Collection of Insect Tissue and Isolation of Poly (A)⁺ RNA

Female OBLR were obtained from a lab colony reared on a semisynthetic medium (Shorey, 1965) in a 16:8 h (L: D) at 25°C, 50% relative humidity. Fat bodies and pheromone glands were carefully dissected from 2-3-day old female moths and stored at −80°C. Poly A⁺ RNA (mRNA) was isolated and purified from fat bodies and pheromone glands by using a mRNA Isolation Kit (Ambion) according to the procedures recommended by the manufacturer.

Construction of cDNA Library

Using a GeneRacer™ Kit (Invitrogen), 1 µg of mRNA from different tissues was dephosphorylated with calf intestinal phosphatase and then decapped with tobacco acid pyrophosphatase. It was then ligated with GeneRacer™ RNA oligo and reverse-transcribed with GeneRacer™ Oligo dT Primer by reverse transcriptase. Two cDNA libraries, Cro-pheromone-gland cDNA library (Cro-PG-cDNA) and Cro fat-body cDNA library (Cro-FB-cDNA) were constructed. The abbreviation Cro uses the first letter of the species (Choristoneura) and first 2 letters of the species name (rosaceana).

Cloning of desaturase cDNAs from OBLR

Two degenerate primers, PR1 and PR2, were described previously (Liu et al., 1999; Liu et al., 2001, Hao et al., 2002 and Liu et al., 20027). These two primers were used to amplify the central region of the desaturase gene from the Cro-FB-cDNA. The PCR products were ligated to PCR2.1 TOPO vector for sequencing. Two fragments were obtained from this cDNA library: Cro-FB1-CR and Cro-FB2-CR. Two pairs of degenerate primers, PR3 plus PR4 and PR5 plus PR6 (Table 1) were designed to amplify the central region of desaturase gene from Cro-PG-cDNA. One fragment (Cro-PG-CR) was amplified from Cro-PG-cDNA. The central regions were compared with other central regions of known desaturase genes to evaluate the possibility that they were desaturase genes.

Based on the sequence information of Cro-FB1-CR and Cro-FB2-CR, gene-specific primers (Table 1) were designed for rapid amplification of cDNA ends (RACE). With Cro-FB-cDNA as template, A1 and GeneRacer 3′-P were used for the first-round PCR and A2 plus GeneRacer 3′-NP were used for the second-round PCR to amplify the 3′-end; A3 and GeneRacer 5′-P were used for the first-round PCR and A4 and GeneRacer 5′-NP were used for the second-round PCR to amplify the 5′-end. With the same cDNA library, B1 and GeneRacer 3′-P were used for the first-round PCR and B2 plus GeneRacer 3′-NP were used for the second-round PCR to amplify the 3′-end; B3 and GeneRacer 5′-P were used for the first-round PCR and B4 plus GeneRacer 5′-NP were used for the second-round PCR to amplify the 5′-end. Similarly, with Cro-PG-cDNA as template, C1 and C2 were used for the 3′-RACE PCR and C3 and C4 were used for 5′-RACE PCR. All the RACE PCR products were cloned to PCR2.1 TOPO vector for sequencing. With the sequencing results of the 5′-end, central region and 3′-end, two full-length cDNA sequences (Cro-FB1 and Cro-FB2) were generated from the Cro-FB-cDNA library and one full-length sequence (Cro-PG) was obtained from Cro-PG-cDNA library.

Functional Assay in YEpOLEX System

Gene-specific primers, A5 plus A6, B5 plus B6 and C5 plus C6 (Table 1) were designed to amplify the open reading frames (ORF) of the Cro-FB1, Cro-FB2 and Cro-PG genes. The PCR products of ORFs were digested and ligated with linearized YEpOLEX, as described previously (Knipple et al., 1998; Liu et al., 1999; Liu et al., 2001, Hao et al., 2002 and Liu et al., 2002a, 2002b). The consensus clone of the recombinant plasmids (YEpOLEX-Cro-FB1, YEpOLEX-Cro-FB2 and YEpOLEX-Cro-PG) were obtained and transformed to mutant yeast cells (strain L8-14C) (Stukey et al., 1990) for functional expression. The yeast cells were inoculated into 50 ml YPD medium and grown at 30°C overnight with shaking (300 rpm). If the cells did not grow overnight, then either Z11-18:Acid (0.5 mM) or Z9-14:Acid (0.5 mM) was added to the medium to promote growth. The induced cells were transferred to a 50-ml sterile centrifuge tube and spun at 1,500 × g for 5 min. The cell pellet was washed two times with 0.2% BSA, transferred to a 1.5-ml tube, and spun briefly to remove as much liquid as possible. The washed yeast cells were lysed with 1 ml of Y-PER (Yeast Protein Extraction Reagent, PIERCE) with brief vortex and 20 min agitation at room temperature. The cell debris was collected by centrifugation at 13,000 × g for 10 min and extracted with 0.5 ml of chloroform/methanol (2:1) at room temperature for 1 hr. The solvent was decanted from the debris and evaporated under nitrogen. The oily residue was extracted twice with 0.5 ml 10% boron trichloride/methanol, and the combined extracts heated at 100°C for 30 min. The resulting fatty acid methyl esters were extracted with 1 ml hexane and the solution concentrated under nitrogen for analyses by GC/MS, using a Hewlett Packard 5890 gas chromatograph (splitless mode) coupled to an HP 5970 B Mass Selective Detector (DB-1MS capillary column, 30 × 0.25 mm ID, 0.25 mm film thickness, J & W Scientific, Folsum, CA). The oven temperature was held at 100°C for 2 min., raised at 10°C/min to 200°C, held for 10 min, and raised at 3°C/min to 300°C. The double bond position in the products was confirmed by mass spectral analysis of the DMDS adducts (Buser et al., 1983) under the same conditions as for the methyl esters.

