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Cotton GhZFP1 gene sequence, its clone and use

A gene and cotton technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as ineffective effects

Inactive Publication Date: 2006-08-16
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, and mainly improved the salt-tolerant ability of transgenic plants by transforming single or multiple functional genes, although the salt-tolerant ability of transgenic plants has improved , but the effect is not obvious

Method used

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  • Cotton GhZFP1 gene sequence, its clone and use
  • Cotton GhZFP1 gene sequence, its clone and use
  • Cotton GhZFP1 gene sequence, its clone and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: the isolation method of cotton GhZFP1 gene

[0065] 1. From 0.3mol· -1 Total RNA was extracted from NaCl-treated cotton leaves, mRNA was isolated, and a cDNA library of cotton under salt stress was constructed.

[0066] 2. Perform differential screening to screen out a large number of positive clones.

[0067] 3. Transform the positive clones into the plasmid pTripLEx2, extract the plasmids for enzyme digestion identification, and pick relatively large positive clones for sequencing.

[0068] 4. Sequence determination: This work was carried out at Shanghai Boya Bioengineering Technology Service Co., Ltd.

[0069] 5. Homology search: use BLAST software to compare the isolated sequence with the sequence in the gene bank.

Embodiment 2

[0070] Embodiment 2: Cotton gene GhZFP1, following sequence:

[0071] (1) Information on SEQ ID NO 1

[0072] (d) Sequential features

[0073] * Length: 1377 bp

[0074] *Type: nucleic acid

[0075] * Chain type: double chain

[0076] *Topology: Linear

[0077] (e) Molecular type: cDNA

[0078] (f) Assumption: No

[0079] (g) Antisense: No

[0080] (h) Original source: Cotton

[0081] (i) Sequence description: SEQ ID NO.1

[0082] 1 acggccgggg gtccccttat cactcagcac tttatcttc tctctccatt aatcttcatt

[0083] 61 gaaagaaaat catctttgct ttcacccatg accactgttt atgacccttc aacaccatctc

[0084] 121 acctccaaaa aattgtgttt caaggacctt gaaatccctc caaggaaaaa gcaactccat

[0085] 181 tgttgccaca acgccgccgc catggaactt ccccaccacg aagctaggct ccacaaatac

[0086] 241 cttccttcca atgaagacga tgacggcacc gatgatccat acggcaccga ccatttccgc

[0087] 301 atgtacgagt tcaaggtaag aaggtgtaca aggagccgta gccatgactg gactgactgt

[0088] 361 ccttttgctc atccaggtga aaaagcccga cgt...

Embodiment 3

[0119] Embodiment 3: Construction of expression vector

[0120] 1. According to the nucleotide sequence of the isolated cotton gene GhZFP1, design primers:

[0121] Forward primer: 5-AtgACCACTgTTTATgACCCTTCAC-3

[0122] Reverse primer: 5-TCTTCTTCCTCTACATCAAgAgC-3

[0123] Using the cotton cDNA containing the gene as a template, polymerase chain reaction was carried out.

[0124] 2. Take 2 μl of PCR and connect it with the pMD18-T vector, and the operation steps are carried out according to the instructions of the product pMD18-TVector system of Promega Company. Then transform E. coli DH5α strain and grow overnight on LB plates coated with 5-bromo-4-chloro-3-indole-β-D-galactoside and X-gal containing ampicillin (100 μg / ml) . Pick white colonies and culture them overnight in LB liquid medium. Plasmid DNA was extracted by alkaline method and sequenced.

[0125] 3. The gene was excised from the pMD18-T vector with two restriction enzymes XbaI and SalI, and connected with PB...

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Abstract

The invention is about the clone, recombination and the salt tolerant analysis of the GhZFP1gene of the zinc finger structure of the CCCH type in the cotton. The invention is to extract the total RNA from the cotton leaves after treating by the 0.3mol / L NaCl to separate the mRNA and construct the cDNA library, then to select the gene encoded the GhZFP1 protein of the zinc finger of the CCCH type. The tobacco transformed by the gene can grow in the concentration of 200mmol / L salt; and the paddy can grow in the in the concentration of 100mmol / L salt. So it can improve the haloduric ability of the plants to plant the plants in the alkaline soil.

Description

(1) Technical field [0001] The invention relates to the analysis and application of the cloning and sequence of the GhZFP1 gene in cotton and the salt tolerance function, and belongs to the fields of molecular biology and biotechnology. (2) Background technology [0002] High concentrations of salt cause ion imbalance and hyperosmotic stress in plants, resulting in poor plant development, slow growth and especially reduced crop yields. Severe salt stress or osmotic stress will cause the second stress in plants - oxidative stress, and even cause plant death. Therefore, people pay more and more attention to salt stress, and people pay more and more attention to improving the salt tolerance of crops. 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, and improve...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82
Inventor 郑成超郭英慧杨国栋吴长艾李新征
Owner SHANDONG AGRICULTURAL UNIVERSITY
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