Isolation And Functional Analysis Of Promoters Of Maize Wound-inducible WIP1and Potato Protease Inhibitor Ⅱ PINⅡGenes

With the development of GM crops, there is an urgent need for useful promoters derived from plants. The WIP1promoter of WIP1gene, a maize wound-inducible gene coding protease inhibitor, and the PIN II promoter of PINⅡ gene, a potato wound-inducible gene coding protease inhibitor, were cloned by PCR. The function of the two promoters was studied by conventional methods, and the acquisition of useful promoters has been expected.The results of studying the function of WIP1promoter are as follows:1. The GUS enzyme activity of3’end deletion fragments wip1231, wip1231A, wip1231C, wip93l transgenic tobacco and Arabidopsis was as high as the one of the control CaMV35S promoter transgenic tobacco and Arabidopsis, but the GUS enzyme activity of the other deletion fragments derived from WIP1transgenic tobacco and Arabidopsis was very weak. Compared with the GUS mRNA level of pCAMBIA1300-221transgenic tobacco, the one of pWip1231, pWip1737, pWipl500transgenic tobacco was very low. It suggests that the high tranalation efficiency of GUS mRNA played an important role to the high GUS enzyme activity of pWip1231transgenic tobacco.2. Transcription start sites (TSSs) were identified by5’-RACE method. Maize WIP1was transcribed from one site, and GUS of pWip1737transgenic tobacco was transcribed from two sites, a major one and a minior one. The minior one was identical to the one of WIP1, the major one was identical to the one of GUS of pWip1231transgenic tobacco. The272bp5’-UTR of GUS mRNA of pWip1231transgenic tobacco was analyzed to find that there were one ACAAAA element, several elements similar to ACAATTAC and several CAA trimers. The feature of the5’-UTR may contribute to the function of high efficient translation. The532bp5’-UTR of GUS mRNA of pWip1737transgenic tobacco was analyzed to find that there were14uATGs, and some of them were in frame with terminal codes to form uORFs. The low efficient translation of GUS mRNA of pWip1737transgenic tobacco may be due to the uORFs.3. The GUS enzyme activity did not alter after pWip1737, pWip1500, pWipl231, p1300-1231-NOS transgenic tobacco were treated by wound. The result showed that the WIP1promoter was not able to respond to wound signals in transgenic tobacco.4. The promoter activity of wip1231fragment is higher in transgenic tobacco stem and leaf than in root and seed.5. Compared with the GUS enzyme activity of pWip1231transgenic tobacco, the one of p1300-1231-NOS transgenic tobacco was very low. The result suggests that the promoter activity of wip1231fragment is enhanced by adjacent CaMV35S promoter.6. The GUS enzyme activity was enhanced after pWip1737, pWip1500, pWip1231transgenic rice were treated by wound, but the activity remained very low. The result showed that the WIP1promoter was able to respond to wound signals in transgenic rice.The results of studying the function of PINII promoter are as follows: 1. Compared with the GUS enzyme activity and the GUS transcript level of pCAMBIA1300-221transgenic tobacco, the GUS enzyme activity of pPinⅡ928transgenic tobacco was very high and the GUS transcript level was very low. It suggests that the high GUS enzyme activity of pPin Ⅱ928transgenic tobacco is due to the highly efficient translation.2. UTR1, UTR2derived from5’-UTR of PINⅡ gene were able to enhance gene expression when they were inserted between CaMV35S promoter and genes. Gene transcript level was analyzed, and the result showed that highly efficient gene expression was due to the highly efficient translation3. Compared with the GUS enzyme activity of pPinⅡ928transgenic tobacco, the one of p1300-928-NOS transgenic tobacco was very low. The result suggests that the promoter activity of piⅡ928fragment is enhanced by adjacent CaMV35S promoter.4. The GUS enzyme activity did not alter after pPin Ⅱ928transgenic tobacco was wounded, but the one was enhanced after p1300-928-NOS transgenic tobacco was wounded. The result showed that the ability of pin11928fragment to respond to wound singnals was impaired by adjacent CaMV35S promoter.5. The analysis of TSS by5’-RACE method showed that GUS of pPinⅡ928transgenic tobacco was transcribed from A, which was4bp downstream the annotation TSS in M29965sequence. GUS of pCAMBIA1300-221, pCAMBIA1300-221-UTR1, pCAMBIA1300-221-UTR2transgenic tobacco was transcribed at the same site, and G2of p3301-SP-G2, p3301-UTR2-SP-G2transgenic tobacco was also transcribed at the same site. The results showed that the insertation of UTR1, UTR2did not alter the primary TSS.W1pl231, wip1231A, wip1231C, wip931, pin1192%fragments are able to drive highly efficient expression of a target gene if there is a nearby CaMV35S promoter. UTR1and UTR2are able to enhance translation of a target mRNA, and there is a high probability that the272bp5’-UTR derived from GUS mRNA of pWipl231transgenic tobacco is able to enhance translation of a target mRNA. The acquisition of these fragments is of important practical significance. Some interesting phenomenons, such as the discordance between mRNA level and protein level, a CaMV35S promoter influencing the characteristics of an adjacent promoter and the functional difference of WIP1promoter in dicotyledon and monocotyledon, were encounted during studying WIP1promoter and PIN II promoter. It is of important theoretical significance to provide guidance on how to study promoter in future.