Application of DT1 proteins to regulating stomatal density of plants and improving drought resistance thereof

A plant and protein technology, applied in the biological field, can solve the problems of reducing leaf area and photosynthetic source, reducing cell turgor, reducing biomass and yield, etc.

Active Publication Date: 2017-05-10
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 proteins to regulating stomatal density of plants and improving drought resistance thereof
  • Application of DT1 proteins to regulating stomatal density of plants and improving drought resistance thereof
  • Application of DT1 proteins to regulating stomatal density of plants and improving drought resistance thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, construction of recombinant plasmid

[0058] 1. Primer design and synthesis

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

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

[0061] 1. Extract total RNA from Arabidopsis thaliana and reverse transcribe it into cDNA.

[0062] 2. Use the cDNA obtained in step 1 as a template, and perform PCR amplification using a primer pair composed of F1 and R1 to obtain a PCR amplification product (after sequencing, the PCR amplification product is shown in sequence 2 of the sequence listing).

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

[0064] 4. Take the gateway clone, use the Gateway Cloning Expression Kit and operate according to the kit instructions, insert the DNA molecule shown in Sequence 2 ...

Embodiment 2

[0065] Embodiment 2, the acquisition of DT1 gene overexpression plant

[0066] 1. Obtaining of DT1 Gene Overexpression Plants

[0067] 1. Introduce the recombinant plasmid pMDC32-DT1 into Agrobacterium tumefaciens C58 to obtain recombinant Agrobacterium.

[0068] 2. Take the recombinant Agrobacterium obtained in step 1, use the inflorescence soaking method to introduce the recombinant plasmid pMDC32-DT1 into the 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 plant and carry out 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 the PCR identification is posit...

Embodiment 3

[0080] Embodiment 3, identification of DT1 gene overexpression plant

[0081] 1. Detection of DT1 gene expression level

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

[0083] Seeds to be tested were sown on MS solid medium and cultured. After 8 days of seed 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 level of DT1 gene. . The eIF4a gene was used as an internal reference gene.

[0084] The expression levels of DT1 gene and eIF4a gene were as follows: fi...

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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 the density of stomata of plants and improving the drought resistance of plants. Background technique [0002] Drought is an important reason affecting crop yield. When the water absorbed by the plant roots is not enough for transpiration, it will cause the plant to lack water. Water scarcity in plants reduces cell turgor, limits cell expansion, reduces leaf area and photosynthetic sources, and therefore reduces biomass and yield. [0003] Reducing the transpiration rate of plants can reduce the consumption of soil water, which is an effective method to improve the drought resistance of plants. [0004] Both stomatal opening and stomatal density can regulate water transpiration and utilization. Reducing stomatal density can improve water use efficiency and plant drought resistance. Contents of the invention [0005] The pu...

Claims

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Application Information

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