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The Functional Analysis Of Salt Tolerance Related Genes Of Thellungiella Halophila

Posted on:2007-12-27Degree:DoctorType:Dissertation
Country:ChinaCandidate:X H GaoFull Text:PDF
GTID:1100360182497789Subject:Botany
Abstract/Summary:PDF Full Text Request
Salinity is one of the major abiotic stresses in the world. High sodium (Na~+)concentrations in soils are toxic to most high plants, severely affect the plant growth anddevelopment, and account for large decreases in the yield of a wide variety of crops all overthe world. The strategies for plants eliminating the Na~+ toxicity include: decreasing Na~+absorption, increasing Na~+ exclusion and Na~+ compartmentalization. Now the process for Na~+absorption is not clear, but the Na~+ exclusion and compartmentalization are indirect activetransport process, which are achieved by the action of Na~+/H~+ antiporters on the plasma andtonoplast membrane. Na~+/H~+ antiporters keep close relationship with the plant stress toleranceand have become the international research hotspot in the recent years. These Na~+/H~+antiporters mediate Na~+ exclusion and compartmentalization, which is driven by the protongradient established by the plasma membrane H~+-ATPases and tonoplast H~+-ATPases andH~+-pyrophosphatases.Up to now, the Arabidopsis is always the preeminent model material for plant molecularbiology research, but it is a true glycophyte and can only reveal a little information for theplant salt tolerance using the Arabidopsis. Salt cress (Thellungiella halophila) is a relative ofthe Arabidopsis and becomes the prospective salinity tolerance model. The major purpose ofour experiment is using salt cress as the research material, studying the function of the saltrelated gene of ThNHX1 and P5CS, elucidating the relationship between these genes andT.halophila's salt tolerance, making primarily research on the establishment of T. halophilaEMS mutant. Here are the major experiment results:1. The cloning and functional analysis of ThNHX1 gene1) According to the conservative region of NHX1 gene in other species the primer wasdesigned, using RT-PCR and RACE methods, the full length of ThNHX1 gene from salt cresswas cloned. The full length of ThNHX1 cDNA sequence was 2031 bp and the open readingframe was 1635 bp, the 5'-untranslated region was 146 bp in length and 3'-UTR was 250 bp.It coded for 545 amino acid residues with calculated weight of 60.4 KD.2) The detailed analysis of the ThNHX1 gene sequence characterization was made.ThNHX1 gene had the highest sequence similarity with the vacuolar-type Na~+/H~+ antiportergene AtNHX1 from Arabidopsis. The deduced amino acid sequence of ThNHX1 had the 12predicted transmenbrane regions. The phylogenetic analysis of various tonoplast Na+/H+antiporters using DNAMAN saftware indicated that ThNHX1, AtNHX1, BnNHX1 andAtNHX2 shared the same cluster.3) The complementation analysis of ThNHX1 in yeast mutant AXT3 (ena1-4 nha1 nhx1)showed that the expression of ThNHX1 enhanced Na+ and Li+ tolerance of yeast mutant andproved that ThNHX1 was a functional tonoplast Na+/H+ antiporter.4) The detailed analysis of ThNHX1 gene expression under different kinds of stresseswas carried out. Northern blotting results showed that the expressing levels of ThNHX1 genewere higher in roots than in shoots and both were induced by NaCl. All the treatments ofNaCl, ABA, high temperature (40℃) and PEG increased the ThNHX1 mRNA expression inthe shoot within a short time ( 1 and 3 hour), while the low temperature (4℃) decreased itsexpression, but the expression had a little increase at the 24-hour of 4℃ treatment.5) The full length of ThNHX1 ORF was constructed into the plant expression vectorpROKII and transformed into Agrobacterium tumefaciens GV3101. The integrated vector wasintroduced into Arabidopsis thaliana using floral dip transformation method. The ThNHX1transformants were continuously screened on the media with kanamycin (30mg/L). 