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Cloning and Functional Analysis of a High Affinity K~+ Transporter Gene SeHAK1 from Salicornia Europaea

Author: WuChuang
Tutor: SuQiao
School: Dalian University of Technology
Course: Biochemistry and Molecular Biology
Keywords: Halophytes Salicornia High- affinity potassium ion transport proteins SeHAK1 Clone Yeast expression
CLC: Q943.2
Type: Master's thesis
Year: 2011
Downloads: 55
Quote: 1
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Abstract


The lack of soil salinization and Potassium important factors that affect plant growth and development. The under the salinization and hypokalemia double stress, maintain plant ion homeostasis in the body, improve plant salt tolerance and resistance to low potassium is critical. The high affinity and potassium ion transporter protein is responsible for the potassium uptake of Na-sensitive potassium ion transport activity of the protein was inhibited in high salt environments, hypokalemia environment, but Na-insensitive high affinity and potassium ion transporter protein affected than small. Speculate, Na-sensitive potassium ion transport proteins in the high salt environment can give better play to the absorption of potassium, while improving salt tolerance in plants. From halophytes isolated to Na-insensitive potassium transporter gene. Salicornia is one of the world's most salt-tolerant dicotyledonous halophytes The purpose of this study is to be obtained from Salicornia Na insensitive potassium transporter gene SeHAKl expect it to efficient low potassium environment mediated potassium absorption, and can improve the salt tolerance of the transgenic plants. This experiment by RT-PCR, the RLM-RACE and anchored PCR technology to obtain a 2706bp of SeHAKl, which contains 249bp of the 5'UTR, the 3'UTR of 96bp and 2361bp coding region, which encodes a 786 amino acid protein. Homology analysis, the HAK family SeHAKl reported homology of 40% -77%. Phylogenetic analysis showed that, SeHAKl belong to the subfamily HAK / KUP / KT. Hydrophobic amino acid analysis showed SeHAKl has 14 transmembrane domains. Patterns of gene expression analysis showed that, SeHAKl the salt angle grassroots organizations, stems organizations have transcriptional level expression; without stress treatment, high salt and low potassium processing Salicornia, can the detected SeHAKl gene expression. The build SeHAKl the coding region into the yeast expression vector pYES2.0, using electroporation The recombinant plasmid is introduced into the yeast strain W △ 3 screened by auxotrophic the transgenic yeast WA3-SeHAKl. Yeast complementation shows that in 100μMK the medium WA3-SeHAKl normal growth, but in the presence of Cs, Rb a medium growth is inhibited, in the environment of high concentrations of Na, WA3-SeHAKl are less affected. The potassium depletion experiment showed that external potassium ion concentration 100μMK conditions W △ 3, The WA3 SeHAKl of potassium ion absorption rate higher than its Km value of 3.8μM, mediated high-affinity K + transporter. The outside potassium ion concentration for 10mMK conditions, WA3-SeHAKl potassium ion absorption rate is higher than the W △ 3, that SeHAKl also mediated by the low affinity absorption and potassium ions.

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CLC: > Biological Sciences > Botany > Plant Cell Genetics > Plant Genetic Engineering
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