Expression patterns and prokaryotic expression vectors screening of laccase genes involved in resistance to woolly apple aphid in different apple varieties

Abstract: 【Objective】Malus domestica is an important economic fruit crop in China, and the woolly apple aphid causes great damage to apple trees. There are differences in the resistance to the woolly apple aphid among different apple varieties. The laccase gene is a key enzyme in the lignin biosynthesis pathway and has an important role in disease and insect resistance. In this study, we analyzed the changes in laccase gene expression patterns caused by woolly apple aphid infestation in different apple variet-ies, identified their differential genes, and explored methods for prokaryotic expression of laccase proteins.【Methods】Three apple varieties, Starkrimson, Ralls Genet and Red Fuji, were used as research materials. Starkrimson and Ralls Genet were resistant to woolly apple aphid, while Red Fuji was susceptible to it. All three varieties were grown at the Fruit Tree Base of Jiaozhou Experimental Farm of Qingdao Agricultural University (120.11° E, 36.44° N). All trees were grafted seedlings. Healthy 1-year-old plants were selected in May 2019 and apple branches infested by woolly apple aphids for 0 h, 12 h and 5 d were collected. Three biological replicates for each treatment were performed. All samples were frozen in the liquid nitrogen and stored at -80 ℃ before being sent to the company for transcriptome testing. Using Arabidopsis laccase protein as the search sequence, a Blastp search was performed in the apple transcriptome to screen out candidate genes with E-value＜1e-5, and finally the laccase candidate genes were further confirmed by NCBI conserved domain database and Pfam database. At the transcriptome level, the expression patterns of laccase genes in different apple varieties before and after being infested by woolly apple aphid were analyzed to screen out candidate genes for aphid resistance. The genes with differential expression between groups was selected by p-value＜0.05 and Fold Change＞2. The full-length coding sequences of three apple laccase genes (MdLac23, MdLac6 and MdLac7) were amplified using high- fidelity enzymes with cDNA as the template. Primers were designed using Premier 5 software. The PCR products were finally ligated into four prokaryotic expression vectors, including pET-28a (+), pET-32a (+), pGEX-TEV and pHAT2, respectively, and transferred into Escherichia coli BL21 (DE3) receptor cells, followed by sequencing. The 20 correctly sequenced recombinant constructs were shaken at 37 ℃ and 200 r·min-1 until the OD600 reached 0.7-0.8, before they were induced at a final concentration of 0.5 mmol·L^-1 IPTG, 16 ℃, and 220 r·min-1 for 24 h. The protein expression was detected by Western-Blot using electrophoresis on a 10% SDS-PAGE gel. The two laccase proteins MdLac23 and MdLac2, which were successfully expressed in the supernatant of pET-28a (+ ) vector, were induced in large quantities. After ultrasonic fragmentation, the supernatant was purified by a Ni2 + column. Concentration and desalination of proteins were determined by using 30 ku ultrafiltration tubes. SDS-PAGE electrophoresis and Bradford method were used to detect the purity and concentration of proteins. The amount of enzyme required to oxidize 1 nmol of substrate ABTS [2, 2’-azinobis- (3-ethylbenzthiazoline-6-sulphonate)] per minute per mg of protein was taken as one unit of enzyme activity. The absorbance values of the two laccase proteins were measured before and after the reaction at 42 ℃ for 3 min at 420 nm with ABTS as the substrate.【Results】A total of 27 differential laccase expression genes were identified, 25 laccase genes had conserved structural domains specific to Cu-oxidase, Cu-oxidase 2 and Cu-oxidase 3 typical laccase, and two laccase MdLac25 (Cu-oxidase 3) and MdLac24 (Cu-oxidase 2) contained only one conserved structural domain. At the transcriptome level, the overall expression pattern of laccase genes was up-regulated in the two resistant varieties, Starkrimson and Ralls Genet, and down-regulated in the susceptible variety, Red Fuji, as the time of infestation by woolly apple aphid increased. Among them, 12 genes were up-regulated and no down-regulated genes existed in the Starkrimson variety; 9 genes were up-regulated and 3 genes were down-regulated in the Ralls Genet; 10 genes were down-regulated and 4 genes were up-regulated in Red Fuji. MdLac20, MdLac21 and MdLac22 were up- regulated in all three varieties; MdLac23 gene was only up- regulated in Starkrimson and Ralls Genet; MdLac24 and MdLac25 were down-regulated in Ralls Genet and up-regulated in Starkrimson; MdLac26 was up-regulated in Ralls Genet and Red Fuji; MdLac27 was down-regulated in Ralls Genet and Red Fuji. There were 6 unique up- regulated genes in Starkrimson, 5 unique up-regulated genes in Ralls Genet, and 9 genes uniquely down-regulated in Red Fuji. In the prokaryoticexpression system, the four laccase genes were not expressed in the pGEX-TEV and pHAT2 vectors; the MdLac7 and MdLac2 genes of the pET-32a (+ ) vector were expressed in the precipitate; the MdLac23 and MdLac2 genes of the pET-28a (+) vector showed clear protein bands in both the supernatant and the precipitate. EGFP (enhanced green fluorescent protein), MdLac23, and MdLac2 enzyme activity values were 408 U·mg^-1 , 427 U·mg^-1 and 433 U·mg^-1 , respectively, and both laccase proteins had no enzyme activity compared to the control EGFP.【Conclusion】In this study, the expression patterns of laccase genes in different apple varieties were clarified from transcriptomics, and laccase differential genes were identified. The laccase gene was expressed and the purified protein was obtained by screening different prokaryotic expression vectors.