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Functional Characterization of Tonoplast Na~+/H~+ Antiporter (NHX1) from Chenopodium Glaucum and Comparative Study of the Putative Regulation Region of K~+ or Na~+ Transportation in C-terminus between CgNHX1 and OsNHX1 (Oryza Sativa)

Author: HuangPing
Tutor: ZhangFuChun
School: Xinjiang University
Course: Biochemistry and Molecular Biology
Keywords: C. glaucum Rice NHX1 C-terminal Transgenic Arabidopsis Plant salt GFP fusion expression vector
CLC: Q943
Type: Master's thesis
Year: 2007
Downloads: 96
Quote: 0
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Abstract


With the Human Genome Project and other modes plants such as Arabidopsis, rice genome plan's completion of, and people gene awareness continues to depth, plant genome research has whole genome sequencing for the goals of the structural gene group learning turning to gene function identification is The goal of functional genomics research. Therefore enter the post-genomic era, the use of gene function in the method of reverse genetics and technology has increasingly become the hotspot in the field of biological research and development. The high salt environment is one of the main environmental factors that affect plant growth and development. Soil salinity is too high can also cause the saline-alkali soil, limiting the use of the soil. Currently, about 20% of the world's cultivated land and nearly half of irrigated land was salt stress hazards. Caused by the excessive concentration of Na ~ ion toxicity, osmotic stress, and K ~ / Na ~ ratio imbalance, plant metabolic abnormalities, salt stress damage to most plants. Therefore, regardless of halophytes or sweet soil plants, plants in response to salt stress the final result is to reduce the concentration of cytoplasmic Na ~ and maintain intracytoplasmic K ~~ Na ~ balance. With the development of molecular genetics and plant transgenic technology, the use of biotechnology to improve the salt tolerance of crops, crop salt stress environment to normal growth and increase crop yields, more and more attention is being achieved certain results. NHX1 a tonoplast Na to the / H ~~ antiporter, can promote the segmentation effect of sodium ions in the vacuole, across tonoplast pH provide energy. The Compartmentalization of sodium ions and chloride ions in the vacuole, not only as effective osmotica also reduced cytotoxicity, thereby enhancing the salt tolerance of plants. Studies have shown that expression in Arabidopsis, canola, tomatoes, rice and cotton Super the the tonoplast Na ~~ / H to antiporter can effectively improve the salt tolerance of the transgenic plants. Based on our laboratory cloned C. glaucum vacuolar membrane Na ~~ / H ~~ the antiporter gene CgNHX1 the cDNA sequence (AY371319), the study by the transformation of Arabidopsis genetic prove that the overexpression of the gene in Arabidopsis improve the salt tolerance of the transgenic plants. Of Chenopodium glaucum CgNHX1 and rice OsNHX1 of gene (AB 021 878) C-terminal sequence analysis, build Agrobacterium transformation of Arabidopsis C-terminal deletion recombinant expression vector, respectively the ultra the expression CgNHX1-histag OsNHX1-histag dcCgNHX1 transgenic plants of the-histag, dcOsNHX1-histag the to study of halophytes and sweet soil plants NHX1 gene C-terminal-control differences laid the foundation. Successfully constructed the study of C. glaucum CgNHX1 and CgVP1 protein subcellular localization the pCAMBIA1301-1-CgNHX1-GFP and pCAMBIA1301-1-CgVP1-GFP fusion expression vector, and the initial initially targeted at the CgNHX1 protein to the membrane system. First, halophyte Chenopodium glaucum cDNA template for PCR amplification, full CgNHX1 cDNA sequence. Dual expression vector carrying the gene pCAMBIA1301-1-CgNHX1, dip method of Agrobacterium tumefaciens into Arabidopsis T2 generation of homozygous transgenic plants obtained after hygromycin. PCR and RT-PCR test results show that the CgNHX1 integrated in the genome of the transgenic Arabidopsis and expression at the transcriptional level. 50 mM NaCl and 100 mM NaCl stress, the transgenic plants and wild-type plants on the ground the Ministry's of Na ~ content increase, the transgenic plants Na ~ content slightly higher than the wild-type plants of Na ~ content, but did not reach a significant difference. 150mM NaCl stress, however, the transgenic plants to significantly improve the salt tolerance than wild-type plants, the root system is more developed, and the content of Na ~ the Na ~ content lower than the wild-type plants transgenic plants. The study results show that overexpression of CgNHX1 able to improve the salt tolerance of transgenic Arabidopsis. The research showed that the Arabidopsis AtNHX1 the C-terminus of the protein has the function of regulating antiporter protein cation transporter. C-terminal regulatory region in comparison halophytes with sweet soil the plant NHX1 gene functional differences, the end of the deletion the C. glaucum CgNHX1 with rice OsNHX1 of protein C by PCR method hydrophilic region of approximately 100 amino acid sequence, and with 6 missing C-terminal histidine tag gene with complete gene after BamH Ⅰ and Sal Ⅰ double digestion at the same time built into the plant expression vector pCAMBIA1301-1, transforming Agrobacterium tumefaciens into Arabidopsis inflorescence dip method, initial screening respectively the the overexpression CgNHX1-histag, OsNHX1-histag, dcCgNHX1-histag dcOsNHX1-histag T1 transgenic plants. After the PCR validation the CgNHX1-histag OsNHX1-histag has been integrated in the genome of the transgenic plants. In addition, the experiment also the laboratory conditions of cultivation and transformation of Arabidopsis preliminary exploration. The positioning of the protein in the cells in the clear and participate in the metabolic pathway plays a very important role. By PCR amplified from pCAMBIA1302 vector length 756bp GFP sequence, Sal Ⅰ and Pst Ⅰ ??double enzyme cloned into the plant expression vector into pCAMBIA1301-1, the recombinant plasmid into pCAMBIA1301-1-GFP. Specific primers were designed according to the cDNA sequence cloned in our laboratory C. glaucum CgNHX1 and the CgVP1 (DQ443731) was amplified by the complete removal of the gene of the stop codon CgNHX1 CgVP1, the product is recovered after BamH Ⅰ and Sal Ⅰ or Kpn Ⅰ and Sal Ⅰ double ligated into the pCAMBIA1301-1-GFP vector, digested and identified by PCR, indicating a successful build the pCAMBIA1301-1-CgNHX1-GFP and pCAMBIA1301-1-CgVP1-GFP fusion expression vector. The sequencing results also show that the two fusion genes in the same reading frame. The transient expression of the test results showed that CgNHX1 protein may be located on the membrane system.

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