| Aquaporins make water-selective channels in plants, facilitating the permeation of water through membranes, and are involved in many important physiological processes as well as the response to stresses. This study aimed at investigating the transport function in plants of seed-germination specific plasma membrane aquaporin BnPIPl from Brassica napus and its meanings to plant drought tolerance. The upstream sequence of BnPIPl was cloned and used for the study of time-space expression pattern of BnPIPl gene. This work has a high academic value and practical significance, which provides a new way for understanding the mechanisms of water transport in plants under water stress and makes a foundation for further study of BnPIPl expression pattern. The followings are the main results:1. A full length cDNA fragment encoding plasma membrane aquaporin BnPIPl was cloned from the leaf total RNA of Brassica napus by RT-PCR method. Sequencing analysis indicated that this fragment showed 99% identity to the previously reported one. There were four mutant sites in this sequence and resulted in only one amino acid change, which was deduced to be the genetic variation between different species.2. Two plant expression vectors with sense or antisense BnPIPlgene, driven by CaMVSSS promoter and terminated by Tnos, were constructed and transformed into tobacco discs via the mediation of Agrobacteriwn tumefaciens to study of physiological functions of BnPIPl in plant. Form the expression of sense or antisense BnPIPl gene in transgenic tobaccos, we could draw a conclusion that antisense expression of BnPIPl could suppress the expression of endoaquaporins in tobaccos, resulting in significant reduction in water transport, longer time needed for leaf protoplasts to burst in hypotonic solution and sensitivity to water stress. Whereas, the lines overexpressing sense BnPIPlgene could fast and finely control the transport of water, leading to quick burst of leaf protoplasts in hypotonic solution, tolerance to water stress and early and even germination of TO seeds in high osmotic pressure soil.3. The study of T0 seeds germination on MS mediums with antibiotic indicated that transgene had different integration patterns in different transgenic lines. In some lines, the foreign gene integrated into one chromosome with one or mutiple copies at one or more loci and segregated in TO seeds with the ratio about 3:1. In some other lines, the foreigngene integrated into different chromosomes with multiple copies at different sites, showing complex segregation ratio.4. The upstream sequence of BnPIPl gene was cloned by genomic walking based on ligation- mediated PCR method. This sequence was 1610bp in length, containing two possible TATA-box, one G-box, four TGAC sequences and several TGAC-like sequences. Blast analysis showed that this was a novel sequence. The GenBank accession number was AF472487.5. Binary vectors pBI121, pCAMBIA1305.1 and binary promoter indicator vector pPR97 were used to construct plant expression vectors, in which the foil length upstream sequence of BnPIPl was fused with gus gene. After the treatment on MS medium with proper hormones, the regenerated shoots from transgenic tobacco calli were detected for GUS activity. The result showed that the full length sequence had promoter activity, the intensity of GUS was as strong as that of GUS driven by CaMVSSS promoter. GUS staining was mainly localized in the parts in which cells grow and proliferate fast and need vast water supply, or in the vessel tissues which function in water transport. At the same time, we deduced that this promoter probably was a seed-germination specific promoter.6. A series of deletion vectors named as p97-0, p97-581, p97-708 and p97-1246 were constructed, in which Obp,580bp, 708bp and 1246bp were deleted from 5' end of the upstream sequence of BnPIPl by PCR method, respectively. Calli from transgenic tobaccos were used for GUS staining and the promoter was divided into three regions accordingly. Region 1: -1...
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