A high‐throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis

Abstract

SummaryIt is desirable to produce high homogeneity of novel fatty acids in oilseeds through genetic engineering to meet the increasing demands of the oleo-chemical industry. However, expression of key enzymes for biosynthesis of industrial fatty acids usually results in low levels of desired fatty acids in transgenic oilseeds. The abundance of derivatized fatty acids in their natural species suggests that additional genes are needed for high production in transgenic plants. We used the model oilseed plant Arabidopsis thaliana expressing a castor fatty acid hydroxylase (FAH12) to identify genes that can boost hydroxy fatty acid accumulation in transgenic seeds. Here we describe a high-throughput approach that, in principle, can allow testing of the entire transcriptome of developing castor seed endosperm by shotgun transforming a full-length cDNA library into an FAH12-expressing Arabidopsis line. The resulting transgenic seeds were screened by high-throughput gas chromatography. We obtained several lines transformed with castor cDNAs that contained increased amounts of hydroxy fatty acids in transgenic Arabidopsis. These cDNAs were then isolated by PCR and retransformed into the FAH12-expressing line, thus confirming their beneficial contributions to hydroxy fatty acid accumulation in transgenic Arabidopsis seeds. Although we describe an approach that is targeted to oilseed engineering, the methods we developed can be applied in many areas of plant biotechnology and functional genomic research.

Development of marker-free transgenic Jatrophaplants with increased levels of seed oleic acid

“…As shown in Additional file 2, the expression of FAD2-1 gene is high in seeds and weaker in vegetative tissues, while the FAD2-2 gene is expressed highly in seeds and not detectable in leaf tissue. The expression pattern of these two FAD2 genes in Jatropha is very similar to others in the Euphorbiaceae: FAD2 and Fatty acid hydroxylase 12 ( FAH12 ) in castor bean [13], FAD2 and FADX in the tung tree ( Aleurites fordii ) [12]. All the above data suggest that JcFAD2-2, similar to FADX and FAH12, may function more like an unusual fatty acid enzyme than a desaturase.…”

A Small Phospholipase A2-α from Castor Catalyzes the Removal of Hydroxy Fatty Acids from Phosphatidylcholine in Transgenic Arabidopsis Seeds

“…A common motif (-LHKP) is found at the C terminus of both the rice and castor sPLA 2 a sequences, constituting a putative ER retention signal (Singh et al, 2012). RcsPLA 2 a and RcsPLA 2 b were cloned from a castor endosperm complementary DNA (cDNA) library, introduced into the expression vector pGate-DsRed-Phas (Lu et al, 2006), and expressed under the control of the seed-specific phaseolin promoter (Slightom et al, 1983) in the Arabidopsis CL37 line. CL37 is an Arabidopsis line expressing RcFAH12 and accumulating 17% HFAs, including ricinoleic acid and densipolic acid (12-hydroxy-octadec-cis-9,15-enoic acid [18:2-OH]; Lu et al, 2006).…”

“…RcsPLA 2 a and RcsPLA 2 b were cloned from a castor endosperm complementary DNA (cDNA) library, introduced into the expression vector pGate-DsRed-Phas (Lu et al, 2006), and expressed under the control of the seed-specific phaseolin promoter (Slightom et al, 1983) in the Arabidopsis CL37 line. CL37 is an Arabidopsis line expressing RcFAH12 and accumulating 17% HFAs, including ricinoleic acid and densipolic acid (12-hydroxy-octadec-cis-9,15-enoic acid [18:2-OH]; Lu et al, 2006). CL37 also is mutated in its fatty acid elongase (fae1) gene (Kunst et al, 1992) and thus is devoid of the verylong-chain HFAs lesquerolic acid (20:1-OH) and auricolic acid (20:2-OH), simplifying the FA profile analysis by gas chromatography (GC).…”

“…The RcsPLA 2 a sequence in pENTR was then sequenced using the BigDye sequencing kit (ABI), primers RcPLA21/f and RcPLA21/r, and the additional primers M13(-21) (59-TGTAAAACGACGGCCAGT-39) and M13r (59-AACAGCTATGACCATG-39). pENTR-RcPLA21-2 was combined with pGateDsRed-Phas (Lu et al, 2006) after an LR clonase reaction (Life Technologies). DNA resulting from minipreparations of positive XL10-Gold colonies was again sequenced with the above-mentioned primers and used to electroporate Agrobacterium tumefaciens strain GV3101 for dipping Arabidopsis flowers (Clough and Bent, 1998).…”

“…With the aim of gaining more insights into the involvement and identities of PLA 2 s involved in acyl editing and HFA metabolism, with potential downstream industrial applications, we made use of the available castor endosperm transcriptome database (Troncoso-Ponce et al, 2011) and the transgenic Arabidopsis seed line CL37 expressing the castor fatty acid hydroxylase FAH12 (Lu et al, 2006). Among the PLA genes examined from the castor endosperm transcriptome, RcsPLA 2 a was the most highly expressed, indicating possible involvement in ricinoleate metabolism in castor.…”