High salt induced dunaliella salina gene fragment and gene of sodium/phosphorus co-transport channel

A technology of co-transportation, Salina, applied in the field of genetic engineering

Inactive Publication Date: 2006-10-04
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the prior art, there is no report on the gene expression induced by

Method used

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  • High salt induced dunaliella salina gene fragment and gene of sodium/phosphorus co-transport channel
  • High salt induced dunaliella salina gene fragment and gene of sodium/phosphorus co-transport channel
  • High salt induced dunaliella salina gene fragment and gene of sodium/phosphorus co-transport channel

Examples

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Embodiment 1

[0044] Example 1 Construction and preliminary identification of a differential cDNA library induced by high-salt stress in Salina salina

[0045] Get in the DM culture medium (Sun Y etc., Expression of foreigngene in Dunaliella by electroporation, Molecular Biotechnology, 30 (3) (2005) 185-92) containing 0.5mol / L NaCl the salina cell of stable culture and again above-mentioned Salina salina cells in culture state were transferred to medium DM with 2mol / L NaCl concentration to continue osmotic shaking and culture for 24 hours to extract mRNA from Salina salina cells (Oligotex TM mRNA Kit, QIAGEN). According to the kit (PCR-selected TM The cDNAsubtraction kit, clontech) requires that each sample needs 2 μg of mRNA. Since there is a small amount of rRNA residue in the purified mRNA sample, 2.4 μg of the mRNA samples obtained in the two states were respectively taken for corresponding cDNA synthesis. In order to check the synthesis efficiency very conveniently, add 1μl of 10mC...

Embodiment 2

[0051] Example 2 Salina Na + / H 2 PO 4 - Isolation and sequence analysis of co-transporting membrane channel genes

[0052] (a) Salina Na + / H 2 PO 4 - Cloning and Sequence Analysis of Full-length cDNA of Cotransporting Membrane Channel Gene

[0053] By using the SSH differential fragment CP1A60 (SEQ ID NO: 1) as a probe to screen the cDNA library of Salina salina, a specific positive clone was obtained. The sequencing identification results showed that the full length of the cDNA was 2649bp (SEQ ID NO: 12), containing the gene of Chlorella The specific tailing signal TGTAA (Andrens HW et al., Primary structure of the plasma membrane H-ATPase from the halotolerant algaDunaliella bioculata. 1995, Plant Mol Biol, 28: 657-666) and poly(A) structure was named DvSPT1. The analysis results of Vector NTI8.0 software showed that the longest reading frame encoded a protein containing 672 amino acids ( image 3 ) (SEQ ID NO: 13), an acidic protein with a protein molecular weigh...

Embodiment 3

[0062] Embodiment 3 Salina Na + / H 2 PO 4- Induced Expression of Cotransport Membrane Channel Genes Under High Salt Stress and Inorganic Phosphorus Starvation

[0063] (a) Salina Na + / H 2 PO 4 - Induced Expression of Cotransported Membrane Channel Genes Under High Salt Stress

[0064] In order to study the correlation between the transcription level of the gene DvSPT1 in Salina cells and the salt concentration, the Salina cells were first cultured in DM medium containing 1.0M NaCl at 2×10 6 Cells / ml were collected, and then transferred to fresh DM medium containing 2.0M NaCl to continue culturing, and continued culturing in fresh DM medium containing 2.0M NaCl for 0 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 9 hours, 12 hours and 24 hours, and then extract the total RNA of Salina cells, using α- 32 The 3' end-specific fragment of P-dCTP-labeled DvSPT1 (2121bp-2633bp) (PCR amplification primers: DvT1RTS: 5'-ccctgttccactcactgc-3' (SEQ ID NO: 16), DvT1RTA: 5'-gaacagg...

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Abstract

The invention provides a dunaliella salina cDNA library induced by high salt as well as screening its cDNA fragment and gene. Wherein, it constructs the library with inhibiting hybridization, and discloses the cDNA fragment and dunaliella salina Na+/H2PO4- co-ttransfer membrane channel protein cDNA, whole gene and its amino acid sequence. This cDNA can screen the specific express of dunaliella salina induced by high salt, and settles foundation for corresponding molecular mechanism research.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to the isolation of a new differential cDNA library of Salina salina induced by high-salt stress, the isolation of gene fragments and a new Dunaliella salina (Dunaliella viridis) Na + / H 2 PO 4 - Co-transport channel protein genes and their encoded proteins. Background technique [0002] Plant response to osmotic stress is a complex process controlled by multiple genes. Different plants respond differently to osmotic stress, with rich diversity. However, the cellular level response to osmolyte stress appears to be conserved throughout the biological kingdom (Yancey et al. Living with water stress: evolution of osmolyte systems. Science 1982, 217: 1214-1222). Quite a few examples have shown that some genes obtained from higher plants are often isolated by indirect use of such homologous genes found in animals, lower plants, and even yeast and bacteria. In fact, these homolog...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/11C12N15/63C07K14/405
Inventor 许政暟李启云高孝舒孙煜张庆琪宋任涛
Owner SHANGHAI UNIV
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