16homozygous T3 transgenic lines were obtained and three of them were selected and used formolecular and physiological analysis. Southern and Northern blotting confirmed that theThNHX1 gene had been integrated into Arabidopsis genome and had the normal transcription.The physiological analysis showed that the transgenic plants of Arabidopsis had moretolerance than wild type plants under NaCl stress. The photosynthetic rate, fresh weight anddry weight were higher than wild type plants, but the Na+ and K+ content of transgenic plantshad no difference with wild type plants. Overexpression of ThNHX1 increased the Li+tolerance of the transgenic plants.6) The gene silencing expression vector of pCAMBIA3301-ThNHX1 was constructedand also transformed into T. halophila using floral dipping method. 52 Basta resistance plantswere selected. Real-time PCR confirmed the expression level of ThNHX1 gene in transgenicplants was lower than in wild type plants. The analysis of the stress tolerance of transgenicplants showed that ThNHX1 gene silencing increased transgenic T. halophila NaCl andmannitol sensitivity. In the transgenic plants, the water content decreased while the prolinecontent increased under salt stress. These results showed that silencing of ThNHX1 genedecreased the stress tolerance of T. halophila.2 The gene silencing research of P5CS geneUsing the already constructed gene silencing expression vector of pGSA1252-P5CS, wetransformed it into T. halophila using floral dipp method. 39 resistance plants were got withthe Basta selection. Real-time PCR confirmed the expression level of P5CS gene in transgenicplants was lower than in wild type plants. 9 transgenic lines were selected randomly forphysiology analysis. The results showed that the silencing of P5CS gene did not affect the salttolerance of transgenic plants under 400 mM NaCl treatment for 7 days. Under normal andsalt treatment, the water content had no difference between the transgenic plants and the wildplants, but there were 6 lines with the significant decrease of proline content. These resultsshowed that silencing of P5CS gene decreased the proline content, but did not affect the salttolerance of the salt cress. Maybe T. halophila synthesized other osmotic protectivesubstances to maintain the homeostasis and benefit for the T. halophila growth.3 The primary research on the establishment of T. halophila EMS mutantsUsing the EMS mutagenesis strategies, the primary research on the establishment of T.halophila EMS mutants was made.1)The sterilization method for the T. halophila seeds was optimized by choosing 0.6%NaClO and appending 0.1% tween to sterilize the seeds for 10 minutes, saving the time andgetting the perfect sterilization,2)The concentration of EMS mutagenesis is 0.4% and the mutagenesis time is 10minutes in the experiment,3)According to the root–bending assay methods, the selection condition for isolating thesalt sensitive mutants was 200 mM NaCl,4)After screening 6000 M2 lines, 208 putative salt sensitive mutants were isolated. Thefurther confirmation is undergoing. Forthermore, we got one probable mutant with the alteredshape of rosette leaf.The main innovation points of this study were generalized as follows:1. First carrying out the detailed research for the ThNHX1 gene of salt cress, proving itwas a functional tonoplast Na+/H+ antiporter gene. The detailed expression profile of ThNHX1gene under different stresses was demonstrated. Overexpression of ThNHX1 gene increasedthe salt tolerance of transgenic Arabidopsis and its silencing increased the salt and mannitolsensitivity of the transgenic salt cress.2. The relationship between the P5CS gene and T. halophila salt tolerance wasdemonstrated using gene silencing methods, the results showed that silencing of P5CS genedecreased the proline content, but did not affect the salt tolerance of the salt cress. Maybe T.halophila synthesized other osmotic protective substances to maintain the homeostasis.3. The primary research on the establishment of T. halophila EMS mutants in our labwas made. The sterilization method for the T. halophila seeds was optimized by choosing0.6% NaClO and appending 0.1% tween to sterilize the seeds for 10 minutes, saving the timeand getting the perfect sterilization. The concentration of EMS mutagenesis was 0.4% and themutagenesis time was 10 minutes in our experiment. Some putative salt sensitive mutantswere isolated and one probabe leaf mutant was got.
Keywords/Search Tags:Thellungiella halophila, Na~+/H~+ antiporter, ThNHX1, P5CS, EMS, mutant
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