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Mutant gene and mutant of corn gibberellin oxidase, expression vector and applications

A technology for mutating genes and expressing vectors, applied in the fields of oxidoreductase, application, genetic engineering, etc.

Active Publication Date: 2020-10-16
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the use of ZmGA2ox-like genes and their alleles to improve plant drought resistance in maize

Method used

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  • Mutant gene and mutant of corn gibberellin oxidase, expression vector and applications
  • Mutant gene and mutant of corn gibberellin oxidase, expression vector and applications
  • Mutant gene and mutant of corn gibberellin oxidase, expression vector and applications

Examples

Experimental program
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Effect test

Embodiment 1

[0030] This example provides a method for cloning the original gene ZmGA2ox6 of zeibberellin oxidase and its mutant gene.

[0031] Wherein, the cloning method of the original gene ZmGA2ox6 of zeibberellin oxidase comprises the following steps:

[0032] S1. Extraction of RNA: The corn inbred line B73 was cultivated to the three-leaf stage, and the total RNA of the leaves was extracted using Kangwei Century's ultra-pure RNA extraction kit (CW0581). The details are as follows:

[0033] S11. Take fresh corn leaves and grind them thoroughly in liquid nitrogen, add 1 mL of TRIzon reagent for every 40 mg of tissue, and mix well.

[0034] S12. The sample after mixing is gently turned upside down several times, and left at room temperature for 5 minutes to fully lyse the sample.

[0035] S13. Add 200 μL of chloroform, cover the centrifuge tube, vibrate vigorously for 15 seconds, and place at room temperature for 2 minutes.

[0036] S14. Place at 4°C and centrifuge at 12,000 rpm for ...

Embodiment 2

[0093] This embodiment provides two mutants of zeibberellin oxidase, which are respectively encoded by the above-mentioned mutant genes ZmGA2ox6-E144A and ZmGA2ox6-A145E, and their corresponding amino acid sequences are as shown in the sequence table SEQ ID NO: 5 and SEQ ID NO: 5 ID NO:6.

[0094] In addition, using the ZmGA2ox6 gene to encode zeiberellin oxidase, its open reading frame has 1089 bp nucleotides and encodes 362 amino acids, and the amino acid sequence of the obtained zeiterin oxidase is shown in the sequence table SEQ ID NO:4 .

[0095] Using the TMHMM Server v.2.0 online website, the transmembrane region of the protein was preliminarily predicted. It was found that there was no transmembrane domain in the protein, and ZmGA2ox6 was preliminarily speculated to be a non-transmembrane protein.

[0096] Such as figure 1 and figure 2 As shown, the zeibberellin oxidase ZmGA2ox6 protein belongs to the 2ODDs (2-ox oglutarate–dependent dioxygenases) family. Through t...

Embodiment 3

[0098] This example provides three recombinant plant expression vectors, which respectively contain the above-mentioned ZmGA2ox6 and its mutant genes ZmGA2ox6-E144A and ZmGA2ox6-A145E. The construction method of the recombinant plant expression vector is as follows:

[0099] S1. According to the corresponding sequence at the multiple cloning site of the intermediate vector, add the corresponding enzyme cutting site when designing the primer.

[0100] S2. The gene amplified by the high-fidelity enzyme is recovered, added A, connected to the T vector, transformed, PCR enzyme digestion identification, sequencing and other steps to determine the correctness of the expressed gene, with a complete open reading frame, No mismatches, no frameshifts.

[0101] S3. Digest the correctly sequenced gene using the designed enzyme cutting site to recover small fragments (genes).

[0102] Specifically, restriction endonucleases Psp XI and XbaI were selected to cut the target fragment with co...

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Abstract

The invention is suitable for the technical field of molecular biology, and provides a mutant gene and mutant of corn gibberellin oxidase, an expression vector and applications. The nucleotide sequence of the original gene of the corn gibberellin oxidase is shown as SEQ ID NO: 1 in a sequence table, and the nucleotide sequence of the mutant gene is shown as SEQ ID NO: 2 or SEQ ID NO: 3 in the sequence table. The mutant gene of the corn gibberellin oxidase provided by the embodiments can be used for building the plant expression vector to convert into crops such as arabidopsis thaliana; the life states such as phenotype and survival rates of transgenic arabidopsis thaliana can be observed before and after drought stress treatment; and through the indication of results, the mutant gene of the corn gibberellin oxidase can lower the plant height of plants such as the arabidopsis thaliana and can also enhance the drought tolerance of the arabidopsis thaliana and other plants.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a mutant gene, mutant, expression vector and application of zeibberellin oxidase. Background technique [0002] Crops are often subject to various abiotic stresses, such as drought, salt, low temperature, etc., which severely restrict the planting area of ​​crops and even lead to a decline in yield. Maize is an important food and economic crop. Although it has been proved to be one of the most adaptable crops to the environment, the growth and yield of maize are still affected by various abiotic stresses. In recent years, my country's maize production has been more and more seriously affected by regional and periodic droughts, which may cause an increase in production costs, or a serious decline in the yield and quality of maize. [0003] The latest research has found that gibberellin metabolism regulation is also directly involved in the response and regul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/82A01H5/00A01H6/20
CPCC12N9/0071C12N15/8273C12N15/8261C12Y114/11013
Inventor 李毅丹郭嘉刘相国刘洋初卫国崔秀兰李辉肖博
Owner JILIN ACAD OF AGRI SCI
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