Cloning and characterization of a calmodulin gene in rice, Oryza sativa
CLONING AND CHARACTERIZATION OF A CALMODULIN GENE IN RICE, Oryza sativa
Submitted by Lee Sbuk Man
for the degree of Master of Philosophy at The University of Hong Kong
in September 1999
Calmodulin is a highly conserved polypeptide of molecular weight 16.7
kilodalton. It consists of 148 amino acids with four calcium-binding domains. It is
widely distributed in animals and plants.
In this project the rice calmodulin gene was isolated from a rice (Oryza sativa
006) genomic library and sequenced. Nucleotide sequence analysis of clone R-3,
which contains the new calmodulin gene, showed that the coding region is interrupted
by a single intron of 828 base pairs (bp) in length. The coding region of the new
calmodulin gene is very similar to that of other calmodulins.
The screening of a rice genomic library with a partial rice calmodulin cDNA
probe of 733 base pairs gave clones that contained most of the coding region of the
rice calmodulin gene. The liquid lysate method was used for amplifying the phage.
The desired DNA fragment in the phage was then subcloned and sequenced. Positive
restriction fragments from Southern blot hybridization were subcloned into the
pBluescript vector. Nested deletion was used to produce shorter fragments for
sequencmg. Sequencing was done by AutoRead sequencmg m an ALFexpress
Clone R-3 which has a 4.5 kb EcoR I fragment in the pBluescript vector
contains the putative rice calmodulin gene. By comparing the amino acid sequences, it
was found that the nucleotide sequence of clone R-3 encodes a typical plant
calmodulin protein of 148 amino acids. Moreover, the difference between most of the
nucleotide sequences is in the third position of each codon. The amino acid sequence
of the rice calmodulin is very conserved and that of clone R-3 is identical to those of
barley and soybean. The partial putative promoter sequence of the rice calmodulin
gene contains a TAlA box (-166) and a GC box (-177). Polymerase Chain Reaction
was used to amplify the 5'-untranslated sequence of the gene for determining the full
sequence of the promoter.
Rice calmodulin clone R-3 has 456 bp in the 5' untranslated region and 409 bp
in the 3' untranslated region. Its coding region is interrupted by a single intron of 828
bp at the 25th amino acid. The intron is highly AT-rich (59% A+T).
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:calmodulin rice genetics
Date of Publication:01/01/2000