Dissertation > Excellent graduate degree dissertation topics show

The Novel Plant Na~+/H~+ Antiporter Gene Evolved by DNA Shuffling Confers Yeast and Arabidopsis Improved Salt Tolerance

Author: XuKai
Tutor: XiaTao
School: East China Normal University
Course: Biochemistry and Molecular Biology
Keywords: DNA shuffling Yeast Salt Tolerance Na ~ / H ~ antiporter Arabidopsis
CLC: Q943.2
Type: PhD thesis
Year: 2010
Downloads: 319
Quote: 1
Read: Download Dissertation

Abstract

The soil salinization limit plant growth and reduce crop yields is an important abiotic stress factors. In response to salt stress, plants usually take a series of adaptive adjustment mechanism, including osmotic adjustment and ion balance. Plants through the vacuolar membrane Na / H antiporter (Na / H antiporter) the cytoplasm excess Na compartmentation into the vacuole, on the one hand, can reduce the concentration of cytoplasmic Na, on the other hand, can also play the role of osmotic adjustment, promote cells absorb water, is a cost-effective way to maintain the balance of Na. The overexpression plants plant Na / H of Antiporter of genes are significantly improve the salt tolerance of the transgenic plants, the plant vacuolar membrane Na / H antiporter plant salt tolerance plays a very important role. DNA the reorganization (DNA shuffling) technology is fast and efficient for the directed evolution of proteins, enzymes and monoclonal antibodies, such as in vitro molecular biology methods, this technology is to improve the activity, change substrate specificity and improved protein (enzyme) The performance has a broad application prospects. In practical application, the Vmax of Na / H antiporter gene cloned in plants is relatively low salt tolerance is not strong, which greatly limits the application of the genes in crop molecular breeding. In view of this, the subject of the use of DNA shuffling technology combined with the restructuring of the gene library was screened to a significant increase in activity, can tolerate the high concentration of salts of the new Na / H-antiporter gene heterologous expression in yeast, plant Na / H-antiporter structure and function the relationship further research. The thesis of this study are summarized as follows: random fragmentation with DNase I AtNHX1 recover the 100-200bp fragment to small fragment restructuring by primer PCR, the final Primer PCR amplification of the recombinant gene library containing recombinant AtNHX1 fragment. Containing a recombinant AtNHX1 The amplified fragment was ligated to the yeast expression vector pYPGE15 Titanium lithium technology transforming yeast Na / H-antiporter the mutant body W303Δena1 4Δnhx1 construct expressing a recombinant gene library. The yeast transformants coating for high throughput screening in the APG-selective plates containing 100mM NaCl to give a salt tolerance significantly improved mutant containing the restructuring of the Na / H antiporter gene we named AtNHXS1 to. Sequence analysis showed that the AtNHXS1 comparing AtNHX1, seven nucleotide mutations, 42 nucleotide deletion and this deletion resulted in early termination of the translation. The expressed protein AtNHXS1 containing four amino acid mutations are L29P, S158P, Y241P and F242L, and in the C-terminal 296 amino acid deletion. Yeast functional complementation test showed that the expression AtNHXS1 Na / h antiporter than AtNHX1 the gene expression products resistant NaCl ability to improve doubled. And the yeast expression AtNHXS1 than the expression AtNHX1 yeast in enriched in Na and slightly more K. And were GFP fusion to AtNHX1 and AtNHXS1 C-terminal transformed into yeast. The confocal laser display the expressed protein AtNHXS1 and AtNHX1 are located in the yeast vacuole and vacuole extremely vesicles. Extraction expression of the yeast mutant AtNHX1 and AtNHXS1 complete vacuole acridine orange marker fluorescence quenching were determined for AtNHX1 AtNHXS1 protein ion-exchange activity found the expressed protein AtNHXS1 Na / H Vmax than AtNHX1 expression higher protein approximately doubled at the same time K / H of Vmax improve slightly. In addition, by PCR-mediated site-directed mutagenesis techniques were built two AtNHX1 single gene mutation and a C-terminal deletion mutant, into the yeast mutant yeast complementation experiments. The results were expressed with L29P, S158P, and the C-terminal 296AA missing AtNHX1 mutant yeast tolerance to NaCl, LiCl, KCl and hygromycin ability AtNHXS1 of similar. Agrobacterium GV3101 were constructed the plant expression vector pCAMBIA1301-35SN-AtNHX1 and vector pCAMBIA1301 were-35SN-AtNHXS1 and transferred by the freeze-thaw method. Penetration method Transformation of Arabidopsis thaliana, screened of the homozygote of the homozygous turn AtNHX1 and turn AtNHXS1, as the next step salt tolerance detection materials. Total genomic DNA was extracted from Arabidopsis strains to turn AtNHX1 and genetically AtNHXS1 gene transgenic Arabidopsis molecular identification by PCR and PCR-Southern blotting technology. Quantitative RT-PCR study found that transferred AtNHX1 Arabidopsis and transferred to AtNHXS1 gene in Arabidopsis Na / H antiporter gene expression was significantly higher than the wild-type plants. 200mm and 300mm NaCl treatment, over-expression of a AtNHX1 the Arabidopsis and overexpression AtNHXS1 Arabidopsis growth conditions significantly better than the wild-type Arabidopsis, and overexpression of AtNHXS1 Arabidopsis growth conditions to some extent better than excessive express AtNHX1 Arabidopsis, its dry weight, fresh weight was significantly higher than the latter, and the aerial parts of Na, K and proline content than the latter. Summarize the results of the above studies, we combine the yeast expression system through DNA shuffling directed molecular evolution and screening, to obtain a new strong salt tolerance gene AtNHXS1 plants Na / H-antiporter, the gene expression of proteins in yeast is still positioned within the vacuole and having a stronger Na forwarding activity, expression of this gene in yeast can be enriched in Na and K, and thus has a stronger salt. Overexpression of the salt tolerance of the transgenic Arabidopsis Arabidopsis than transferred to wild-type AtNHX1 gene, to a certain extent be raised. This study provides new information to further understand the relationship between the structure and function of AtNHX1 new strong salt tolerance gene can be applied to cultivate strong salt-tolerant transgenic plant molecular breeding.

Related Dissertations

CLC: > Biological Sciences > Botany > Plant Cell Genetics > Plant Genetic Engineering
© 2012 www.DissertationTopic.Net Mobile