Coding sequence and application of epoxy carotene dioxygenase OsNCED3 gene of rice

A rice and gene technology, applied in the field of genetic engineering, can solve the problem of unclear mechanism of leaf senescence

Active Publication Date: 2018-03-23
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although there have been many studies on the biosynthetic pathway of ABA, the downstream signaling mechanism, and the induction of leaf senescence, the upstream transcriptional regulatory network and the mechanism associated with leaf senescence are still unclear.

Method used

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  • Coding sequence and application of epoxy carotene dioxygenase OsNCED3 gene of rice
  • Coding sequence and application of epoxy carotene dioxygenase OsNCED3 gene of rice
  • Coding sequence and application of epoxy carotene dioxygenase OsNCED3 gene of rice

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, rice gene OsNCED3 clone

[0044] 1. The rice variety Zhonghua11 was cultured in an incubator (SPX-250-GB, Shanghai, China): the growth conditions were photoperiod 16h / 8h (L / D), 28°C;

[0045] 2. RNA extraction. Take about 100 mg of fresh rice plant tissue material and fully grind it 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;

[0046] 3. Cloning of genes. by corresponding Arabidopsis AtNCED3 Bioinformatics ...

Embodiment 2

[0047] Example 2, Rice OsNCED3 Gene organ expression pattern analysis

[0048] Rice stems, roots, two leaves, three leaves, four leaves, five leaves, six leaves ( figure 1 , A, B) total RNA, the total RNA was reverse-transcribed into cDNA using a reverse transcription kit, and real-time fluorescent quantitative PCR was detected using primers SEQ ID NO.5 and SEQ ID NO.6 ( figure 1 , C). The results showed that the gene was expressed constitutively, with the highest expression level in the second leaf and the least expression level in the root; there was no significant difference in the expression level among the stem, fifth leaf, and sixth leaf.

Embodiment 3

[0049] Embodiment 3, rice gene OsNCED3 Expression pattern analysis under drought, high-salt stress and plant hormone treatment

[0050] Two-week-old rice seedlings were treated with 150mM NaCl, drought and 20µM ABA for 24h, and the total RNA in the leaves after 0, 2, 4, 8, 12, and 24h were extracted, and the total RNA was extracted using a reverse transcription kit. Reverse transcribed into cDNA, using primers SEQ ID NO.5 and SEQ ID NO.6, for real-time fluorescent quantitative PCR detection. The results showed that the expression level was significantly increased after NaCl treatment for 12h ( figure 2 , A), the expression level increased significantly at 4h under drought ( figure 2 , B), the expression level decreased significantly after 20 μM ABA treatment for 24 h ( figure 2 , C).

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Abstract

The invention belongs to the technical field of genetic engineering and particularly relates to a coding sequence and an application of an epoxy carotene dioxygenase OsNCED3 gene of rice. A method specifically comprises steps as follows: a nucleotide coding sequence of the OsNCED3 gene is cloned, isogeny comparison of the sequence is performed, space expression modes of different endogenous organsand tissue of the gene of the rice and expression mode change after drought, salt stress and plant hormone treatment are analyzed and identified, a Tilling mutant is obtained on the basis of rice Zhonghua11, molecular identification and stress experiments are performed, gene expression change and resistance change are detected. The OsNCED3 gene can be used for plant variety improvement includingimprovement of rice senescence resistance and improvement of the capability of rice for coping with the dark condition, so that the rice yield is improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a gene expressed in rice OsNCED3 Gene coding sequence and its application. Specifically including: 9-cis-epoxycarotenoid dioxygenase OsNCED3 Cloning of the nucleotide coding sequence of the gene, analysis and identification of the spatial expression patterns of different organs and tissues endogenous to this gene in rice, and the expression pattern changes after drought, high-salt stress and plant hormone treatment, and obtained in rice Zhonghua11 For Tilling mutants, carry out molecular identification and stress experiments to detect changes in gene expression and resistance. Background technique [0002] In higher plants, the content of the plant hormone ABA is regulated and balanced by its synthesis and decomposition reactions (Nambara and Marion-Poll, 2005). Key enzymes regulating ABA production include 9-cis-epoxycarotenoid dioxygenases (NCEDs) involved in sca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/11C12Q1/6851
CPCC12N9/0069C12Q1/6851C12Y113/11051C12Q2531/113C12Q2561/113C12Q2521/107
Inventor 明凤丁佳琳毛婵娟卢松冲吕波罗莉琼
Owner FUDAN UNIV
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