Molecular cloning and characterization of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (hmgr1) gene from rubber tree (Hevea brasiliensis Muell. Arg.): A key gene involved in isoprenoid biosynthesis

Abstract

Natural rubber (cis-1,4-polyisoprene) is a secondary metabolite produced in the laticiferous tissue of Hevea tree. Mevalonate synthesis, which is the first step in isoprenoid biosynthesis, is catalyzed by the enzyme 3-hydroxy-3-methylglutarylcoenzyme A reductase 1 (hmgr1). We have cloned and characterized a full-length cDNA as well as genomic DNA for hmgr1 gene from an elite Indian rubber clone (RRII 105). The nucleotide sequence of the genomic clone comprises 4 exons and 3 introns, giving a total length of 2440 bp. The sequences of 42 bp 5' UTR and 69 bp of the 3' UTR were also determined. The hmgr1 cDNA contained an open reading frame of 1838 bp coding for 575 amino acid protein with a theoretical pI value of 6.6 and the calculated protein M W was 61.6 kDa. The deduced amino acid sequence showed high identity with other plant hmgr1 sequences. The amino acid sequence of the Hevea hmgr1 revealed several motifs which are highly conserved and common to the other plant species. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural and/or catalytic properties of the enzyme. Southern blot analysis of genomic DNA from Hevea probed with a genomic fragment indicated that there were at least three isoforms of hmgr in Hevea. This result reveals that hmgr1 is one of the members of a small gene family. (Northern blot analysis showed that hmgr1 mRNA transcripts were noticed in all tissues - latex, leaf, immature leaf, and seedlings), however, the abundance of transcript level was higher in latex cells. As one step towards a better understanding of the role that this enzyme plays in coordinating isoprenoid biosynthesis in plants, hmgr1 cDNA was over expressed in transgenic Arabidopsis plants. Transgenic plants were morphologically distinguishable from control wild-type plants and an increased expression level of hmgr1 mRNA was detected. These data suggest that hmgr1 gene expression is playing an important role in isoprenoid biosynthesis.