid synthesis and other metabolic activities.However,whether NTT participates in the regulation of seed oil synthesis through transporting ATP has not been reported.Based on the relationship between energy metabolism and lipid biosynthesis,this study explores the mechanism of BnNTT gene involved in regulation of fatty acid biosynthesis using molecular biology methods in Brassica napus L.Firstly,this study constructed the vector of overexpression and CRISPR/Cas9 gene editing system,respectively.After that,the vector was transformed into the Westar material of Brassica napus L.,and transgenic plants were obtained.Oil content and fatty acid composition of Brassica napus L.seeds in transgenic plants were analyzed by GC-MS.Finally,we combined with the data of gene expression during seed oil accumulation in Brassica napus L.,to comprehensively analyze the regulation of fatty acid synthesis by BnNTT genes.It was found that overexpression of BnNTTA7-1 gene driven by CaMV 35S promoter in transgenic plants reduced the oil content of mature seeds,suggesting that overexpression of BnNTTA7-1 had a certain negative effect on fatty acid synthesis in Brassica napus L.seeds.In addition,CaMV 35S promoter was used to drive the overexpression of BnNTTC6-1,BnNTTC6-2 and BnNTTC8 genes,respectively.Oil content of mature seeds increased significantly,indicating that BnNTTC6-1,BnNTTC6-2and BnNTTC8 positively regulated the accumulation of oil in Brassica napus L.seeds.In addition,CRISPR mutants of BnNTTA7-1,BnNTTC6-1 and BnNTTC6-2,were obtained by CRISPR/Cas9 gene editing technology.It was found that the oil content was not significantly different between the mutant A7C6~Mutantutant plants and wild type.At the same time,the oil content of the CRISPR mutant plants of BnNTTC8 gene was significantly lower than that of the wild type,which confirmed that BnNTTC8 positively regulates the anabolic pathway of fatty acids in seeds.The results also showed that the fatty acid composition of CRISPR mutant material was significantly different from that of transgenic plant material with 35S overexpression vector,indicating that the four gene of BnNTTA7-1?BnNTTC6-1?BnNTTC6-2 and BnNTTC8 had different regulatory mechanisms for oil synthesis in Brassica napus L.In this study,we investigated the regulation of NTT transporters in the oil biosynthesis of Brassica napus L.seeds.The finds fill the gap in the molecular mechanism of NTT transporters in lipid anabolism,and also provide a theoretical basis for rapeseed high-oil breeding and a new idea for oil synthesis of plant.