CLONING OF GENES ENCODING S-ADENOSYL-L-METHIONINE SYNTHETASE AND 1-AMINO-CYCLOPROPANE-1-CARBOXYLATE SYNTHASE IN MUSTARD (BRASSICA JUNCEA (L.) CZERN & COSS)

Abstract

The objective of this study is to clone genes encoding two major enzymes of ethylene biosynthesis, namely, S-adenosyl-L-methionine (SAM) synthetase and 1-aminocyclopropane-1-carboxylate (ACC) synthase, which catalyse the conversion of methionine to SAM and of SAM to ACC, respectively. For cloning of gene encoding ACC synthase, a 300 bp DNA sequence was first amplified from a mustard cDNA library using polymerase chain reaction (PCR) with the mixed oligonucleotide primers synthesized based on the sequence of the conserved regions of the ACC synthase gene. After the library was screened using CM1 as a homologous hybridization probe, one cDNA clone with 1.8 kb insert, designated as MACC, was obtained. Sequence analysis showed that MACC was 1768 nucleotides in length consisting of a single open reading frame (ORF) of 1491 bp coding for a protein of 497 amino acids, with an estimated molecular mass of 55.5 kDa. The deduced amino acid sequence of the coding region showed extensive homology with ACC synthase in Arabidopsis thaliana (60%), tomato (52%), carnation (52%), zucchini squash (52%), winter squash (51%), rice (51%) and orchid (51 %). Results of northern analysis showed that MACC was strongly hybridized to an endogenous 1.9 kb transcript in stems and leaves of mustard. However, no transcript was detected in the root, indicating that expression of ACC synthase gene in mustard may be regulated in the organ-specific manner. MACC was used as a probe to examine the ACC synthase homologs in the genome of different members of Brassica (broccoli, cauliflower, Chinese kale, oilseed rape, Chinese cabbage, pak choy, choy sum and mustard). Results showed that the DNA banding patterns appeared to be related to the phylogenetic relationship among Brassica members, indicating that MACC can be used as a marker for the study of genetics and evolution of this genus. In addition, the presence of multiple DNA hybridized bands in mustard suggests that ACC synthase is encoded by a multigene family. For isolation of gene encoding SAM synthetase, the mustard cDNA library was screened using a 1.4 kb cDNA clone encoding SAM synthetase of A. thaliana as a heterologous hybridization probe. One cDNA clone, designated as pMSAMS was isolated. Sequence analysis showed that pMSAMS contained 1474 bp insert with an ORF of 1179 bp encoding a protein of 393 amino acids, with the calculated molecular mass of 43.0 kDa. Comparison of deduced amino acid sequences between msams and SAM synthetase genes from other plant species revealed that msams was 96% homologous with SAM-2 of A. thaliana and 89% with SAS-I of parsley and PdxPt of poplar. Extensive homology also occurred between mustard and rat (62%), yeast (59%) and E. coli (50% ). Northern analysis showed that msams was exclusively hybridized to a 1.5 kb transcript in leaf, stem and root of mustard, indicating that msams may be a housekeeping gene in mustard.