The Cloning Of Part Of CDNA Encoding Chloroplast Ribosomal Protein PSRP-1

In the chloroplast of land plants, a large amount of proteins are synthesized by chloroplast ribosomes, including about 30 components of the four energy transduction complexes, approximately 20 ribosomal proteins, as well as ribulose 1,5-bisphosphate carboxylase. Thus the chloroplast ribosomes play a dominant role in protein metabolism. It is thought that several plastid-specific ribosomal proteins (PSRPs) are involved in the expression regulation of the chloroplast genes on the level of translation. PSRP-1 encoded by the nuclear genome constitutes the 30S subunit of chloroplast ribosomes. In order to study the functions and expression of PSRP-1 in Cruciferae, cDNA libraries were constructed and screened to get the cDNA encoding PSRP-1.Five cDNA library were constructed using SMART?cDNA Library Construction Kit, including B. oleracea var. acephala, Cheiranthus Cheiri L., Orychophragmus violaceu.sd. )0. E. Schulz, Brassica aloglabra, B. campestris. A 1.02×10 6 pfu/ml cDNA library was constructed from the leaf tissue of B. campestris. The ratio of recombination was 82%. The cDNA inserts of the cDNA library were amplified by PCR and found to be between 500bp~l. 5Kb. The titer of Cheiranthus Cheiri L. cDNA library was 5. 72 × 105pfu/ml. Seven pi agues were picked at random. After convertingλTriplEx2 to the corresponding pTriplEx2, the seven plasmids were purified and digested by the endonuclease -Sfi I. Four of the seven plasmids contained cDNA inserts which were between 500bp~1.0Kb. The five cDNA libraries were amplified separately and the amplified cDNA libraries were stored at -70癈.The oligonucleotide encoding part of the polypeptide of spinach PSRP-1 was labeled by DIG system and used as the probe to screen two cDNA libraries. Twelve clones were obtained from the cDNA library of Cheiranthus cheiri L.. Then after the second screening, one positive clone was obtained. The cDNA insert of the positive clone was sequenced. After nucleotide sequence analysis, one 506bp cDNA was identified, which edcoded a polypeptide of 99 amino acids. It showed 68% sequence identity to part of the spinach Psrp-1 gene and 91% sequence identity to part of the reported nucleotide sequence possibly encoding S30 in Arabidopsis thaiiana. Further protein sequence alignment showed that the putative amino acid sequence shared substantial sequence identity with PSRP-1. It was thought that the cDNA encoded the NH2 terminus of the homologous protein of PSRP-1 in Cheiranthus cheiri L. .After screening the cDNA library of B. campestris with the same probe, no cDNA inserts was found with similarity to the sequence encoding PSRP-1.