Application of OsKTN80b gene in terms of reducing rice plant height

A rice and gene technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of unfavorable agronomic traits and difficult application, and achieve the effect of increasing planting density, good effect, and improving rice yield

Inactive Publication Date: 2017-11-03
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among these dwarf genes, except for sd1, most of them have the genetic phenotype of "one cause and multiple effec

Method used

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  • Application of OsKTN80b gene in terms of reducing rice plant height
  • Application of OsKTN80b gene in terms of reducing rice plant height
  • Application of OsKTN80b gene in terms of reducing rice plant height

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The construction of embodiment 1OsKTN80b CRISPR / Cas9 knockout vector

[0037] The inventor screened a small mutant material in the EMS mutagenesis mutant library of Shuhui 498 background, and obtained 5 candidate genes through whole genome resequencing and MutMap positioning analysis, including the microtubule splicing protein encoded in it The LOC_Os04g58130 gene of the p80 subunit OsKTN80b; the candidate gene OsKTN80b was knocked out by CRISPR / Cas9 gene editing, and the function of this gene in regulating the phenotype of rice plants was unexpectedly discovered.

[0038] A cas9 knockout vector was constructed using Baigegene CRISPR / Cas Vector Construction Kit.

[0039] (1) Target sequence design: sgRNA online design software website in CRISPR / Cas9 system: http: / / www.e-crisp.org / E-CRISP / designcrispr.html Input the OsKTN80b gene sequence (see SEQ ID NO.1), design and generate gRNA target sequences SG1 and SG2. The recognition sequence is 19bp, and the target sequenc...

Embodiment 2

[0058] Example 2: Transformation of rice with OsKTN80b cas9

[0059] (1) The two recombinant plasmids C1 and C2 obtained in Example 1 were respectively introduced into Agrobacterium strain EHA105 by freeze-thaw method. Mix each 100 μl of EHA105 competent cells with 0.5-1 μg (about 2 μl) of plasmid DNA, place on ice, liquid nitrogen and 37°C water bath for 5 minutes; dilute to 1ml with fresh LB liquid medium, shake at 28°C The bed was incubated for 2-4 hours; 200 μl was taken out and spread on an LB plate containing antibiotics Kan (50 μg / ml) and Rif (50 μg / ml), and incubated at 28° C. for 2-3 days. Pick a single colony of Agrobacterium from the LB plate and inoculate it into 3ml of LB liquid medium containing antibiotics, cultivate overnight on a shaker at 28°C, and transfer it to 50ml of LB liquid medium containing antibiotics the next day at 1% inoculum size. Continue shaking culture at 200rpm to OD 600 When it is about 0.6 to 0.8, centrifuge the fresh Agrobacterium broth ...

Embodiment 3

[0064] Example 3: Genomic DNA extraction of transgenic rice, PCR detection and sequencing of mutants

[0065] Proceed as follows:

[0066]The CTAB method was used to extract the genomic DNA of the transgenic rice leaves of the T0 generation obtained in Example 2; PCR sequencing analysis was performed on the knockout sites of the transgenic plants of the T0 generation. The PCR amplification system is: 10×buffer for KOD-Plus 2.5μl, KOD plus polymerase (5U / μl) 0.25μl, 25mmol / L MgSO4 1μl, dNTPs (2mmol / L) 2.5μl, each primer (10μmol / L , Cas9-p80-1F and Cas9-p80-1R) 0.5 μl, and (10 μmol / L, Cas9-p80-2F and Cas9-p80-2R) template DNA 0.5 μl, made up to 25 μl with ultrapure water. The reaction conditions were 94°C for 2min; 30 cycles of 94°C for 20s, 58°C for 30s, and 68°C for 60s; 68°C for 5min. The amplified product was subjected to 1% agarose gel electrophoresis (electrophoresis buffer 0.5×TBE), observed and photographed by the BIORAD gel imaging system (see image 3 and Figure 5...

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Abstract

The invention discloses application of an OsKTN80b gene in terms of reducing rice plant height, belonging to the field of rice gene engineering. A plant with reduced plant height is finally obtained mainly by performing gene editing on the OsKTN80b gene through a CRISPR/Cas9 system, so as to obtain a transgenic plant subjected to OsKTN80b gene knockout, and selecting a homozygous mutant. The invention further discloses two target spot sequences SG1 or SG2 used for the OsKTN80b gene knockout; the target spot sequences are respectively formed by nucleotide sequences shown as SEQ ID No. 4 and SEQ ID No. 5. Compared with wild type rice, the plant height of the transgenic plant subjected to the OsKTN80b gene knockout is reduced by 13.56 to 16.33 percent, and the effect of reducing the rice plant height is good; secondly, a method provided by the invention is not limited to a genetic background, and the problems linked with mal-characters do not exist.

Description

technical field [0001] The invention relates to the field of rice genetic engineering, in particular to the application of OsKTN80b gene in reducing the plant height of rice. Background technique [0002] In the 1960s, the successful breeding of dwarf rice increased the yield of rice by 20%-30%, which was called the "green revolution" of rice breeding. Dwarf breeding not only improves the lodging resistance of rice, but also plays an important role in increasing the planting density and improving the comprehensive traits of rice. At present, the rice dwarf gene used in production is mainly sd1, which has the disadvantages of single dwarf gene and narrow genetic background, which can easily cause genetic fragility and limitations. In addition, most of the other known dwarf and semi-dwarf genes have negative effects on rice agronomic traits, that is, rice varieties containing these dwarf genes have poor agronomic traits and are difficult to apply in actual production, thus li...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/113C12N15/82A01H5/00
CPCC12N9/14C12N15/113C12N15/8213C12N15/8261C12N2310/10
Inventor 钦鹏胡彬华李仕贵张国华陈微兰涂斌王玉平马炳田
Owner SICHUAN AGRI UNIV
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