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Application of arabidopsis microRNA400 in regulating and controlling cadmium resistance of plants

A technology of Arabidopsis and plants, applied in the field of molecular biology and genetic engineering, to achieve the effect of improving cadmium resistance

Active Publication Date: 2018-12-11
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

miR400 participates in plant defense against pathogenic fungi and bacteria through alternative splicing in response to high temperature and targeted inhibition of PPR1 and PPR2 mRNA expression, but how miR400 participates in Cd stress has not been reported

Method used

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  • Application of arabidopsis microRNA400 in regulating and controlling cadmium resistance of plants
  • Application of arabidopsis microRNA400 in regulating and controlling cadmium resistance of plants
  • Application of arabidopsis microRNA400 in regulating and controlling cadmium resistance of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Cloning of Arabidopsis MIR400

[0026] (1) Extraction of Arabidopsis genome.

[0027] (2) PCR amplification of MIR400 gene: the Arabidopsis genome is used as a template, primers are designed according to the MIR400 gene sequence, PCR amplification is carried out, the PCR amplification products are recovered and purified, and sequenced to obtain the precursor MIR400. The primers are:

[0028] Forward primer: 5'-GTTCTGAGGATTGTTTATGAGAGT-3' (SEQ ID NO. 3).

[0029] Reverse primer: 5'-GTTAAATGGAAGATGCTT-3' (SEQ ID NO. 4).

[0030] (2) The PCR reaction system and amplification conditions are shown in Table 1.

[0031] Table 1

[0032]

[0033] (4) Sequencing is sent to Shenggong Company for sequencing. The specific operations for recovering and purifying PCR amplification products are as follows: After gel electrophoresis, use a clean blade to cut the gel with the target fragments and put them in a centrifuge tube. Do not cut the gel too large to avoid DNA recovery. The fr...

Embodiment 2

[0034] Example 2: Construction of recombinant expression vector and preparation of transgenic plants

[0035] (1) Construction of recombinant expression vector

[0036] Primers were designed according to the Arabidopsis MIR400 gene sequence, and the PCR reaction was performed with EVO high-fidelity enzyme for PCR cloning to obtain the Arabidopsis MIR400 gene. And ligated to pEASY-Blunt simple Cloning Kit vector (purchased from TransGen company) via T4DNA. After the ligation product is transformed into Escherichia coli, take the transformed bacteria solution and spread it on the LB plate containing Kanna resistance, pick the colonies into the corresponding antibiotic medium solution, shake culture and confirm, extract the plasmid of the positive clone, digest by enzyme, and electrophoresis And the destination strip is recycled. The recovered target band was ligated into the dicot binary expression vector PBI121. After the reaction product is transformed into Escherichia coli, it ...

Embodiment 3

[0043] Example 3: Comparison of the growth characteristics of miR400Knock down strain and wild type under 95μM cadmium concentration

[0044] miR400Knock down plants have strong cadmium stress tolerance. Using short tandem targetmimic (STTM) technology (the existing conventional technology in the prior art) to reduce the expression of miR400 and hinder its function, the two miR400 Knock down strains obtained were named STTM3 and STTM5 respectively. The expression level of miR400 was detected by qRT-PCR and identified as miR400Knock down strain, see Figure 5 A. Put wild-type Arabidopsis, STTM3, STTM5 in normal 1 / 2MS medium, containing 95μM CdSO 4 Cultured on 1 / 2MS medium, the results are as follows: wild-type and STTM strains grow the same on normal 1 / 2MS medium, see figure 1 A. figure 2 A, it means that the decrease in the expression of this gene will not affect the normal growth and development of plants. Wild-type and STTM strains are grown on 95μM CdSO 4 When on 1 / 2MS medi...

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Abstract

The invention discloses an application of arabidopsis microRNA400 / arabidopsis microRNA400 precursor MIR400 in regulating and controlling cadmium resistance of plants / preparing transgenic plants, wherein the nucleotide sequence of arabidopsis microRNA400 is as shown in SEQ ID NO.1; the nucleotide sequence of arabidopsis microRNA400 precursor MIR400 is as shown in SEQ ID NO.2. According to the invention, MIR400 containing microRNA400 intron is cloned from arabidopsis, microRNA playing a role in regulating plant cadmium resistance in the seedling period of arabidopsis is verified. After verification, transgenic arabidopsis with higher cadmium resistance than a wild type can be obtained by reducing expression of microRNA. The microRNA in the invention can provide theoretic basis and gene sources for breeding new species of crops.

Description

Technical field [0001] The invention relates to the application of Arabidopsis microRNA400 in regulating plant cadmium tolerance, and belongs to the fields of molecular biology and genetic engineering. Background technique [0002] With the development of industry, industrial waste generated by industrial and mining enterprises and the large-scale application of pesticides in agricultural production have severely polluted the soil and water sources that humans rely on for survival. Cadmium in farmland soil not only affects the yield and quality of crops, but also affects human health through the food chain. The residue and concentration of cadmium in agricultural products bring great safety risks to human health. Even more dangerous is that soil cadmium pollution is irreversible, and it may take 100 to 200 years or even longer for soil contaminated by cadmium to recover. Cadmium is a toxic heavy metal in these wastes and pesticides, which is very harmful to animal and plant hea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/20
CPCC12N15/113C12N15/8271C12N2310/141
Inventor 颜康徐维博郭骞欢郑成超
Owner SHANDONG AGRICULTURAL UNIVERSITY
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