Functional Study Of Lysophosphatidic Acid Acyltransferase 2 & 5 Underlying Seed Oil Synthesis In Brassica Napus

Brassica napus is one of the important oil crops,and rapeseed oil is an important source for edible vegetable oil and industrial oil.However,most of vegetable oil in china relies on imports.Therefore,analyzing the regulation mechanism of oil synthesis and metabolism and increasing the oil content of rapeseed are of important guiding significance for the cultivation of high-quality and high-yield rapeseed varieties.In the present study,lysophosphatidic acid acyltransferase 2&5(LPAT2&5),which are the key enzymes in the Kennedy pathway regulating TAG biosynthesis,were used to reveal the regulation mechanisms on seed oil biosynthesis of B.napus.The main findings are as follows:(1)The establishment of an efficient and convenient genetic transformation system in B.napus.DsRed,which encodes red fluorescent protein,was integrated into six plant expression vectors and transformed into the spring,semi-winter and winter rapeseed varieties successively by Agrobacterium-mediated hypocotyl transformation.Through tissue culture,calli and regenerated shoots with red fluorescence could be conveniently and quickly selected by visual screening based on red fluorescent protein,with an average efficiency of 59.9% and an average accuracy of 96.1%.Among 214 T1 transgenic plants obtained,the transgenic-positive efficiency was 80%?100%.This visual screening technology could also be applied to screen the seedlings at seed germination stage,thus is,all the cotyledon,hypocotyl,seed coat and root could be used for positive screening.This indicated that the efficient genetic transformation method combined with the convenient positive screening method could bring great convenience for efficiently obtaining transgenic-positive plants.(2)Effective editing of BnLPAT2 and BnLPAT5 in allotetraploid rapeseed.Four single-g RNAs and two multi-g RNAs were constructed into vectors carrying CRISPR-Cas9 for knocking out BnLPAT2 and BnLPAT5,and all the homologous copies on the A and C genomes corresponding to these two genes were successfully knocked out.The editing types at these targets were mainly consist of base insertion and base deletion,most of which were single base insertion,single base deletion and large fragment deletion.No gene editing was detected in the fourteen-potential off-target sites corresponding to these target sites.Cas9 also remained strong cleavage activity in progeny,indicating the high efficiency of CRISPR-Cas9 mediated gene editing.The T1 mutant seeds were wizened in different degrees companied with enlarged oil bodies in contrast to the wild type seeds.It was found that the seed oil content was decreased by 32% and 29% in the single-g RNA mediated Bnlpat2 and Bnlpat5 mutant,respectively;and an average of 39%decrease in the multi-g RNA mediated double mutant lines.These results lay the foundation for simultaneously knocking out multiple copies of multiple genes using CRISPR-Cas9 gene editing technology in allotetraploid rapeseed or other polyploid plants.(3)BnLPAT2 and BnLPAT5 promote the increase of seed oil content.Phenotypic analysis of the three type(i.e.,overexpression,RNAi,and CRISPR-Cas9-mediated knock-out)of T3 BnLPAT2 and BnLPAT5 transgenic plants showed an increase in oil content from 38.3% to 46.0% in BnLPAT2 and BnLPAT5 overexpression plants,but the oil content was significantly decreased to 26.8% and 25.5% in Bnlpat2 and Bnlpat5 mutant plants,respectively.In addition,the total sugar content and total protein content in mature seed were negative with the seed oil content.The microstructure of mature seeds revealed that the cell size in the BnLPAT2 and BnLPAT5 overexpression lines was much larger,and the number of oil body was also increased.The cell size of the Bnlpat2 and Bnlpat5 mutant lines was decreased,and the number of oil body was decreased significantly companied with significantly increased size for oil body.These results indicated that BnLPAT2 and BnLPAT5 genes could significantly promote the seed oil content in B.napus.(4)The mechanism of BnLPAT2 and BnLPAT5 regulating seed oil synthesis.Transcriptomics analysis was performed for the early,middle and late seed development stages in aforementioned BnLPAT2 and BnLPAT5 transgenic lines.It was showed that the seed development of the overexpression lines was lagging behind the mutant lines in the middle and late stages,and fatty acid and lipid synthesis related metabolic pathways were mainly involved in overexpression lines,which was conducive to seed oil accumulation.Besides,the synthesis of secondary metabolites and various biological stress related metabolic pathways were mainly involved in the mutant lines,which resulted in fatty acids degradation in seeds.In addition,the seed development of BnLPAT2 overexpression line always lags behind the BnLPAT5 overexpression line at the three seed development stages.It was further found that the former was involved in fatty acid/lipid synthesis and carbohydrate metabolic pathways,and BnLPAT2 gene tended to participate in promoting the synthesis of seed oil whereas BnLPAT5 gene tended to participate in promoting the synthesis of membrane lipids.These results indicated that BnLPAT2 and BnLPAT5 could promote the flow of carbon sources into lipid synthesis,and then differentially stored in mature seeds.In summary,a rapid,simple and efficient technology was established for screening transgenic positive plants,and effective gene editing for loss-of-function was achieved by CRISPR-Cas9 gene editing technology in allotetraploid rapeseed.Combined with the transcriptomics analysis at different stages of seed development,the regulation mechanism of BnLPAT2 and BnLPAT5 on seed oil accumulation was illuminated,which lay the foundation for the improvement and cultivation for new rapeseed varieties with high oil content.