Dwarf gene in paddy rice

A dwarf gene and rice technology, applied in genetic engineering, plant genetic improvement, biochemical equipment and methods, etc., can solve the problems of few research reports

Inactive Publication Date: 2009-12-23
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are not many reports on gene cloning by insertion mutagenesis at this stage.

Method used

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  • Dwarf gene in paddy rice
  • Dwarf gene in paddy rice
  • Dwarf gene in paddy rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Using the genomic DNA of rice variety Nipponbare as a template and the nucleic acid sequence published on NCBI as the basis, two pairs of primers were designed for PCR amplification. The amplified product was recovered, and 8 μl of the recovered product was ligated with 1 μl of T-vector overnight at 16°C. The ligated product was transformed into Escherichia coli competent cells, and recombinants were selected for identification by PCR and enzyme digestion.

[0123] The positive recombinants were picked and sent to Boya Company for sequencing.

Embodiment 2

[0125]The 416bp nucleic acid sequence at the 3-end of the osDw gene was selected, two pairs of primers were synthesized, and Kpn 1 and BamH I, Sac I and Spe I restriction sites were introduced into the primers respectively. Then use the genomic DNA as a template to carry out PCR amplification. The PCR amplification conditions are 94°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 55°C annealing for 1 minute, 72°C for 1 minute, 35 cycles; 72°C extension for 5 minutes. The PCR product was recovered, and double-digested with Kpn 1 and BamH I, and Sac I and SpeI, respectively, to recover the digested product. The ds1301 was double-digested with Kpn1 and BamH I, and the digested product was recovered. Take 1 μl of T4 ligase buffer, 7 μl of Kpn1 and BamH I double enzyme digested PCR product, 1 μL of Kpn 1 and BamH I double enzyme ds1301, 10 U of T4 ligase, and ligate overnight at 16°C, and transform the ligated product into Escherichia coli competent Cells, reco...

Embodiment 3

[0128] Preparation of co-culture medium: Pick glycerol bacteria stored at -20°C in 1.5ml LB (containing Kan 50μg / ml), shake at 28°C for 32h, then pipette 150μl of the stalk liquid into 10ml LB (containing Kan 50μg / mL) After culturing at 28°C for 24 hours, centrifuge to collect the cells, suspend the cells with an equal volume of AA liquid medium, and add acetosyringone (AS) to the cells to make the concentration reach 100 μmol / L.

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Abstract

The rice dwarf gene relates to a rice gene, in particular to a rice dwarf gene obtained by T-DNA insertion mutation method. Provided are an osDw gene obtained by a method of T-DNA insertion mutation and its cloning method; an osDw gene RNAi expression vector and its construction method; a genetic transformation method of the osDw gene and its application in genetic transformation. The full length of the gene is 2720 bases, in which ATG at the 33rd to 35th is the start codon, and the TGA sequence at the 2253rd to 2255th is the stop codon. The cloning method is to amplify a 2.7kb nucleic acid fragment by PCR method based on the nucleic acid sequence published on NCBI based on the results of blast analysis; the nucleic acid fragment is recovered and confirmed by sequencing after being connected to the T carrier. It has the function of controlling the plant height of rice. The RNAi vector can be constructed according to its sequence, and dwarf plants can be obtained by transgenic method.

Description

technical field [0001] The invention relates to a rice gene, in particular to a rice dwarf (Oryza sativa Dwarf, abbreviated as osDw) gene obtained by T-DNA insertion mutation method. Background technique [0002] With the completion of the rice genome sequencing project and the establishment of a saturated physical map (Feng Q, Zhang Y, Hao P, et al. Sequence and analysis of rice chomosome 4. Nature. 2002 21; 420(6913): 316-320), The study of its functional genome has become one of the hotspots. There are many methods for cloning functional genes, among which the insertion tag method is one of the effective methods for cloning plant functional genes. If T-DNA is inserted into a functional gene that regulates plant growth and development, it may cause the inactivation of the functional gene, resulting in Physiological or phenotypic mutations. Using the T-DNA tag in the T-DNA insertion mutant, after cloning the adjacent sequence of the T-DNA by PCR or plasmid rescue method, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/10C12N15/63C12N15/82A01H4/00
Inventor 陈亮张红心林涛郭小玲苏勇波夏令
Owner XIAMEN UNIV
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