|
Vacuoles of higher plant cells contain two primary electrogenic
proton pumps, a vacuolar H+-ATPase (V-ATPase) and a H+-
translocating inorganic pyrophosphatase (V-PPase), for the
regulation of cell turgor, cytoplasmic homeostasis, and the
storage of metabolites. In this work, the structure of the mung
bean V-ATPase A subunit gene was investigated. In order to
clone the cDNA, primers were synthesized according to the
conserved sequences of cotton and carrot V-ATPase A subunit to
conduct polymerase chain reaction (PCR) using the total DNA
extracted from mung bean cDNA library as a template. The PCR
product was then employed as a probe to screen the constructed
mung bean cDNA library. The cloned V-ATPase A subunit cDNA
exhibits 83.8, 83.4, and 81.1% nucleotide homology to those of
cotton, carrot, and Brassica napus, respectively. The cDNA
sequence encodes 623 amino acids with a predicted Mr of
68,664and a predicted isoelectric point of 5.17. The amino acid
sequence of V-ATPase A subunit from mung bean seedlings shares
94.0~95.0% identity and 96.6~97.0% similarity to those from
carrot, cotton, and Brassica napus. Expression of the A subunit
gene was also investigated by Northern hybridization. It was
found that the leave expressed the most abundance of the
transcript, followed by hypocotyl and roots. Genomic Southern
analyses reveal a simple reaction pattern of the gene,
referring the lack of isoform for the A subunit. We also took
further step to explore the genomic structure of the A subunit.
To construct the DNA library, mung bean genomic DNA was
partially digested by Sau3AI, and fractionated by sucrose
gradient centrifugation. The 9 to 20 kb DNAs were ligated with
l/Dash II DNA and packaged. The constructed library was
screened using A subunit cDNA as a probe, and several positive
clones were obtained. Some of these clones contain both 5?and
3?regions of the gene.
|