Application of dt1 protein in regulation of plant stomatal density and improvement of plant drought resistance

A protein and plant technology, applied in the application field of DT1 protein in regulating plant stomatal density and improving plant drought resistance, can solve problems such as reducing cell turgor, reducing leaf area and photosynthetic source, and limiting cell expansion.

Active Publication Date: 2019-08-02
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Water scarcity in plants reduces cell turgor, limits cell expansion, reduces leaf area and photosynthetic sources, and therefore reduces biomass and yield

Method used

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  • Application of dt1 protein in regulation of plant stomatal density and improvement of plant drought resistance
  • Application of dt1 protein in regulation of plant stomatal density and improvement of plant drought resistance
  • Application of dt1 protein in regulation of plant stomatal density and improvement of plant drought resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1. Construction of recombinant plasmid

[0058] 1. Design and synthesis of primers

[0059] F1: 5'-CACCATGATACATTACGAACAAAACA-3';

[0060] R1: 5'-TCACTCGCATTCTCCTTCAGT-3'.

[0061] 1. Extract total RNA from Colombian ecotype Arabidopsis and reverse transcribed into cDNA.

[0062] 2. Using the cDNA obtained in step 1 as a template, a primer pair consisting of F1 and R1 is used for PCR amplification to obtain a PCR amplification product (after sequencing, the PCR amplification product is shown in sequence 2 of the sequence list).

[0063] 3. Take the PCR amplification product obtained in step 2 and use the Gateway cloning entry kit and operate according to the kit instructions to obtain the entry clone (the PCR amplification product is introduced into the pENTR / TEV / D- in the kit vector, get the entry clone).

[0064] 4. Take the entry clone, use the Gateway cloning expression kit and operate according to the kit instructions, insert the DNA molecule shown in sequence 2 betwe...

Embodiment 2

[0065] Example 2. Obtaining of DT1 gene overexpression plants

[0066] 1. Obtaining DT1 gene overexpression plants

[0067] 1. The recombinant plasmid pMDC32-DT1 was introduced into Agrobacterium tumefaciens C58 to obtain recombinant Agrobacterium.

[0068] 2. Take the recombinant Agrobacterium obtained in step 1, and use the inflorescence soaking method to introduce the recombinant plasmid pMDC32-DT1 into Colombian ecotype Arabidopsis thaliana, and harvest the seeds.

[0069] 3. Sow the seeds obtained in step 2 on MS solid medium containing 60 mg / L hygromycin, and select resistant plants (T1 generation plants).

[0070] 4. Extract the genomic DNA of the T1 generation plants and perform PCR identification.

[0071] The primer pairs identified by PCR are as follows (F2 corresponds to the vector backbone, R1 corresponds to the DT1 gene):

[0072] F2: 5'-AAGTTCATTTCATTTGGAGAGGACC-3';

[0073] R1: 5'-TCACTCGCATTCTCCTTCAGT-3'.

[0074] If PCR is identified as positive (PCR amplification yields a...

Embodiment 3

[0080] Example 3. Identification of DT1 gene overexpression plants

[0081] 1. DT1 gene expression level detection

[0082] The seeds to be tested are as follows: L4 strain (30 T3 seeds), L6 strain (30 T3 seeds), L9 strain (30 T3 seeds), L10 strain (30 T3 seeds), L11 Strain (30 T3 seeds), L13 strain (30 T3 seeds), L14 strain (30 T3 seeds), L15 strain (30 T3 seeds), L16 strain (30 T3 seeds) Generation seeds) and Colombian ecotype Arabidopsis (30 seeds).

[0083] The test seeds were sown on MS solid medium and cultured. After 8 days of germination, RNA was extracted and reverse transcribed into cDNA. Using cDNA as a template, PCR amplification was performed using a primer pair composed of F1 and R1 to detect the expression of DT1 gene. . The eIF4a gene is used as the internal reference gene.

[0084] The expression levels of DT1 gene and eIF4a gene are as figure 2 Shown. The results showed that the background expression of DT1 gene in Colombian ecotype Arabidopsis was very low. Co...

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Abstract

The invention discloses application of DT1 proteins to regulating the stomatal density of plants and improving the drought resistance thereof, and provides a method for cultivating transgenic plants. The method for cultivating the transgenic plants includes a step of transferring DT1 protein encoding genes into starting plants to obtain the transgenic plants with the drought resistance higher than the drought resistance of the starting plants. The DT1 proteins are proteins (a) or (b); the proteins (a) comprise amino acid sequences shown as sequences 1 in sequence tables; the amino acid sequences shown as the sequences 1 are subjected to substitution and / or deletion and / or addition by the aid of an amino acid residue or a plurality of amino acid residues, so that the proteins (b) can be derived from the sequences 1 and are related to the drought resistance of the plants. The application has the advantages that as discovered by the inventor, the stomatal density of leaf epidermis can be effectively reduced if the expression level of DT1 genes is improved in Arabidopsis thaliana, accordingly, the transpiration rates of the transgenic plants can be reduced, and the drought resistance of the plants can be improved; important effects can be realized for controlling the stomatal density of leaves of the plants and improving the drought resistance of the plants.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the application of DT1 protein in regulating plant stomatal density and improving plant drought resistance. Background technique [0002] Drought is an important reason affecting crop yields. When the water absorbed by the roots of plants is not enough for transpiration, the plants will be short of water. The lack of water in plants will reduce cell turgor pressure, limit cell swelling, reduce leaf area and photosynthetic source, and thus reduce biomass and yield. [0003] Reducing the transpiration rate of plants can reduce the consumption of soil water and is an effective method to improve the drought resistance of plants. [0004] Both stomata opening and stomata density can regulate the transpiration and utilization of water. Reducing stomata density can improve water use efficiency and drought resistance of plants. Summary of the invention [0005] The purpose of the presen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/20
Inventor 华学军齐世连林清芳张敏
Owner INST OF BOTANY CHINESE ACAD OF SCI
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