CRISPR/Cas9-Targeted Mutagenesis of BnaFAE1 Genes Confers Low-Erucic Acid in Brassica napus.

Yunhao Liu,Shan Tang,Shaoping Lu,Haoming Li,Shengli Lin,Liang Guo,Zhuolin Du

doi:10.3389/fpls.2022.848723

Yunhao Liu, Shan Tang + Show 5 more

Open Access

https://doi.org/10.3389/fpls.2022.848723

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Abstract

Rapeseed (Brassica napus) is an important oilseed crop widely planted in the world, providing substantial edible oil and other nutrients for mankind. The composition of fatty acids affects the edible and processing quality of vegetable oils, among which erucic acid (EA) is potentially to cause health problems. Therefore, low erucic acid (LEA) has always been a breeding trait of B. napus. Fatty acid elongase 1 (FAE1) plays a decisive role in the synthesis of EA. There are two functional homologous copies of FAE1 on the A08 and C03 chromosomes in B. napus. In this study, we used CRISPR/Cas9 technology to create targeted mutations on these two homologous copies of BnaFAE1 in three B. napus germplasms with high EA (>30%) and high oil (>50%). Our results show that the EA content was significantly reduced by more than 10 percentage points in the mutant of BnaC03.FAE1 (c03), while the double mutation of BnaA08.FAE1 and BnaC03.FAE1 (a08c03) resulted in nearly zero EA in three BnaFAE1-edited germplasms, and the oleic acid content was increased in different degrees. In addition, knockout of BnaA08.FAE1 or/and BnaC03.FAE1 mildly decreased seed oil content, but had no significant effect on other agronomic traits. In general, we successfully created low EA germplasms of B. napus, which provides a feasible way for future low EA breeding.

Highlights

In oilseeds, the de novo synthesis of fatty acids occurs in plastids with acetyl-coenzyme A (CoA) as substrates
Fatty acids were determined by gas chromatograph (GC) analysis, and the results show that erucic acid (EA) of these three germplasms were between 31.05 and 34.95 mol% (Figure 1A)
There are four homologous copies of BnaFAE1 in B. napus and the expression data in different tissues showed that BnaA03.Fatty acid elongase 1 (FAE1) and BnaC03.FAE1-2 were barely expressed in different tissues, while BnaA08.FAE1 and BnaC03.FAE1 were mainly expressed in the developing seeds, especially in the middle and late periods of seed development (Figure 2B)

Summary

Introduction

The de novo synthesis of fatty acids occurs in plastids with acetyl-coenzyme A (CoA) as substrates. Acetyl-CoA carboxylase (ACC) catalyzes the condensation of acetyl-CoA and CO2 to form malonyl-CoA, the malonyl group of malonyl-CoA is transferred from CoA to acyl carrier protein (ACP) under the catalysis of malonyl-CoA:acyl carrier protein malonyltransferase (MCMT). Acetyl-CoA and malonyl-ACP, respectively, enter the fatty acid synthesis complex (FAS) and undergo a series of reactions including condensation, reduction, dehydration, and re-reduction to form C4:0-ACP. Going through the same cycle reaction, C4:0-ACP reacts with acetyl-CoA, adding two carbons every cycle, to produce C16:0-ACP eventually. Each cycle uses malonyl-ACP as a source of 2C units

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