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Brachypodium distachyon drought-resistant gene and expression vector as well as coding protein and application thereof

A technology for expressing vectors and proteins, which is applied in the direction of introducing foreign genetic material, application, and genetic engineering by using vectors. It can solve the problems of crop yield reduction and harm to the normal growth and development of plants, so as to reduce oxidative damage, improve tolerance, and osmotic pressure. Improved effect

Active Publication Date: 2018-06-01
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Adverse conditions such as drought, high salinity, and extreme temperature seriously endanger the normal growth and development of plants and cause crop yield reduction

Method used

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  • Brachypodium distachyon drought-resistant gene and expression vector as well as coding protein and application thereof
  • Brachypodium distachyon drought-resistant gene and expression vector as well as coding protein and application thereof
  • Brachypodium distachyon drought-resistant gene and expression vector as well as coding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Isolation of the Brachypodium distachyon BdGF14g gene

[0034] 1. Cloning of BdGF14g gene

[0035] The 14-3-3 gene sequence obtained by Phytozome v11.0 (https: / / phytozome.jgi.doe.gov / pz / portal.html) Blast is submitted to NCBI (https: / / www.ncbi.nlm.nih.gov) ) for BLAST comparison analysis. Use the Oligo7 primer design software to specifically amplify the BdGF14g gene of Brachypodium distachyon. The primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. The PCR reaction system is shown in Table 1. The PCR program is as follows: pre-denaturation at 98°C for 2 minutes; Denaturation at 98°C for 10s; annealing at 65°C for 5s; extension at 72°C for 11s; final extension at 72°C for 10min; storage at 4°C. Among them, the three consecutive steps of denaturation, annealing and extension set up 35 cyclic reactions.

[0036] Table 1 PCR reaction system

[0037]

[0038] 2. Gel recovery of target fragments

[0039] Spot the PCR reaction product on 1% agarose ge...

Embodiment 2

[0062] Example 2: Construction of pBI121-BdGF14g-GFP eukaryotic expression vector

[0063] 1. PCR amplification of BdGF14g gene

[0064] Using the cloning vector pMD18-T-BdGF14g containing the correctly sequenced Brachypodium distachyon BdGF14g gene as a template, use the Oligo7 primer design software to design the XbaI restriction site and BamHI enzyme in the 5' end of the pBI121 vector multiple cloning site region The gene-specific primers at the cleavage site were used for PCR amplification. The primer sequences are shown in Tables SEQ ID NO.5 and SEQ ID NO.6. The PCR reaction system is shown in Table 3 / same as Table 1. The PCR program is as follows: pre-denaturation at 98°C for 2 minutes; 98°C Denaturation at ℃ for 10s; annealing at 65℃ for 5s; extension at 72℃ for 11s; final extension at 72℃ for 10min; storage at 4℃. Among them, the three consecutive steps of denaturation, annealing and extension set up 35 cyclic reactions.

[0065] Table 3 PCR reaction system

[0066]...

Embodiment 3

[0113] Example 3: Subcellular localization of Brachypodium distachyon BdGF14g protein

[0114] 1. Preparation and transformation of Agrobacterium competent

[0115] All the operation steps of the preparation and transformation experiment of Agrobacterium competent cells were carried out aseptically in the ultra-clean workbench. The specific process is as follows:

[0116] 1) Use a micropipette to draw 10 μL of the preserved Agrobacterium strain EHA105 from a -80°C ultra-low temperature refrigerator into a 2mL sterilized centrifuge tube, add 1mL of YEB liquid medium containing 50mg / L streptomycin, and store at 28°C Cultivate overnight on a shaker at 200 rpm;

[0117] 2) Take 1 mL of the above-mentioned bacterial solution and transfer it to 100 mL of YEB (containing 50 mg / L streptomycin), expand the culture on a shaker at 28 ° C at 200 rpm, and measure its OD value to OD with a UV / Vis spectrophotometer 600 = about 0.4;

[0118] 3) Ice-bath the above-mentioned Agrobacterium ba...

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Abstract

The invention provides a brachypodium distachyon drought-resistant gene and an expression vector as well as coding protein and application thereof. The nucleotide sequence of the gene is the sequenceshown by SEQ ID NO.1 or a nucleotide sequence complementary to the sequence shown by SEQ ID NO.1. The protein is obtained by coding the gene of claim 1, and the amino acid sequence of the protein is shown by SEQ ID NO.2. The gene is used for improving application of the plant tolerance to drought stress. The gene provided by the invention regulates the plant to close pores to reduce the moisture lost in transpiration; a stronger root system is obtained in a drought condition so as to guarantee moisture absorption; moreover, the gene can improve the activity of such enzymes as catalase, peroxidase and superoxide dismutase in the antioxidase system and eliminate reactive oxygen species (ROS) in time, thereby reducing the oxidative damage of cells caused by the ROS and improving the plant tolerance to drought.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a brachypodium distachyon drought-resistant gene, an expression vector and its encoded protein and application. Background technique [0002] Adverse conditions such as drought, high salinity, and extreme temperature seriously endanger the normal growth and development of plants and cause crop yield reduction. In recent years, in the face of the severe reality of environmental climate deterioration, how to improve plant resistance to cope with abiotic stress has attracted more and more attention. During the long-term adaptation to the external environment, plants have evolved dynamic gene regulatory networks and complex physiological change mechanisms. The identification and functional study of stress resistance genes are of great significance for elucidating the molecular mechanism of plant stress resistance and its breeding practice. Compared with t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/70C12N1/21C12N15/82A01H5/00A01H6/82
CPCC07K14/415C12N15/8273
Inventor 何光源杨广笑常俊丽何圆张扬
Owner HUAZHONG UNIV OF SCI & TECH
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