Cloning of hmg1 and hmg2 cDNAs encoding 3-hydroxy-3-methylglutarylcoenzyme A reductase and their expression and activity in relation toalpha-farnesene synthesis in apple

摘要

In plants, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyses the synthesis of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A. It has been reported to be the rate-limiting enzyme in sesquiterpene and triterpene biosynthesis and is encoded by a small gene family. The accumulation of alpha -farnesene in the skin tissue of apple fruit during storage appears to be predominantly through the classical mevalonate pathway and not through the novel glyceraldehyde-3-phosphate/pyruvate pathway independent of HMGR action. The content of a-farnesene in the skin tissue increased during the first 8 weeks of storage at 0 degreesC in air, and started declining after 12 weeks. In contrast, HMGR activity in the total membrane and soluble fractions, was the highest at the time of harvest, but decreased during the first 8 weeks in storage and remained stable thereafter. The potent ethylene action inhibitor 1-methylcyclopropene inhibited alpha -farnesene evolution and HMGR activity by 97 and 30 %, respectively. As a first step in studying the molecular mechanism of apple HMGR regulation, we have isolated and cloned a full-length cDNA (hmg1) as well as a fragment (hmg2), using apple skin mRNA and RT-PCR in the presence of degenerate oligonucleotides designed against conserved regions of plant HMGR genes. Genomic Southern analysis using probes designed for the 3'-end of the two cDNA clones confirmed the presence of at least two HMGR genes in apple. The cDNA for hmgl has an open reading frame of 1 767 nucleotides. Analysis of the nucleotide sequence revealed that the cDNA encodes a polypeptide of 589 residues with a relative molecular mass of 62.7 kDa. The hydropathy profile of the putative polypeptide indicated the presence of two highly hydrophobic domains near the amino terminus. Northern blot analysis confirmed that both hmg1 and hmg2 transcripts possessed a size of 2.4 kb. The two genes are differentially expressed during low temperature storage and in response to C2H4, with hmg1] being expressed constitutively and hmg2 being relatively more sensitive to developmental stimuli and ethylene.