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Couchgrass sodium ion reverse transport protein gene and its cloning method and use

A technology of antiporter and wheatgrass, which is applied in the fields of molecular biology and biology, and can solve the problems of less obvious effect and so on.

Inactive Publication Date: 2003-04-09
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Early salt-tolerant genetic engineering mainly focused on scavenging free radicals and increasing osmotic adjustment substances. The salt-tolerant ability of transgenic plants has improved, but the effect is not obvious

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach ： Embodiment approach 1

[0040] According to the technique described above, the coding Na + Antiporter gene ENHX1, overexpression of this gene can lead to higher salt tolerance in transgenic Arabidopsis. Echinopsis is a monocotyledonous gramineous plant. Expressing this gene in plants such as wheat, rice, and corn, which are closely related to Echinopsis, will improve the salt tolerance of these plants, and will allow these plants to survive on saline-alkali land. Planting has very important economic benefits and social benefits. (four) specific invention implementation modes: implementation mode 1: Thinopyrum Na + Cloning method of antiporter gene

[0041] 1. Extraction of total RNA: use one-step method or RNA kit to extract total RNA

[0042] 2. Synthesis of the first strand of cDNA: Take 2 micrograms of total RNA, add 4 μl of 5× reaction buffer, 2 μl of 10 mM deoxyribonucleic acid (dNTP), 0.5 μl of ribonuclease inhibitor (40-200u / μl), primer oligodT (1μg / μl) 1μl, reverse transcriptase (10u / μl) 2...

Embodiment approach 2

[0057] 8. Homology search: use BLAST software to compare the isolated sequence with the sequence in the gene bank. Embodiment 2: Thinopyrum Na + Antiporter gene ENHX1, the following sequence: (1) Information of SEQ ID NO1

[0058] (a) Sequential features

[0059] * Length: 1955 bp

[0060] *Type: nucleic acid

[0061] * Chain type: double chain

[0062] *Topology: Linear

[0063] (b) Molecular type: cDNA

[0064] (c) Assumption: No

[0065] (d) Antisense: no

[0066] (e) Original source: Thinopyrum

[0067] (f)序列描述:SEQ IN NO.11 ATCCGCCGAG GTGGCGACCG GCATGGGGCT CGATTTGGGA GCCATCGCTC TCAAGTACAC-6061 GGGGCTGGCG GTGTCGGACC ACGGCTCCAT CGTCGCCATC AACATCTTCA TCGCGCTGCT-120121 CTGCGGCTGC ATTGTCTTCG GCCACCTGCT CGAGGGGAAC CGCTGGGTCA ATGAGTCCAC-180181 CACCGCGCTT GTCCTGGGGC TCATCACCGG TGGTGTGATT CTGATCTGCT CCAAAGGGGT-240241 GAATTCGCGC ATCCTTATCT TCAGCGAGGA TATTTTCTTC ATCTACTTGC TCCCGCCCAT- 300301 CATTTTTAAC GCCGGGTTTC AAGTAAAGAA AAAGCAATTC TTCCGCAACT TTGCGACAAT-360361...

Embodiment approach 3

[0074](c)序列描述1 MGLDLGAIAL KYTGLAVSDH GSIVAINIFI ALLCGCIVFG HLLEGNRWVN ESTTALVLGL 6061 ITGGVILICS KGVNSRILIF SEDIFFIYLL PPIIFNAGFQ VKKKQFFRNF ATIILFGAAG 120121 TLISFVIITF GAMGLFSKLG VGPLELGDYL AIGAIFSATD SVCTLRVLNQ DEAPLLYSLV 180181 FGEGVVNDAT SVVLFNAIQN IDINHFDVFV LLQFIGKFLY LFFTSTVLGV AAGLLSAYII 240241 KKLCSARHST DREVAIMILM AYLSYMLSML LDLSGILTVF FCGIVMSHYT WHNVTESSRV 300301 TTKHTFATLS LIAEIFLFLY VGMDALDIDK WKLASSSPKK PIALSAVILG LVMVGRAAFV 360361 FPLSFLSNLS KKESHPKISF NQQVIIWWAG LMRGAVSIAL AYNKFTTSGH TAVRVNAVMI 420421 TSTIIVVLFS TMVFGLLTKP LINLLIPPRP GTAADISSQS FLDPLTASLL GSDFDVGQLT 480481 PQTNLQYLLT MPTRSVHRVW RKFDDKFMRP MFGGRGFVPF VPGSPIERSV HGPGLLGTVT 540541 EAEDRS 546实施方式3:表达载体的构建

[0075] 1. According to the separated Na + Nucleotide sequence of antiporter gene, design primers:

[0076] Forward primer: 5′-TATTCTAGACGAGGTGGCGACCGGCATGG-3′

[0077] Reverse primer: 5′-GACGAGCTCCTTAACTACGGTCTTCTGC-...

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Abstract

The present invention belongs to the cloning, recombination and salt endurance function analysis of couchgrass sodium ion reverse transport protein ENHX1 gene, and belongs to the field of molecular biology and biological technology. General RNA is first extracted from couchgrass root and then inverse transcripted into cDNA. A pair of facultative primer is designed based on the conservative amino acid sequence of other plant's sodium ion reverse transport protein CycD2 for conventional PCR, land the PCR product is connected to pGEM-The vector to convert DH5 alpha cell and determine sequence. Then, whole length cDNA is obtained via fast amplification in 3' and 5' ends. Further, positive-sense expression vector is constituted to convert pseudomustard.

Description

(1) Technical field: [0001] The present invention relates to Na in Thinopyrum + The invention relates to the cloning and recombination of the antiporter ENHX1 gene and the analysis and application of the salt tolerance function, belonging to the field of molecular biology and biotechnology. (two) background technology: [0002] Salt stress can cause ion osmotic stress, generate active oxygen, destroy the osmotic potential of plants and the balance of ion distribution, resulting in plant damage, especially affecting the yield of crops. Therefore, increasing the salt tolerance of crops has attracted more and more attention. Under high salinity, plants mitigate the toxicity caused by salt stress by producing stress proteins and soluble osmoregulatory substances. Early salt-tolerant genetic engineering mainly focused on scavenging free radicals and increasing osmotic adjustment substances. The salt-tolerant ability of transgenic plants was improved, but the effect was not obvi...

Claims

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Application Information

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IPC IPC(8): A01H1/00C07H21/04C12N15/10C12N15/29C12P19/34
Inventor 张宪省乔卫华李兴国李全梓
Owner SHANDONG AGRICULTURAL UNIVERSITY
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