Paddy rice miR399d and application thereof

A technology of rice and transgenic rice, applied in the field of genetic engineering, can solve problems such as differences in microRNA regulation

Inactive Publication Date: 2012-09-19
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

in Arabidopsis miR399 The expression of microRNA did not change significantly before and after salt treatment (Fujii et al., 2005), indicating tha

Method used

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  • Paddy rice miR399d and application thereof
  • Paddy rice miR399d and application thereof
  • Paddy rice miR399d and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 rice miR399d clone

[0048] 1. Rice variety Nipponbare ( Oryza sativa Japonica) were cultured in an incubator (SPX-250-GB, Shanghai, China): the growth conditions were photoperiod 16h / 8h (L / D), 28°C.

[0049] 2. Genomic DNA extraction. Take 0.05g tender leaves, add 500μl 65°C preheated extraction buffer, and mix quickly; 65°C water bath for 20min (inverted and mixed 1-2 times during the period), cool to room temperature; add 500μl chloroform:isoamyl alcohol (24: 1), mix gently, let stand for 1-2min; centrifuge at 12000rpm at room temperature for 10min, transfer the supernatant to a new tube; add an equal volume of chloroform:isoamyl alcohol (24:1), mix gently, let stand for 1-2min ; Centrifuge as above, transfer the supernatant to a new tube; add 1 / 10 volume of 3M NaAC (pH5.2), 2 times the volume of ice-cold absolute ethanol (-20°C), mix gently, and stand at -20°C for 40 minutes; same as above Centrifuge, discard the supernatant, wash the precipitate...

Embodiment 2

[0051] Example 2 Relationship between expression of miR399d and phosphorus deficiency treatment

[0052] After 20 days of Nippon Harenae and phosphorus deficiency treatment for 14 days, total RNA was extracted from stems and leaves of rice plants and reverse-transcribed. Semi-quantitative PCR was performed with specific primer pair (SEQ ID NO.2 and SEQ ID NO.3). OsmiR399d The precursor of is induced by phosphorus deficiency, while OsmiR399d The high expression of OsPHO2 Decrease in mRNA level ( figure 1 ).

Embodiment 3

[0053] Example 3 OsmiR399 The relationship between the expression of and salt treatment

[0054] (1) Pre- OsmiR399d expression

[0055] The seedlings cultivated in Nipponbare for 20 days were treated with 50, 100, 150, and 200 mM NaCl for 24 hours, respectively, and RNA was extracted from shoot and root, reverse-transcribed into cDNA, and carried out with specific primer sequences such as SEQ ID NO.4 and SEQ ID NO.5. real-time RT-PCR detection miR399d Precursor expression. The results show that in Shoot, miR399d The expression of began to increase with the increase of NaCl concentration, reached the highest at 150mM, and slightly decreased at 200mM ( figure 2 a). Root, miR399d The expression of the precursor increased with increasing concentration ( figure 2 b).

[0056] (2) The expression of pre-miR399d at different treatment time under the same salt treatment concentration

[0057] The seedlings cultivated in Nipponbare for 20 days were treated with 200mM N...

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Abstract

The invention belongs to the technical field of gene engineering, in particular to paddy rice miR399d and application thereof. Functions of a paddy rice miR399d gene are verified through overexpression of transgenic rice plants of the paddy rice miR399d (short for OsmiR399d). The paddy rice miR399d comprises a clone of a paddy rice miR399d precursor gene, construction of an expression carrier containing the gene, a primer sequence, an Osmir399d overexpression plant, response of the gene OsmiR399d on low phosphorus stress and salt stress, and verification of a target gene of the gene OsmiR399d. Semiquantitative and quantitative PCR (Polymerase Chain Reaction) detection results show that the expression of phosphorus deficiency treatment and salt treatment in roots and stem leaves can be improved. The target gene of the OsmiR399d is proved to be OsPHO2 through 5'RACE. The invention also discloses the application of the paddy rice miR399d, which is characterized in that the content of phosphorus in the stem leaves of the transgenic plants can be obviously improved after the gene OsmiR399d is overexpressed in the paddy rice, so that the plants are shown as phosphorus poisoning. The gene OsmiR399d can be used for variety improving of plants.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a rice miR399d (abbreviated as OsmiR399d ) and its applications. Background technique [0002] Rice is one of the main food crops in the world. Rice soil areas in my country are mainly concentrated in the plains of the middle and lower reaches of the Yangtze River, the Sichuan Basin, the Pearl River Delta and the western plains of Taiwan Province. These areas are mostly acidic and slightly acidic soils. The main form of phosphorus in the soil is iron phosphate, and the content of iron phosphate in paddy soil can generally reach 50-80% of that in the non-closed storage state (China Soil, 1978). Phosphorus is poorly absorbed by crops. The shortage of phosphate fertilizer in paddy soil greatly limits the yield of rice, so improving the nutrient characteristics of rice is of great significance for increasing rice yield. [0003] MicroRNA is a class of endogenously e...

Claims

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Application Information

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IPC IPC(8): C12N15/113A01H5/00C12N15/11C12Q1/68
Inventor 明凤张璇吕波金津张睿
Owner FUDAN UNIV
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