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Gene OsCKX11 for controlling number of rice grains per spike and application of gene OsCKX11 for controlling number of rice grains per spike

A technology of rice panicle number and rice, which is applied in rice panicle number control gene and its application field, can solve the problem that the function of OsCKX has not been reported, and achieve the effect of improving rice varieties and increasing rice panicle number

Active Publication Date: 2020-09-18
ZHEJIANG NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some technical problems in this field: 1. In addition to the reported mutants of osckx2, osckx4, and osckx9, there is no related report of osckx11 mutant materials; 2. Among the 11 members of the rice cytokinin oxidase family , only OsCKX2 has been reported to be related to the regulation of grain number, the functions of the other 10 members have not been resolved or have nothing to do with the regulation of grain number, most of the functions of OsCKX have not been reported

Method used

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  • Gene OsCKX11 for controlling number of rice grains per spike and application of gene OsCKX11 for controlling number of rice grains per spike
  • Gene OsCKX11 for controlling number of rice grains per spike and application of gene OsCKX11 for controlling number of rice grains per spike
  • Gene OsCKX11 for controlling number of rice grains per spike and application of gene OsCKX11 for controlling number of rice grains per spike

Examples

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Effect test

example 1

[0083] Example 1: OsCKX11 knockout target design and vector construction

[0084] The OsCKX11 gene accession number is: LOC_Os08g35860, and its gene function has not been described yet. Query the deoxynucleotide sequence of the gene through the RiceGenome Browser (http: / / rice.plantbiology.msu.edu) as shown in SEQ ID NO:1 in the sequence list, and the partial deoxynucleotide sequence of the protein encoded by the gene is as shown in the sequence list The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2 in the sequence table. The nucleotide sequence of the OsCKX11 gene is 2949bp, including four exons and three introns, as shown in SEQ ID NO:1 in the sequence table.

[0085] (1) Specific knockout target design

[0086] According to the queried OsCKX11 gene deoxynucleotide sequence, log in to the CRISPR-PLANT website (https: / / www.genome.arizona.edu / crispr / CRISPRsearch.html) to design specific knockout primers. Design a knockout target in the first exon. ...

example 2

[0114] Example 2: Identification of homozygous mutants of osckx11

[0115] (1) DNA extraction of transgenic rice seedlings

[0116] Obtain 24 transgenic T1 seedlings in a transgenic cycle of about three months. After refining the seedlings, extract DNA from rice leaves. The kit used is a plant genomic DNA extraction kit (Shanghai Shenggong Bioengineering Co., Ltd., for specific usage and dosage, refer to the product instructions) .

[0117] (2) Amplification of fragments near the OsCKX11 gene target

[0118] The OsCKX11 DNA fragments near the target site were amplified by PCR technology. The PCR amplification primers SEQ ID NO: 7 are as follows:

[0119] Identify the forward primer: 5’-ATGGCTGTTTTGGAGGTCCG-3’

[0120] Identification of reverse primer: 5’-AGCAGACATGGCACTCGCCG-3’

[0121] The total volume of the PCR reaction is 50μL, containing 5μL of template DNA, 2×KOD Buffer 25μL, dNTP 7μL, ddH 2 O2μL, forward and reverse primers each 5μL, KOD FX enzyme 1μL. All KOD Buffer, dNTP, and ...

example 3

[0129] Example 3: Determination of cytokinin content in homozygous mutants of osckx11

[0130] (1) Cytokinin extraction

[0131] T1 generation osckx11 homozygous mutants were harvested and planted in the field. Field sampling was performed on the T2 generation mutants and wild-type flag leaves at the young leaf stage, including three independent mutant lines and 3 biological lines for each independent line. repeat. The sample was ground with liquid nitrogen, weighed about 100 mg of the ground sample, placed in a 2 mL centrifuge tube (Eppendorf), and recorded the accurate mass. Quickly add 1mL of 80% methanol and the corresponding internal standard ([ 2 H5]tZ,[ 2 H5]tZR,[ 15 N4]cZ,[ 15 N4]cZR,[ 2 H6]iP,[ 2 H6] iPR 45pg each). Rotate and mix at 4℃ for 2h. Centrifuge in a centrifuge at 4°C, 13000g, 10min. Pipette the supernatant to a new 2mL centrifuge tube and blow dry with nitrogen. Add the remaining precipitate to 1mL of 80% methanol solution at 4°C and mix well, and then suck...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and discloses a gene for controlling the number of rice grains per spike and application of the gene for controlling the number of rice grains per spike. A nucleotide sequence of OsCKX11 is SEQ ID NO:1 as shown in the description; a protein-coding region nucleotide sequence is SEQ ID NO:2 as shown in the description; and aprotein-coding amino acid sequence is SEQ ID NO:3 as shown in the description. According to the gene for controlling the number of rice grains per spike and application of the gene for controlling thenumber of rice grains per spike, an OsCKX11CRISPR / Cas9 knockout vector is constructed, multiple independent homozygous lines are identified by means of PCR amplification and sequencing, and a mutantwith the rice OsCKX11 gene specifically knocked out to cause rise of a cytokinin level and increase of the number of grains per spike is provided. On the basis of the biological function of increasingthe number of rice grains per spike through function loss of OsCKX11, by means of gene editing, RNA interference, molecular assisted breeding and the like, an existing rice variety can be improved, the number of rice grains per spike can be increased, and a theoretical basis can be provided for breeding of high-yield rice varieties.

Description

Technical field [0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to a rice grain number control gene per panicle and its application. Background technique [0002] With the increase of the global population, the food crisis facing mankind has become increasingly severe. Rice is one of the three major food crops in the world. Nearly half of the population uses rice as the main food. Its output has always been an important feature in production and breeding. Rice yield is mainly determined by tillers, panicle grains and grain weight, among which panicle grains are a key factor in rice yield. Therefore, research on genes related to rice grain number per ear can provide an important theoretical basis for increasing food production and ensuring national food security. [0003] Cytokinins are a class of N 6 -Small molecular plant hormones composed of adenine derivatives play an important role in plant growth and development, sen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/06C12N15/82A01H5/10A01H6/46
CPCC12N9/0026C12N15/8261C12N15/8216C12Y105/99012C12N15/8213C12N15/8295C12N15/8262
Inventor 张可伟彭凯轩章薇崔付斌赵江哲
Owner ZHEJIANG NORMAL UNIVERSITY
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