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Ammopiptanthus mongolicus guard cell specific expression promoter and application thereof

A guard cell-specific and promoter technology, which is applied in the field of specific expression of promoters in Mongolian holly guard cells, to achieve strong specificity

Inactive Publication Date: 2018-09-28
INST OF SOIL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current research on it is limited to species that grow in conventional habitats, such as Arabidopsis, and there are no reports on species that grow in extreme drought conditions.

Method used

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  • Ammopiptanthus mongolicus guard cell specific expression promoter and application thereof
  • Ammopiptanthus mongolicus guard cell specific expression promoter and application thereof
  • Ammopiptanthus mongolicus guard cell specific expression promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: A kind of Ilex chinensis guard cell specific expression gene promoter P AmSLAC1 clone method

[0049] The used Ilex Ammopiptanthus mongolicus (Maxim.) Cheng f. of the present invention, test material is planted in plant growth box, incubator condition, light 100 μ mol.m -2 the s -1 ; Photoperiod: 16h light / 8h dark; Temperature: 23°C±0.5°C; Relative air humidity 70%.

[0050] 1. Sand Holly P AmSLAC1 Promoter clone:

[0051] Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method, and chromosome walking was performed as described by Liu et al. , splicing the PCR product sequences obtained in the two steps, and finally obtaining S. hollyensis P AmSLAC1 Promoter (1455bp). The primers used are listed in Table 1.

[0052] Table 1 Chromosome walking clone P AmSLAC1 Primers for the promoter

[0053]

[0054] Sand Holly P AmSLAC1 The specific steps for cloning the promoter are as follows:

[0055] Use the CTAB method to extrac...

Embodiment 2

[0061] Example 2: Sand Holly P AmSLAC1 Construction of plant expression vectors and transformation of Agrobacterium tumefaciens strain GV3101:

[0062] The recombinant vector constructed was to use the cloned P AmSLAC1 Fragment replacement. For this purpose, first cut the cloning vector pEasy-P with SacI and NcoI AmSLAC1 , At the same time, the pCambia1301 plasmid was digested with SacI and NcoI. The enzyme digestion reaction was carried out in a water bath at 37° C., and after 8 hours, it was detected by electrophoresis on 1% (w / v) agarose gel. The cloning vector pEasy-P AmSLAC1 The small fragment of about 1.5kb digested by the enzyme and the large fragment digested by pCambia1301 were recovered with a DNA gel recovery kit. Mix pEasy-P in a ratio of 3:1 (molar concentration ratio) AmSLAC1 Mix the small fragments excised by enzymes and the large fragments excised by pCambia1301, add 2 units of T4 DNA ligase (NEB), 10ⅹ reaction buffer 1 μL, and connect in a 16°C water bat...

Embodiment 3

[0065] Example 3: Plant expression vector pCambia1301-P AmSLAC1 Genetic Transformation in Arabidopsis and Screening of Transgenic Plants

[0066] Refer to the method in the Arabidopsis Experiment Manual for Arabidopsis transformation, the operation steps are as follows:

[0067]1) When Arabidopsis thaliana blooms for the first time, cut off the flower buds to promote the proliferation of more flower branches on the side branches, and wait for use; 2) Pick the positive clones that are verified to be correct, first shake the bacteria in a small amount in a 100mL Erlenmeyer flask, and culture overnight at 28°C and 200rpm; 3) Transfer to a 500mL Erlenmeyer flask (inoculate into 200mL liquid LB medium containing 50mg / L kanamycin, 25mg / L gentamicin and 50mg / L rifampicin at a ratio of 1:100) ) Expanded culture, 28°C, 200rpm, 8-12 hours, 4) Measure the OD of the bacterial solution with a spectrophotometer (BIO-RAD) 600 The value is 0.8-1.2; put the bacterial liquid into a 50mL centr...

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Abstract

Disclosed are an ammopiptanthus mongolicus guard cell specific expression promoter and application thereof. The ammopiptanthus mongolicus guard cell specific expression promoter is a nucleotide sequence shown in SEQ ID No.1 and can be used for screening specific genes related to guard cells. The promoter capable of realizing specific expression in the ammopiptanthus mongolicus guard cells is provided for the first time and is quite high in specificity, and thus an effective tool is provided for research of the functions of plant guard cells.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering and biotechnology, in particular to a specific expression promoter (named P AmSLAC1 ) and its applications. Background technique [0002] The expression of plant genes is finely regulated by the internal and external environment of cells, and has strict temporal and spatial order. Gene expression regulation includes five levels: pre-transcriptional, transcriptional, post-transcriptional, translational, and post-translational. Among them, the regulation of transcription level is the most critical. Promoter is an important element of transcriptional regulation. According to the different functions and modes of action, promoters can be divided into three categories: constitutive, inducible and tissue-specific promoters. The target gene driven by the constitutive promoter can be stably expressed in all growth stages and tissues of transgenic plants. The most commonly used ones contain ca...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82C12N15/74C12N1/21C12N15/10C12Q1/6876
CPCC07K14/415C12N15/10C12Q1/6876
Inventor 苏彦华韩蕾金曼杨顺瑛
Owner INST OF SOIL SCI CHINESE ACAD OF SCI
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