Cloning And Functional Analysis Of Laticiferious Specific Expression Promoter From Carica Papaya L.

The study of the promoter was a hotspot in the world at present. A lot of tissue specific promoters and induced promoters had been found by now. As an ideal plant acceptor for bioreacter, papaya had more advantages, such as getting and purifying of a foreign proteins expressed in its laticiferious system easily. As a result, it was important to isolate the laticiferious specific promoters from papaya.5 ' flanking sequence and partial gene sequence of proteinase omega and chymopapain were isolated from papaya (cv. Sui Zhong Hong) genomic DNA via chromosome walking technology in this paper. Though genetic engineering technique, the plant chimeric expression vectors contained 5 ' deleted promoter were constructed, and then were transferred into leaves of papaya and tobacco via particle bombardment and agrobacterium-mediated transformation technology. Transient GUS expressions were observed to analyse the function of deleted promoters. And the results were as follows:1. A 5 ' flanking sequence and partial gene sequence of proteinase omega were isolated from the genomic DNA via chromosome walking technology at first. A 1,505 bp fragment was obtained though 3 times chromosome walking, and then submitted to GenBank, the accession number was AY695444. The results of DNA sequence analysis and homology comparison indicated that 3 ' flanking of the l,505bp fragment had 96 % homology with the proteinase omega gene which had been submitted to GenBank. Compared with the datas in GenBank, 5 ' flanking sequence of the 1,505 bp fragment, 1,039 bp, had no homological region, and this indicated that a novel proteinase omega promoter was obtained from papaya. The two core promoter regions and some upstream regulatory elements were found in the fragment using the PROMOTER PREDICTION and PLANT CARE softwares. Transcriptional start sites (TSS) were A, T, respectively. TATA-box, CAAT-box, G-box, I-box, AT-rich regions and other cis-elements were found in the promoter in identical positions common to all promoter sequence regions.2. A 5 ' flanking sequence and partial gene sequence of chymopapain were also isolated from the genomic DNA. The 719 bp fragment was submitted to GenBank, accession number was AY803756. The results of the DNA sequence analysis and homology comparison of the 3 ' flanking of the 719 bp fragment indicated that it had 99 % homology with the chymopapain gene which had been submitted to GenBank. Compared with the datas, its 5 ' flanking sequence, 556 bp, had no homological region, and this indicated that a novel chymopapain promoter was obtained. A core promoter region and some upstream regulatory elements in the fragment were analysed using the PROMOTER PREDICTION and PLANT CARE softwares. Transcriptional start sites (TSS) was T. Atl-motif, HSE, I-box, WUN-motif, P-box, G-box and other cis-elements were found in the promoter .3. A 5 ' flanking sequence and partial gene sequence of papain were isolated from the genomic DNA at last. The 1,135 bp fragment was submitted to GenBank The results of the DNA sequence analysis and homology comparison of the 3 ' flanking of the l,135bp fragment indicated that it had 91 % homology with the papain gene which had been submitted to GenBank. Compared with the datas in GenBank, 5 ' flanking sequence of the l,135bp fragment, 972 bp, had no homological region, and this indicated that a novel papain promoter from papaya was possible obtained. Two core promoter regions and some upstream regulatory elements in the fragment were analysed using the PROMOTER PREDICTION and PLANT CARE softwares. Transcriptional start sites (TSS) was A. Some cis-elements were found in the promoter.4. The homology comparisons of the promoter sequence of proteinase omega gene and the chymopapain gene's were analysed though the software of CLUSTAL W (1.82). The same TATATAAA-box, core promoter region and a transcriptional start sites (TSS) were found.5. The l,039bp fragment of 5 ' flanking region of proteinase omega gene were deleted into 4 fragments by PCR methods, which replaced the 35S promoter of pCAMBIA 1301 and constructed the plant chimeric vectors, they were: pCAMBIA 1(1039 bp), pCAMBIA 2(735 bp)> pCAMBIA 3(460 bp) and pCAMBIA 4(271 bp). So did the 556 bp fragment of 5 ' flanking region of chymopapain gene, and two chimeric vectors were alsoconstructed, they were pCAMBIA 5(556 bp)and pCAMBIA 6 (252 bp) .6. Transient GUS expressions of four binary vectors from the promoter of proteinase omega were observed in the papaya leaves via particle bombardment. The blue color of pCAMBIA 1, pCAMBIA 3, pCAMBIA 4 were brighter than that of 35 S from pCAMBIA 1301 (positive control) , whereas the negative control, non-transferred leaves, had no blue color. And once more, the blue color of pCAMBIA 2 was more faint than that of 35 S from pCAMBIA 1301. As for the blue color location, the results of the transient expression in papaya leaves indicated that the laticifers bombarded by the microcarries coated with pCAMBIA 1 , pCAMBIA 2, pCAMBIA 3, pCAMBIA 4 showed bright blue, but only pCAMBIA 1301 also showed bright blue both in the laticifers and the parenchma cells, and this indicated that the promoters of proteinase omega was laticiferious specific. Based on the results of the transient GUS expressions, there was a enhencer or positive regulatory element in the region of 1 bp -301 bp in 5 ' flanking of proteinase omega gene. There was a negative regulatory element in the region of 301 bp-578 bp. The AT-rech sequence (TAAAatatt) had possible important effect in the region 830bp-838bp of reverse sequence. Though the sequence of 578 bp-768 bp had no distinct effect on the gene expression, it was indispensable to perform constitutive expression. Because the sequence of 768 bp-1,039 bp had important effect on laticiferious specific expression, it showed that this sequence contained the whole information of controlling gene laticiferious specific expression, and it was indispensable to promote gene expression.The results of the GUS stable expression transferred into tobacco mediated by agrobacterium transformation indicated that four chimeric plasmids contained 5 ' deleted flanking of the promoter of proteinase omega gene were laticiferious specific, because of the blue color showing in the veins (or fibrovascular tissue) of tobacco leaves only. And this was the same as the rusults of particle bombardment's.7. In the research of the 5 ' deleted flanking region of chymopapain gene , the pCAMBIA 5 , pCAMBIA 6 were transferred into tobacco leaf disks mediated by agrobacterium transformation. The results of the steady GUS activities were close to the 35S promoter of pCAMBIA 1301 (positive control) . As for the color location, the results of the steady expression in tobacco leaves indicated that the veins (or fibrovascular tissue)...