Cloning Of A Potato Proteinase Inhibitor Gene PINâ…¡-2x And Its Transformation Into Rice

Proteinase inhibitors are widly distributed in the plant kingdom. They can form complexes with proteinase to inhibit proteolytic activity, and serve as storage protein in plant storage organs. Most importantly, plant proteinase inhibitor play important roles in defense against herbivore and pathogens. Potato proteinase inhibitor II (PINII) belongs to serine proteinase inhibitor family. Compared with other plant proteinase inhibitor, it has several merits. Firstly, Potato PINII from potato has two active sites that inhibit trypsin and chymotrypsin simultaneously. So it can defense more kinds of insects or pathogens and be more competitive. Secondly, potato PINII is an original component of plant-derived food for human and animal, so it is safe to people. Thirdly, some researches had showed the potato PINII has anticarcinogenic properties. Moreover, potato PINII is tissue-specific expression in potato tuber and induced expression in leaf by many factors, which making potato PINII be a good model for studying the plant gene expression regulation. So it is meaningful to deeply study the potato PINII in both theory and practical use. In this paper, we report cloning of a new potato proteinase inhibitor II gene PINII-2x from dipoloid potato and its transformation into rice. At same time, we transferred the tomato prosystemin gene into rice and get some transformed plants. The results are as following.1. Based on the published gene sequence of tetraploid potato (Solarium. tuberosum) proteinase inhibitor II, A genomic DNA and a cDNA sequence of potato PINII gene was obtained from the cDNA library and the genomic DNA of a diploid potato IVP101 (Solanum.phurejia) using PCR method and named PINII-2x. Nucleotide sequencing confirmed that the genomic DNA of PINII-2x is 580 bp, including a 115 bp intron and two exons. cDNA is 462 bp (excluding the stop codon TGA) and have 88% similarity to the tetraploid potato PINII. The PINII-2x open reading frame encode a 154-amino acid polypeptide with a predicated size of 16.6 KD and a calculated PI of 6.08. The deduced proteins from PINII-2x cDNA had 93% homology with other tetraploid potato PINII, which contain the intact signal peptide and two active sites similar to the potato PINII family. RT-PCR and Northern blot indicated that PINII-2x mRNA is wound induced expression in potato leaves. These indicate the potato PINII-2x belongs to the potato PINII family.