Coding sequence of paddy rice indole-3-acetic acid-amido synthetase gene OsGH3.6 and applications thereof

A nucleotide sequence, rice technology, applied in the field of genetic engineering, can solve problems such as affecting the initiation of main root growth, crown root initiation, blocking auxin signaling pathway, etc.

Inactive Publication Date: 2018-04-06
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It was recently found that overexpression of miR393a or miR393b in rice leads to auxin receptor gene OsTIR1 and OsAFB2 Down-regulatio

Method used

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  • Coding sequence of paddy rice indole-3-acetic acid-amido synthetase gene OsGH3.6 and applications thereof
  • Coding sequence of paddy rice indole-3-acetic acid-amido synthetase gene OsGH3.6 and applications thereof
  • Coding sequence of paddy rice indole-3-acetic acid-amido synthetase gene OsGH3.6 and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, rice gene OsGH3.6 clone

[0047] 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;

[0048] 2. RNA extraction. Take about 100 mg of fresh rice plant tissue material and grind it fully with liquid nitrogen. Add 1 ml Trizol reagent, vortex for 15 s, and place at room temperature for 5 min. Add 0.2 ml chloroform, remove protein, centrifuge at 12000rpm for 10min, transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, mix thoroughly, place at room temperature for 10min, centrifuge at 12k rpm for 10min, discard the supernatant, and treat with DEPC Wash the precipitate with 1 ml of 75% ethanol prepared in water, and repeat once. Dry at room temperature for 5-10 min, dissolve in 20 μl DEPC water, measure OD value, and detect by electrophoresis;

[0049] 3. Cloning of genes. Using the first strand of reve...

Embodiment 2

[0050] Embodiment 2, rice OsGH3.6 Gene organ expression pattern analysis

[0051] Extract total RNA from rice stems, young leaves, old leaves, roots, etc., use a reverse transcription kit to reverse transcribe the total RNA into cDNA, and use primers SEQ ID NO.5 and SEQ ID NO.6 for real-time fluorescence Quantitative PCR detection ( figure 1 ). The results show, OsGH3.6 The gene was constitutively expressed in various organs, but there was no significant difference in the expression of leaves and roots in different parts, and the expression level in stems was significantly increased.

Embodiment 4

[0054] Example 4, OsGH3.6 Molecular characterization of tilling mutants

[0055] The total DNA in the mutant and the wild-type control group was extracted respectively, and real-time fluorescent quantitative PCR detection and sequencing were performed using primers SEQ ID NO.7 and SEQ ID NO.8 ( Figure 5 ). according to osgh3-6 mutant g19 with g20 respectively with WT's GH3.6 DNA sequences were compared and it was found that: g19 At 452bp, G is mutated to A;g20 At 452bp, G was mutated to A ( Figure 5 A, B). All of the above are in OsGH3-6 A mutation occurred in the coding region, which is a sense mutation. OsGH3-6 two mutants of g19 with g20 with WT GH4 protein ( Figure 5 C, D) Comparison found that the histidine (H) at position 87 was mutated to arginine (R).

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Abstract

The invention belongs to the technical field of gene engineering, and specifically relates to a coding sequence of an indole-3-acetic acid-amido synthetase gene OsGH3.6 expressed in paddy rice and applications thereof. The applications are as follows: the nucleotide coding sequence of the indole-3-acetic acid-amido synthetase gene OsGH3.6 is cloned; the spatial expression pattern of different endogenous organs and tissues of the gene of paddy rice is analyzed and identified; after treatments such as drought treatment, high salt stress, plant hormone treatment, and the like, the change of expression pattern is analyzed and identified; the tilling mutant of OsGH3.6 is detected, and the root length and output are analyzed.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to an indole acetic acid aminosynthetase gene expressed in rice OsGH3.6 coding sequence and its application. Including: indole acetic acid amino synthetase gene OsGH3.6 Cloning of the nucleotide coding sequence of the gene, analysis and identification of the spatial expression pattern of the gene in different organs and tissues of rice endogenous, and the expression pattern changes after drought, high-salt stress and plant hormone treatment, and OsGH3.6 Detection of tilling mutants and analysis of root length and yield. Background technique [0002] Auxin is widely distributed in plants, such as roots, stems, leaves, flowers, fruits, seeds and coleoptiles. However, it is mainly concentrated in the vigorously growing parts (coleoptile, young stem tips, root tips, leaves, fertilized ovary and young seeds, etc.), while in tissues and organs that tend to age, the content...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N15/11C12Q1/6858C12Q1/6851C12Q1/6895A01H5/00A01H6/46
CPCC12N9/93C12N15/8273C12N15/8293C12Q1/6851C12Q1/6858C12Q1/6895C12Q2600/158C12Q2531/113C12Q2563/107
Inventor 明凤毛婵娟何建美丁佳琳吕波张彬
Owner FUDAN UNIV
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