Construction of a rice immature seeds cDNA library and molecular cloning of oryzacystatin cDNA

来源 :Science in China(Series C:Life Sciences) | 被引量 : 0次 | 上传用户:applebanana0204
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Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5’-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5’-end non-coding region and 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 DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3’ region after poly(A) signal. Total RNA was extracted from rice immature seeds harvested for 2 weeks after flowering; then mRNA was purified. CDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5 × 106 pfu. A 21 -mer oligodeoxynucleotide was synthesized according to the 5’-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contains the entire coding region of oryzacystatin. λ OC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5’-end non-coding region and 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 DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3 ’region afte r poly (A) signal.
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