Use of miR397 in drought resistance plant cultivation

A drought resistance and plant technology, applied in the direction of introducing foreign genetic material using a vector, recombinant DNA technology, etc., can solve the problems of large water demand for plant growth and easy to be affected by drought, achieve good drought resistance effect, improve drought resistance, edible safe effect

Inactive Publication Date: 2009-07-22
SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to address the major problem that plants, especially crops, require large amounts of water for growth and are susceptible to drought, and introduce miRNA regulation technology into plant variety improvement, thereby providing the application of miR397 in cultivating drought-resistant plants

Method used

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  • Use of miR397 in drought resistance plant cultivation
  • Use of miR397 in drought resistance plant cultivation
  • Use of miR397 in drought resistance plant cultivation

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1 Rice drought treatment and total RNA extraction

[0033] In this example, 2-week-old rice seedlings (using Yoshida culture fluid) were subjected to drought treatment, that is, the rice seedlings were treated with 20% PEG6000 simulated drought conditions for 5 hours and 12 hours respectively, and then the total RNA of the treated rice seedlings was extracted. The total RNA of rice seedlings subjected to drought treatment was used as the control group.

[0034] In this embodiment, the phenol chloroform isoamyl alcohol method commonly used in the art is used to extract rice total RNA, and the specific steps are as follows:

[0035] (1) Weigh 1g of rice sample and grind it into fine powder with liquid nitrogen, add 10mL RNA zol (100ml contains 47.2g of guanidine isothiocyanate, 2.5ml of 1M sodium citrate at pH7.0, 5ml of 10% sodium lauroyl azolate) and 1mL sodium acetate (2M, pH4.0), continue grinding;

[0036] (2) Add 10mL of water-saturated phenol, 4mL of chlo...

Embodiment 2

[0046] Example 2 Northern hybridization of miRNA

[0047] In this example, Northern blot detection was performed on the total RNA of the treated sample group and the total RNA of the control group extracted in Example 1, and the methods used can refer to the routine operations of Northern hybridization in the art.

[0048] Take 1 μL of the rice total RNA prepared in Example 1 and perform formaldehyde denaturing gel electrophoresis, and the denaturing electrophoresis adopts routine operations in the art.

[0049] After the electrophoresis is completed, remove the electrophoresis plate and uncover it, cover the gel sample area with a piece of dry filter paper that is about the same size as the target area, cut off the excess gel with a blade, turn the gel over with the filter paper, and peel off another piece of electrophoresis gel. plate. Cover the glue with an equal area of ​​HybondN pre-soaked with 0.5×TBE + Nylon membrane, put the filter paper-gel-nylon membrane between th...

Embodiment 3

[0051] Example 3 Construction of rice mutants overexpressing miR397

[0052] In this example, on the basis of the detection results in Example 2, a rice mutant strain overexpressing miR397 was further constructed, and the specific steps are as follows:

[0053] 1. Construction of miR397 constitutive expression plasmid

[0054] In this embodiment, the vector pCAMBIA1390 (commercially available) was selected, and the CaMV35S promoter was inserted between the restriction sites HindIII and SalI of the vector, which can be used as a constitutive promoter for controlling the expression of miR397 in the future. The experimental operation here adopts the routine technique in this field.

[0055] From the total RNA of the processed sample prepared in Example 1 as a template, clone the miR397 gene, the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2, and Add XbaI and EcoRI enzyme cutting sit...

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Abstract

The invention discloses application of miR397 in the cultivation of drought-resistant plants, and by realizing the drought processing of paddy rice and detecting the miRNA expression level of processed samples, the invention discovers that the miR397 expression of the samples after the drought processing lies in a significant increasing trend, and the miR397 expression rise after the drought processing within 5 hours is more significant than the miR397 expression rise after the drought processing within 12 hours. The invention constructs a miR397 over-expression paddy rice mutant strain, which has better growth situation than wide paddy rice at relatively lower temperature and does not have obvious burliness difference with wide paddy rice, and results of simulated draught and post-draught recovery processing show that the paddy rice mutant strain has better drought resistance than wide paddy rice. The results provide a novel, simple and effective method for cultivating drought-resistant paddy rice and plants, which can be applied into large-scale popularization and application, does not cause environmental pollution and ensures food safety.

Description

technical field [0001] The present invention relates to plant breeding, in particular to the application of miR397 in cultivating drought-resistant plants. Background technique [0002] The food problem is one of several difficult problems facing the world today. Increasing grain production by increasing unit yield or expanding planting area, increasing agricultural input to transform low-yielding fields, etc. will encounter the problem of overcoming adversity constraints or mitigating adversity hazards. Therefore, understanding the response mechanism of plants to adversity and improving the stress resistance of plants has become an important basic research for further increasing agricultural production. It has attracted the attention of governments and scientists around the world, and is also a hot spot in current life science research. [0003] With the economic development and population expansion of human beings, the salinization of soil and the shortage of water resour...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
Inventor 陈月琴张玉婵王聪颖刘清李洪清王小菁徐杰
Owner SUN YAT SEN UNIV
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