Drought resisting and salt resisting gene of brachypodium distachyon and coded proteins and application thereof

A protein and gene technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as deterioration, quality decline, and crop yield reduction, and achieve the effects of reducing oxidative damage, improving tolerance, and enhancing stability

Active Publication Date: 2019-03-26
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Unfavorable environments such as drought and high salinity affect the normal growth and development of plants by affecting photosynthesis, lipid metabolism and protein synthesis and metabolism of plants,...

Method used

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  • Drought resisting and salt resisting gene of brachypodium distachyon and coded proteins and application thereof
  • Drought resisting and salt resisting gene of brachypodium distachyon and coded proteins and application thereof
  • Drought resisting and salt resisting gene of brachypodium distachyon and coded proteins and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Isolation of the BdGF14a gene of Brachypodium urticae

[0037] 1. BdGF14a gene cloning

[0038] The 14-3-3 gene sequence (BRADI1G11290.1, GenBank:KU933262.1) obtained in Phytozome v12.1 (https: / / phytozome.jgi.doe.gov / pz / portal.html) Blast was submitted to NCBI (https: / / www.ncbi.nlm.nih.gov) for BLAST comparison analysis. Use Primer6 primer design software to specifically amplify the primers of Brachypodium dilatum BdGF14a. 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: 98°C pre-denaturation for 3 minutes; Denaturation at 98℃ for 15s; annealing at 65℃ for 5s; extension at 72℃ for 30s; final extension at 72℃ for 10min; storage at 4℃. Among them, three consecutive steps of denaturation, annealing, and extension set up 35 cyclic reactions.

[0039] Table 1 PCR reaction system

[0040]

[0041] 2. Glue recovery of the target segment

[0042] The PCR reaction product was spotted ...

Embodiment 2

[0065] Example 2: Construction of pBI121-BdGF14a-GFP eukaryotic expression vector

[0066] 1. PCR amplification of BdGF14a gene

[0067] Using the cloning vector pMD18-T-BdGF14a containing the correctly sequenced BdGF14a gene of Brachypodium diffracta as the template, the Oligo7 primer design software was used to design the 5'end with the XbaI restriction site and BamHI enzyme in the multiple cloning site region of the pBI121 vector Gene-specific primers at the cut site are amplified by PCR. The primer sequences are shown in Tables SEQ ID NO. 5 and SEQ ID NO. 6, and the PCR reaction system is shown in Table 3 / Same as Table 1. The PCR program is as follows: 98°C pre-denaturation for 2 min; 98 Denaturation at ℃ for 10s; annealing at 65℃ for 5s; extension at 72℃ for 30s; 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.

[0068] Table 3 PCR reaction system

[0069]

[0070] 2. ...

Embodiment 3

[0116] Example 3: Subcellular localization of BdGF14a protein of Brachypodium ergatum

[0117] 1. Preparation and transformation of Agrobacterium competence

[0118] The preparation of Agrobacterium cell competence and all operation steps of the transformation experiment are carried out aseptically in an ultra-clean workbench. The specific process is as follows:

[0119] 1) Inoculate the Agrobacterium EHA105 strain preserved from the ultra-low temperature refrigerator at -80°C into YEB solid medium containing 50 mg / L Str (streaking), and cultivate in the dark at 28°C for 2 to 3 days.

[0120] 2) Pick a large and round monoclonal colony and inoculate it into 1 mL YEB liquid medium containing 50 mg / L Str, and cultivate in the dark at 28°C and 200 rpm for 12-16 hours.

[0121] 3) Transfer 1 mL of the above-obtained bacterial solution to 100 mL of YEB (containing 50 mg / L streptomycin), expand the culture at 28°C on a shaker at 200 rpm, and measure the OD value to OD with an ultraviolet / visi...

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Abstract

The invention provides an application of coded proteins of drought resisting and salt resisting gene of brachypodium distachyon. The nucleotide sequence of the gene is a sequence as shown in SEQ ID NO.1 or a nucleotide sequence complementary as shown in SEQ ID NO.1. The protein is obtained by coding the gene according to the claim 1, and the amino acid sequence of the protein is as shown in SEQ IDNO.2. The gene is applied to improving tolerance of plants to drought and salt resistance. The gene adjusted plant keeps cell moisture by accumulating through matters. ROS accumulation is reduced andthe cytomembrane oxidizing damage caused by active oxygen is reduced by improving the activity of an antioxidant enzyme system. By accelerating closing of pores under drought, the drought and salt resistance of transgenic tobacco is improved by way of reducing water damaged by means of transpiration, improving the expression level of stress related genes and the like. Drought and salt resisncve of the transgenic tobacco with the gene is dependent on adjustment of ABA synthesis and an ABA signal channel.

Description

Technical field [0001] The present invention belongs to the technical field of biological genetic engineering, and particularly relates to a drought- and salt-tolerant gene of Brachypodium urticae, an expression vector, and its coded protein and application. Background technique [0002] Unfavorable environments such as drought and high salinity affect the normal growth and development of plants through processes such as photosynthesis, lipid metabolism, and protein synthesis and metabolism, and cause crop yields and quality decline. Ecological imbalance and environmental pollution make the growth environment of plants more complex and deteriorating, and the normal growth of plants is seriously threatened. Because they cannot evade environmental stress by moving to a favorable position like animals, plants have formed a complete system that senses, transmits and responds to various stresses at the molecular, cellular and physiological levels in the process of evolution. To clari...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/70C12N1/21C12N15/82A01H5/00A01H6/82C12N1/19
CPCC07K14/415C12N15/70C12N15/8273
Inventor 何光源杨广笑常俊丽张扬赵洪艳何圆
Owner HUAZHONG UNIV OF SCI & TECH
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