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Effects Of Overexpression Of Suaeda Salsa CDNA Library And Co-overexpression Of SsNHX1-AVP1 On Salt Tolerance In Arabidopsis Thaliana Cloning And Expression Of FAD7 Gene Of Suaeda Salsa

Posted on:2005-09-16Degree:DoctorType:Dissertation
Country:ChinaCandidate:X J ZhangFull Text:PDF
GTID:1100360125462334Subject:Botany
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Salt stress consisting of osmotic and ionic stress, and then the secondary stress such as oxidative stress arising by them, can bring on .growth inhibition and even death of plants. High concentrations of salt in soils account for large decreases in the yield of a wild variety of crops all over the world. Plant salt-tolerant genetic engineering was considered as the most effective and economical mean to bring up salt-tolerant crops and to exploit salt wasteland. The lack of efficient genes has been the bottleneck to limit its development. The main objective of the dissertation was to do some work to find new efficient genes for plant salt-tolerant genetic engineering.1. Effects of overexpression ofSuaeda salsa cDNA library on the salt tolerance in Arabidopsis thalianaWe have developed a system named sense/antisense RNA expression (SARE) to over-express or co-suppress random Suaeda salsa cDNAs. The binary vector containing a novel Suaeda cDNA library driven by the cauliflower mosaic virus (CaMV) 35S promoter consisted of the part of pCAMBIA1200 and pRT105. The vector offers selection with hygromycin-B and the ability to identify the cDNA insert using PCR with flanking primers. We introduced this overexpression library into Arabidopsis by floral-dip upon Agrobacterium /wme/ac/'ens-mediated transformation and selected over 2000 transformants. Only about 15.7% of Tl plants have one insert. The insert size is ca 800-2000bp. There were three lines, 19, 27 and 185, which exhibited higher salt tolerance during the period of seeds germination and seedling. The transgenic Arabidopsis plants grew much better than wild type plants under 200 mmol/L NaCl condition. Three insert fragments, which sizes are ca SOObp, lOOObp and 1200bp respectively, have been obtained from the three transgenic lines by PCR amplification. The DNA sequence and analysis is under goning.2. Effects of co-overexpression ofSsNHXJ and AVPl on the salt tolerance in ArabidopsisThe bivalence vector carrying both SsNHXI and AVPl have been constructed and thenintroduced into Arabidopsis. We have got 343 pure lines. PCRs RT-PCR, Southern Northernand Western blotting analysis testified that both extraneous genes had been integrated into all of the transgenic plant genomes. Under normal condition, the PPase activities of transgenic plants were obviously higher than that of wild type plants, which indicated that the extraneous genes had played its role on transgenic plants. No difference was observed in growth between transgenic plants and wild type plants in the absence of NaCl stress conditions, which indicated that the insertion and overexpression ofSsNHXI and AVPl had no effect on the normal growth of Arabidopsis plants.The growth of both transgenic and wild type Arabidopsis plants were suppressed after a 200mmol/L NaCl treatment, but the transgenic plants had higher salt tolerance than wild type plants. The expression of extraneous genes can improve seeds germination, increase fresh and and dry weight, accumulate more K"1", maintain a high cytosolic K/Na ratio and photosynthetic rate (Pn), increase the content of proline and then reduce the osmotic potential. These results indicated that transgenic Arabidopsis plants co-overexpressed ofSsNHXI and AVPl had greater salt tolerance than wild type plants, which further indicated the importance of Na compartmentation in plant salt tolerance. This can be used in genetic engineering for improving salt tolerance of crop plants.3. cDNA cloning and expression of Suaeda salsa FAD7 gene (plastid w -3 fatty acid desaturase)Full cDN A sequence encoding plastid w -3 fatty acid desaturase (FAD7) were isolated from the cDNA library of halophyte Suaeda salsa through EST analysis. The cDNA contained a 1470-bp putative open reading frame encoding for 489 amino acids with a predicted molecular mass of 54.29 kDa and a predicted isoelectric point of 9.04. The deduced amino acid sequence shows 80%, 79% and 78% identity with Nicotiana tabacum, Sesamum indicum and Perilla frutescens w -3 fatty acid desaturases respect...
Keywords/Search Tags:Suaeda salsa (L.) Pall, sense/antisense RNA expression (SARE) system, SsNHXI, AVPI, ω -3 fatty acid desaturase, plant salt tolerance
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