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Rice CYP81A6 gene mutant CYP81A6-m2 and application thereof

A CYP81A6 and cyp81a6-m2 technology, applied in the field of genetic engineering, can solve the problems of loss of biochemical functions, inability to detect, difficult molecular marker assisted breeding, etc., and achieve obvious screening effect and huge economic value

Active Publication Date: 2015-09-30
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It caused the deletion of 49 amino acids in the encoded enzyme protein, and mutated the key structural domain at positions 441-450, so that it basically lost its biochemical function (Zhang Jiwen (2010) Research progress on sensitive mutations of rice bentazolone. Chinese rice Science 24:551-558)
However, both of the above two mutants have only 1 bp deletion, which cannot be detected by agarose electrophoresis commonly used in the laboratory, and are difficult to be used in molecular marker-assisted breeding, and special technical methods for detecting single-base mutations are required
In addition, among the two mutants, 8077S from indica rice was not highly sensitive to bendazone

Method used

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  • Rice CYP81A6 gene mutant CYP81A6-m2 and application thereof
  • Rice CYP81A6 gene mutant CYP81A6-m2 and application thereof
  • Rice CYP81A6 gene mutant CYP81A6-m2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Screening of rice mutants sensitive to bentazone

[0039] 1. Cobalt 60 Radiation Mutation Mutant Library and M1, M2 Generation Planting and Character Observation

[0040] In the summer of 2013 in Changsha via cobalt 60 Irradiate 10 kg of 93-11 seeds and plant them in the Lingao field in Hainan in summer. This is the M1 generation. The M1 generation seeds are harvested by individual plants, and a total of about 4557 seeds are harvested. 3617 lines of M1 generation seeds were selected, and 50 individual plants were planted for each line. They were planted in the Lingao field in Hainan in the spring of 2014. This is the M2 generation. After transplanting, carefully observe the field traits at the tillering stage, booting stage, heading stage, flowering stage, and filling stage, and screen various types of mutants such as plant type, panicle type, fertility, and yield. A single plant was harvested and stored as a special mutant; 6 individual plants were harvest...

Embodiment 2

[0043] Example 2 Determination of Rice CYP81A6 Gene Mutant CYP81A6-m2

[0044] After confirming bentazone-sensitive mutants, get 3522m leaves, extract DNA, and design primers covering the full length of the gene (SEQ ID NO.2-3, SEQ ID NO.4-5, SEQ ID NO.6-7 and SEQ ID NO. NO.8-9), perform PCR amplification and sequence the product. DNA extraction is carried out as follows: Take 3522m rice leaves about 2cm long and put them in a 2ml centrifuge tube; add 800μl 1.5×CTAB to the mortar, grind the leaves until homogenized and pour them back into the centrifuge tube; 65℃ water bath for 20-30min , and mix once every 5 minutes; add an equal volume of chloroform / isoamyl alcohol (24:1), mix up and down for 10 minutes; Double the volume of ice-cooled 95% ethanol, place on ice at -20°C for 20 minutes; centrifuge at 12,000 rpm for 15 minutes; discard the supernatant, add 500 μl of 75% ethanol, rinse upside down, and centrifuge at 12,000 rpm for 5 minutes; discard the supernatant, and place ...

Embodiment 3

[0050] Example 3 Screening for detection of CYP81A6-m2 molecular markers

[0051] After the DNA mutation site of 3522m was identified by sequencing, a co-dominant primer marker was designed around this site for molecular marker-assisted selection breeding. The PCR amplification conditions were the same as in Example 2, and the amplified products were detected by agarose electrophoresis and polyacrylamide (PAGE) electrophoresis respectively. The result is as Figure 5 and Figure 6 shown. 2% agarose electrophoresis results showed ( Figure 5 Figure A), the primer pair BM1, BM3, and BM4 amplified a single target fragment with clear bands, which can be used as molecular markers for subsequent molecular marker-assisted selection breeding to distinguish wild-type CYP81A6 and mutant CYP81A6-m2; 6% PAGE electrophoresis results show( Figure 5 Figure B), the primer pair BM1-BM4 can be used as a molecular marker for subsequent assisted selection breeding to distinguish wild-type C...

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Abstract

The invention provides a rice CYP81A6 gene mutant CYP81A6-m2 and application thereof and belongs to the technical field of genetic engineering. Indica variety 93-11 undergoes radiation-induced mutation through cobalt-60, so that 23 base pairs in a second exon of a rice CYP81A6 gene are deleted, the CYP81A6 gene mutant is named as CYP81A6-m2, the nucleotide sequence of the CYP81A6 gene mutant is as shown in SEQ ID No.1, the high sensitivity of CYP81A6-m2 to bentazone herbicide is further verified, and the CYP81A6-m2 can be used for preparing genetically modified rice sensitive to bentazone and have significant effects in the genetic improvement and seed breeding of rice germplasm resources. The invention further provides a method for molecular marker identification of the mutant and application thereof to seed breeding and seed production.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a rice CYP81A6 gene mutant CYP81A6-m2 and its application. Background technique [0002] Rice is the core food crop in my country, and hybrid rice has played an important role in increasing my country's food production. Hybrid rice has obvious heterosis phenomenon. At present, the cumulative planting area of ​​hybrid rice in my country exceeds 4.5 billion mu, and the planting area of ​​hybrid rice has accounted for about 55% of the country's rice planting area. Hybrid rice can be divided into three-line hybrid rice and two-line hybrid rice. The efficiency of hybrid rice seed production in my country is not high, mainly in the aspects of high labor intensity, unstable seed yield and quality. Therefore, realizing the mechanization of the whole process of hybrid rice seed production is the only way to further improve the production of hybrid rice in my country. At p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/82C12N5/10C12Q1/68A01H5/00
Inventor 黄培劲吴永忠安保光欧阳超李新鹏龙湍张维
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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