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Dwarf gene in paddy rice

A technology of dwarf genes and genes, applied in the field of rice genes, can solve the problems of few research reports

Inactive Publication Date: 2006-10-04
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 the rice variety Nipponbare as the template, and based on the nucleic acid sequence published on NCBI, 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 competent cells of S. coli, and the recombinants were selected for PCR and enzyme digestion identification.

[0123] Pick the positive recombinants and send them to Boya for sequencing.

Embodiment 2

[0125]The 3-terminal 416bp nucleic acid sequence of the osDw gene was selected, two pairs of primers were synthesized, and Kpn 1 and BamH I and Sac I and Spe I restriction sites were introduced into the primers respectively. Then, using the genomic DNA template, PCR amplification was carried out. The amplification conditions of PCR were as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 45 sec, annealing at 55 °C for 1 min, 72 °C for 1 min, 35 cycles; extension at 72 °C for 5 min. The PCR products were recovered and digested with Kpn 1 and BamH I and Sac I and SpeI respectively, and the digested products were recovered. The ds1301 was double digested with Kpn 1 and BamH I, and the digested product was recovered. Take 1 μl T 4 Ligase Buffer, 7 μl Kpn 1 and BamH I double-digested PCR product, 1 μL Kpn 1 and BamH I double enzyme ds1301, 10U T 4 Ligase was used overnight at 16°C, and the ligation product was transformed into competent cells of S. coli. Th...

Embodiment 3

[0128] Preparation of co-culture medium; pick glycerol bacteria stored at -20°C in 1.5ml LB (containing 50μg / ml Kan), and after shaking at 28°C for 32h, pipette 150μl of agricultural straw bacteria solution into 10ml LB (containing 50μg / ml Kan) 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 invention discloses an osDw gene obtained by insertion mutation of T-DNA with its RNAi expression vector and construction method and genetic transformation. Wherein, the gene includes 2720 alkaline groups with 33-35 ATGs as initiator codon and 2253-2255 TGAs as terminator codon. The clone method comprises: according the blast analysis result, basing on nucleic acid sequency of NCBI, amplifying 2.7kb nueleic acid fragment with PCR method for recovery to connect to T vector for sequenching; then, constructing RNAi vector to obtain short-stem plant.

Description

technical field [0001] The present invention relates to a rice gene, in particular to a rice dwarf (Oryza sativa Dwarf, abbreviated as osDw) gene obtained by a method of T-DNA insertion mutation. 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 them, the insertion tag method is one of the effective methods for cloning plant functional genes. If T-DNA is inserted into functional genes that regulate plant growth and development, it may cause the inactivation of functional genes, resulting in Physiological or phenotypic mutation. Using T-DNA to insert the T-DNA tag in the mutant, after cloning the adjacent sequence of T-DNA by PCR or plasmid rescue metho...

Claims

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Application Information

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Patent Type & Authority Applications(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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