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cDNA Cloning of Resistance Genes from Alfalfa and Primary Functional Verification of Transgenic Tobacco

Author: ZengGuang
Tutor: ShiQingHua；ZhangZuo
School: Xinjiang Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: alfalfa NHX gene P5CS gene PCR-Southern blot transgenic tobacco
CLC: S541.9;S572
Type: Master's thesis
Year: 2010
Downloads: 164
Quote: 5
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Abstract


Stress is one of the major factors that affect crop growth and production. Plants accumulate excessive sodium irons that cause ions toxicity and osmotic stress then abnormal metabolism if they vegetate in a high ion environment for some time, which have serous effect on plants growth and even result in death. The Na~+/H~+ antiporter protein that mainly locates in vacuole membrane is responsible for the exchange of Na~+/H~+ across the membrane. The Na~+/H~+ antiporter protein also play an important role in the plant salt tolerance. The metabolism physiologically changes when plants undergo drought stress. Water deficiency and celluar physiological drought occur in plants if they consume more water than absorbed water. Proline in plants is commonly free. The contents of proline increase under stress, which strengthens the plants resistance to stress. Na~+/H~+ antiporter protein gene and proline synthase gene were cloned from Xinjing Medicago falcata L. for the first time. These genes were MfNHX and MfP5CS respectively. The MfNHX (GenBank accession number: GU265772) which encoded 541 amino acids was 1626 bp cDNA, the MfP5CS (GenBank accession number: GU180149) which encoded 715 amino acids was 2148 bp cDNA, and the identifications of MfNHX and MfP5CS were extremely close to Medicago.sativa and Medicago.varia. xinmu No.1. Bioinformatics analysis indicated that Na~+/H~+ antiporter protein from Medicago falcata L. was a transmembrane protein and that proline synthase from Medicago falcata L. probably was a plasma protein. xmNHX and xmP5CS from Medicago.varia. xinmu No.1 were transferred into tobacco via agrobacterium mediation, and then Kanamycin–resistance transgenic plants were obtained. The results of PCR and PCR-Southern blot analysis indicated that the xmNHX and xmP5CS were integrated into tobacco genome and transcripted. Experiments results showed as below: The xmNHX and xmP5CS worked as expected under stress, which made the T1 transgenic tobacco acquire stess resistance. The T1 transgenic tobacco could survive and grow better than control group. The xmNHX had more effects on salt stress than the xmP5CS, and the xmP5CS played more roles on drought stress.

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CLC: > Agricultural Sciences > Crop > Economic crops > Tobacco ( tobacco )
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