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Gene for controlling female properties of corn, and kit, mutant genotype and method for creating female sterile line of corn

A technology for mutating genes and maize, applied in the field of molecular genetics, can solve the problems of few female sterile lines, unclear expression and regulation mechanism, and restricting the efficiency and application scope of maize hybrid seed production.

Active Publication Date: 2020-03-24
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the genes controlling female development in maize and the gene expression and regulation mechanism of female sterility are still unclear, and there are few related female sterile lines, which seriously restrict the efficiency and application range of maize hybrid seed production

Method used

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  • Gene for controlling female properties of corn, and kit, mutant genotype and method for creating female sterile line of corn
  • Gene for controlling female properties of corn, and kit, mutant genotype and method for creating female sterile line of corn
  • Gene for controlling female properties of corn, and kit, mutant genotype and method for creating female sterile line of corn

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Maize Zm00001d000204 gene sequence and gRNA sequence

[0059] In the maizeGDB database (https: / / www.maizegdb.org / ), the maize Zm00001d000204 (GRMZM2G444801) gene was found, the CDS nucleic acid sequence in B73 is shown in SEQ ID NO.3, and the gene function is annotated as a sulfate transporter (sulfate transporter, sfp5), its encoded protein contains 681 amino acids, and its sequence is shown in SEQ ID NO.1.

[0060]After further querying the sequence information of the gene in other maize materials (DK105, Mo17, EP1) in the database and the self-selected maize material KN5585, it was found that the amino acid sequence encoded by the gene was very similar to B73 in these 4 maize materials. High, both encode 681 amino acids. Among them, the protein sequences in Mo17 and DK105 are the same as B73, and the sequences in KN5585 and EP1 have a P31A variation compared with B73, and the sequences are shown in SEQ ID NO.2.

[0061] The CDS nucleic acid sequence of th...

Embodiment 2

[0064] Example 2 The function of the maize Zm00001d000204 gene and the creation of maize female sterile lines using CRISPR / Cas9 method

[0065] 1. Construction of CRISPR / Cas9 gene editing vector.

[0066] The gene editing vector of the present invention is GX-CPB-ZmUbi-hspCas9. The basic vector of this vector is CPB-ZmUbi-hspCas9. In the present invention, U6-sgRNA is obtained by Overlap PCR and then cloned into the basic vector by homologous recombination. The specific construction process is as follows:

[0067] (1) Cloning of U6 promoter. The U6 promoter was cloned from B73, and the sequence information of the U6 promoter is shown in SEQ ID NO.16.

[0068] (2) Design of target gRNA. Enter the genome sequence of the recipient material KN5585 into http: / / cbi.hzau.edu.cn / crispr / for target design. The DNA sequences of the two preferred target regions of the present invention are shown in SEQ ID NO.7 or SEQ ID NO.8. The sgRNA backbone sequence of the present invention is ...

Embodiment 3

[0095] Example 3 Sequences of Maize Zm00001d000204 Gene in Different Maize Inbred Lines and Creation of Maize Female Sterile Lines under Different Genetic Backgrounds

[0096] 1. Introduce the mutant genotype into other maize materials by backcrossing.

[0097] The mutants obtained in the present invention and the functional markers (SEQ ID NO.3-SEQ ID NO.8) of the above-mentioned mutant genes can be used in various molecular marker-assisted selection methods, and the mutant genotypes are bred to In other maize genetic backgrounds, the approximate process is:

[0098] Hybridization: use the mutant as the male parent, and cross the recipient corn material as the female parent to obtain F1 seeds; obtain F1 after the first round of backcrossing, obtain F1 plants after sowing, and cross the F1 plants with recurrent parents to obtain BC1 seeds; BC1 Sterility gene selection (foreground selection): sow BC1 seeds, obtain no less than 500 seedlings, collect each individual plant leaf ...

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Abstract

The invention discloses a gene for controlling female properties of corn, and a kit, a mutant genotype and a method for creating a female sterile line of corn. The invention provides a Zm00001d000204gene nucleic acid and amino acid sequence, and CRISPR-Cas9 method for editing the Zm00001d000204 gene sequence. The invention discloses the method for creating the female sterile line of corn, and discloses an edited corn genotype sequence with female sterility properties. According to the invention, the corn sulfate transport protein coding gene Zm00001d000204 is mutated by using the CRISPR / Cas9method, and regulation and control functions of the Zm00001d000204 gene on the female properties of the corn are found. By utilizing the CRISPR / Cas9 gene editing method, the corn female sterile line can be created, so that the method is used for corn hybrid seed production, and the seed production efficiency is improved. The invention also provides Zm00001d000204 mutant genotypes with female sterility properties, the sequences of the mutant genotype can cause female sterility of corn, and can be used for creating the new female sterile line.

Description

technical field [0001] The invention belongs to the field of molecular genetics, and specifically relates to a gene controlling maize female traits, a method for creating a maize female sterile line, a kit, and a mutant genotype. The invention provides a method for editing the Zm00001d000204 gene sequence by using CRISPR-Cas9 to create a maize female sterile line, and discloses a genotype sequence having maize female sterility traits after editing. Background technique [0002] Fertility is a key agronomic trait of crops, and its mechanism and genetic basis are research hotspots in developmental biology. Infertility can be divided into male infertility and female infertility. The study of female sterility lags far behind that of male sterility because of the difficulty in identification of traits, difficulty in obtaining mutants, and complex internal mechanisms. [0003] Female sterility has great application value in genetic breeding and hybrid seed production. In recent...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/113C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/113C12N15/8218C12N15/829C12N2310/20
Inventor 简六梅刘相国许洁婷严建兵
Owner HUAZHONG AGRI UNIV
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