CONSTRUCTION OF A RICE IMMATURE SEEDS CDNA LIBRARY AND MOLECULAR-CLONING OF ORYZACYSTATIN CDNA

Total RNA was extracted from rice immature seeds harvested 2 weeks aft er flowering; then mRNA was purified. cDNA with NotI and SalI cohesive ends was synthesized and inserted into lambda gt22A. After packaged i n vitro, the cDNA library was constructed with 1.5 x 10(6) pfu. A 21-m er oligodeoxynucleotide was synthesized according to the 5'-end conser ved coding sequence of oryzacystatin (a thiol proteinase inhibitor) an d labeled as a probe. From 2.1 x 10(4) pfu, 9 positive clones have bee n isolated, 8 of which contain the entire coding region of oryzacystat in. lambda OCl has the longest cDNA insert, which contains an open rea ding frame of 309 bp coding sequence, 84 bp 5'end non-coding region an d a poly(A) signal AATAAA at the 3'-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DN A sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsuccess ive insertion in the 3' region after poly(A) signal.