Functional Assay in pYES2 System

Since Cro-FB2 was not expressed, and Cro-PG not expressed well, in the YEpOLEX system, a pYES2 expression system (Invitrogen) (Liu et al., 2002a; 2002b) was used for the functional assay. Gene-specific primers, B7 plus B8, C7 plus C8 (Table 1) were designed to amplify the ORFs of Cro-FB2 and Cro-PG for pYES2 vector construction. The pYES2 plasmid containing consensus ORF was transformed to Saccharomyces cerevisiae strain elo1 competent cells (Toke and Martin, 1996) with the methods described previously (Liu et al., 2002a; 2002b). A single elo1 colony transformed with pYES2-Cro-FB2 or pYES2-Cro-PG, which grew on an SC-U plate (0.67% YNB, 0.19% SC-U amino-acid mixture, 2% glucose and 2% agar) (Bio 101), was inoculated into 30 ml SC-U liquid medium (0.67% YNB, 0.19% SC-U amino-acid mixture, 2% glucose) in a 250-ml flask. The yeast cells were grown at 30°C overnight with shaking (300 rpm). After 16–24 hrs, the cells were collected by spinning at 500 × g for 5 min. After discarding the supernatant, the cell pellet was washed one time with induction medium (0.67% YNB, 0.19% SC-U amino-acid mixture, 1% raffinose and 1% galactose) and suspended with induction medium containing 1% tergitol with a cell density of 5 × 10⁷/ml. Thirty ml of the cell suspension was added to each 250-ml flask with or without addition of 0.5 mM myristic acid methyl ester (MAME) for desaturase induction at 18°C with shaking (300 rpm) for three days. The induced cells were harvested and extracted with 2:1 chloroform/methanol for GC/MS analysis with the same methods described above.

Cloning and functional assay of Cro-FB1 [Cro-Z9(16)]

RT-PCR including RACE PCR with Cro-FB-cDNA library produced a full-length cDNA sequence (Cro-FB1) that spans 2171 nt encoding a protein with 352 amino acids (aa), The deduced aa sequence has high identity to other known moth Z9-desaturases from Tricoplusia ni, Epiphyas postvittana, Argyrotaenia velutinana, Heliothis zea, and Planotortrix octo (Liu et al., 1999; 2002a; 2002b; Rosenfield et al., 2001; and Hao et al., 2002) (Fig. 1).

Mutant ole1 yeast cells (L8-14C) were successfully used for the functional assay of Cro-FB1. After transformation with YEpOLEX-Cro-FB1 the yeast cells grew well in YPD medium without addition of UFAs. GC/MS analysis of the unsaturated fatty acid methyl esters generated by transmethylation of acyl compounds extracted from the yeast cells showed that this desaturase produced Z9-16:Acid and Z9-18:Acid in a 2:1 ratio (Fig. 2). It was labeled Cro-Z9(16) to differentiate it from the Z9-desaturases that produce 18:Acid>16:Acid.

Cloning and functional assay of Cro-FB2

RT-PCR including RACE PCR with Cro-FB-cDNA library produced a full-length cDNA sequence (Cro-FB2) that spans 1142 bp encoding a protein with 328 amino acids (aa). The deduced aa (Fig. 3) has low identity to other known moth Z9-desaturases (44% to Cro-FB1, 41% to T. ni, 43% to A. velutinana, 46% to H. zea and 45% to P. oct) and ▵11-desaturases (48% to T. ni, 53% to H. zea, 49% to A. velutinana, and 49% to E. postvittana. This deduced aa sequence has 51% identity to that of Cro-PG.

Yeast cells L8-14C transformed with YEpOLEX-Cro-FB2 were not able to grow without addition of UFAs. With addition of Z9-14, the transformants grew in YPD, but the GC/MS assay did not reveal any new products. Yeast cells elo1 transformed with pYES2-Cro-FB2 also did not yield any new fatty-acid products. Attempts to obtain any unsaturated product from this clone with various saturated and mono-unsaturated precursors, such as 14:Acid, E9-14:Acid, Z11-14:Acid, E11-14:Acid, in the YEpOLEX and the pYES2 expression systems all failed.

Cloning and functional assay of Cro-PG (Cro-Z/E11)

Cro-PG was amplified from Cro-PG-cDNA library and the full-length cDNA sequence spans 1300 nt and contains an ORF encoding a 335-aa protein. This deduced aa has high identity to other Z11-, Z/E11- and E11-desaturases (57% to T. ni; 66% to H. zea; 81% to A. velutinana, 75% to E. postvittana; 62% to O. nubilalis) (Knipple et al., 1998; Rosenfield et al., 2001; Liu et al., 2002a; 2002b; Roelofs et al., 2002) and low identity (ca. 48%) to Z9-desaturases (Fig. 1).

The ole1 cells transformed with YEpOLEX-Cro-PG did not grow in YPD medium, and with the addition of UFAs, a trace amount of Z/E11-14:Acids was detected from the YEpOLEX system (data not shown). With the pYES2 system, GC/MS analysis of the unsaturated fatty acid methyl esters generated by transmethylation of acyl compounds extracted from yeast cells elo1 transformed with pYES2-Cro-PG showed that Cro-PG desaturase produced both Z11-14:Acid and E11-14:Acid with a ratio of 7:1 when MAME was added into the induction medium. Control elo1 cells with pYES2 plasmid only produced normal Z9-UFAs, but no Z/E11: acids were produced under the same conditions (Fig. 4). The elo1 cells are deficient in the chain-elongation enzyme and so the products were not elongated to Z/E13-16:Acids as in normal yeast cells.