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Paddy rice gene of synthetase of coded beta - keto acyl coenzyme A

A technology of ketoacyl coenzyme and synthetase, which can be used in genetic engineering, plant gene improvement, recombinant DNA technology, etc., and can solve problems such as difficulties

Inactive Publication Date: 2007-10-10
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is still difficult to achieve resistance breeding in rice by changing the structure of leaf epidermis at the molecular level

Method used

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  • Paddy rice gene of synthetase of coded beta - keto acyl coenzyme A
  • Paddy rice gene of synthetase of coded beta - keto acyl coenzyme A
  • Paddy rice gene of synthetase of coded beta - keto acyl coenzyme A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Obtainment of Nipponbare T-DNA insertion mutant P1424

[0042] Rice mutant P1424 was obtained by Agrobacterium tumefaciens-mediated transformation (Hiei, et al., 1994). The specific method is as follows:

[0043] 1. Induction of Rice Callus

[0044] Get mature paddy rice (Nipponbare) seeds (International Rice Research Institute, IRRI), shell, with 70% ethanol treatment 1-2min, 0.1% mercuric chloride treatment 15-20min, sterilized deionized water wash 4-6 times . After the surface moisture was blotted by sterile filter paper, inoculate into NB containing 2mg / L 2,4-D (Fluka Company) 0 On the culture medium (see attached table 1 for specific components), seal with parafilm membrane, and culture in dark in an incubator at 25°C to induce embryogenic callus. After 7-10 days, the callus induced by the scutellum was stripped and subcultured on the same medium. After dark culture in an incubator at 25° C. for about 20 days, vigorously growing embryogenic calli th...

Embodiment 2

[0054]Example 2: Genetic Analysis of Mutant P1424

[0055] The mutant phenotypes of transgenic rice plants are not all caused by T-DNA insertions (An et al., 2005), so it is necessary to confirm whether the mutant phenotypes and T-DNA insertions co-segregate through genetic analysis.

[0056] 1. Extraction and quantification of rice genomic DNA

[0057] Rice DNA extraction was performed as described by Dellaporta et al. (Malmberg et al., 1985). Specific steps are as follows:

[0058] (1) Take 3-5g of fresh rice leaves, quick-freeze in liquid nitrogen, quickly grind them into powder in a mortar, and transfer them to a 50ml centrifuge tube;

[0059] (2) Immediately add 20ml of extraction buffer preheated to 65°C (1M Tris-HCl (pH8.0) 100ml / L, 0.5M EDTA (pH8.0) 100ml / L, 5M NaCl 100ml / L, 10% SDS 125ml / L, β-mercaptoethanol 1.5ml / L), in a water bath at 65°C for 30min;

[0060] (3) Add 7.5ml of 5M potassium acetate, mix gently by inversion, place on ice for 20min, centrifuge at 40...

Embodiment 3

[0071] Example 3: T-DNA insertion site analysis

[0072] T-DNA is randomly inserted into genomic DNA. The insertion site of T-DNA in mutant P1424 was determined by thermal heterogeneous interleaved polymerase chain reaction (TAIL-PCR) method (Liu et al., 1995). The three specific primers in the TAIL-PCR reaction are:

[0073] SP1: 5'-GGTGACCAGCTCGAATTTCCC-3' (SEQ ID NO: 9)

[0074] SP2: 5'-TGAATCCTGTTGCCGGTCTTG-3' (SEQ ID NO: 10)

[0075] SP3: 5'-GCGCGCGGTGTCATCTATGT-3' (SEQ ID NO: 11)

[0076] The random primer was AD4: 5'-TG(A / T)GNAG(A / T)ANCA(G / C)AGA-3' (SEQ ID NO: 12). Amplification was performed using P1424 genomic DNA as a template, and the amplification procedure was referred to the literature (Liu et al., 1995). The amplified product was subjected to agarose gel electrophoresis, and a specific amplification band of about 800 bp was recovered with a gel extraction kit (Shenergy Gaming Biotechnology Co., Ltd.), and connected to the pGEM-T easy vector (Promega Company...

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Abstract

This invention provides a rice gene coding beta-keto co A synthetase, OsKCS1, which comprises 2186bp and 1560bp coding zones at the upstream of the start codon with promoter function, codes 519 amino acid, and contains typical FAE1 CUT1 RppA domain. The function-deficient mutant P1424 obtained by inserting T-DNA into rice OsKCS1 gene has short plants, occasionally curled leaves, and low fruiting rate. Further research shows that the wax at the surfaces of the mutant gas-generating organs is obviously reduced, and the draught resistance is seriously influenced. The expression of OsKCS1 gene in the mutant can recover its wild phenotype, thus can confirm the direction relationship between OsKCS1 gene and synthesis and growth of wax on rice surfaces. The identification of the gene is important to research on synthesis of wax on rice surfaces, and breeding of draught-resistant rice or other monocotyledon crops.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering. Specifically, the present invention relates to a rice gene encoding a β-ketoacyl-CoA synthetase related to rice epidermis structure, growth and development, including a recombinant plasmid of the gene, a host and applications thereof. Background technique [0002] In plants, very long-chain fatty acids (≥18C, very long-chain fatty acids, VLCFAs) and their derivatives are important components of cuticle wax, sphingolipid, seed oil and root suberin. The epidermis is the first barrier between the plant and the environment. Because there are wax crystals covering the surface and amorphous wax embedded in it, it can reduce non-stomatal water evaporation and block the invasion of fungi, bacteria, viruses and herbivorous insects. It plays an important protective role for terrestrial plants against ultraviolet rays, frost, etc. (Hamilton, 1995). [0003] The fatty acid de novo synthesis pathway...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/82C12N15/63C12N5/10C12N15/29C12N1/21
Inventor 何朝族于冬梅
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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