Cloning and characterization of a calmodulin gene in rice, Oryza sativa

by Lee, Shuk-man

(Uncorrected OCR) Abstract of thesis entitled

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.

i.

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

sequencing machine.

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).

11.