Cloning and bacterial expression of a 3-hydroxy-3-methylglutaryl-CoA reductase cDNA (HMG1) from peel tissue of apple fruit

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Summary

As part of an effort to elucidate the mechanism of superficial scald in apple fruit, and ultimately to devise a molecular genetic strategy for control of this storage disorder, we have begun to clone and characterize gene products that regulate production of α-farnesene in peel tissue. Oxidation products of this sesquiterpene are thought to induce necrosis of cell layers just beneath the fruit skin, leading to development of scald symptoms. Here we report the cloning and bacterial expression of a cDNA encoding 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), the initial, rate-limiting enzyme in sesquiterpene biosynthesis via the mevalonic acid pathway. RT-PCR cloning based on the conserved catalytic domains of plant HMGRs yielded two different cDNA fragments from peel tissue mRNA, designated HMG1 and HMG3. The 3′ untranslated regions of the two cDNAs, obtained using 3′ RACE, were used to screen an apple peel cDNA library. A full-length transcript of HMG1 was obtained (2334 bp), with an open reading frame of 1827 bp, encoding a protein 608 amino acids in length. A partial HMG3 clone was isolated (1192 bp), composed of a 1143-bp coding region and a 49-bp 3′ untranslated region. RNA-gel blots of HMG1 showed a high level of expression in peel tissue at harvest and after 4 and 8 weeks of storage at 0.5 °C, even when tissue responsiveness to ethylene was blocked by prestorage treatment of fruit with 1-methylcyclopropene, which suppresses α-farnesene synthesis. Hence, the rapid rise in α-farnesene production that occurs in the initial weeks of storage cannot be attributed to increased expression of HMG1. E. coli cells transformed with the HMG1 transcript showed over 10-fold higher HMGR activity than those with the control vector. Bacterial expression was confirmed by immunoblots of HMG1 protein fused to a C-terminal myc tag, which showed a single immunoreactive band with a molecular mass of ∼69 kDa.

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