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Male Sterility Gene zmphd11 and Its Application in Creating Male Sterile Lines of Maize

A male sterile line and male sterile technology, applied in the field of plant biotechnology breeding, can solve the problems of slow breeding efficiency of new varieties, difficult to guarantee seed quality, single cytoplasm of sterile lines, etc.

Active Publication Date: 2021-06-22
BEIJING SHOU JIA LI HUA SCI TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, due to factors such as the lack of fundamental breakthroughs in the basic research on maize male sterility, it is difficult to protect the intellectual property rights of maize inbred lines. Efficiency is slow
Second, the corn seed production industry is still in a labor-intensive stage relying mainly on manual detasseling, with high costs, huge resource consumption, and difficulty in guaranteeing seed quality
CMS is jointly controlled by mitochondrial genes and nuclear genes. Although it has been used in maize breeding and hybrid production, it has problems such as low resource utilization, single cytoplasm of sterile lines, and susceptibility to diseases.
GMS is controlled solely by nuclear genes, which can overcome CMS defects, but it is difficult to reproduce homozygous sterile lines in large numbers through conventional breeding methods

Method used

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  • Male Sterility Gene zmphd11 and Its Application in Creating Male Sterile Lines of Maize
  • Male Sterility Gene zmphd11 and Its Application in Creating Male Sterile Lines of Maize
  • Male Sterility Gene zmphd11 and Its Application in Creating Male Sterile Lines of Maize

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Maize ZmPHD11 (Zm00001d013416) Gene Sequence and Expression Pattern Analysis

[0034] In the maizeGDB library (https: / / www.maizegdb.org / ), the maize ZmPHD11 (Zm00001d013416, GRMZM2G408897) gene was queried. The nucleic acid sequence of the gene in B73 is shown in SEQ ID NO.1, and the gene function is annotated as PHD11 transcription factor (PHD-transcription factor 11, PHD11), its coded protein comprises 689 amino acids, and its sequence is shown in SEQ ID NO.2.

[0035] PHD transcription factors are involved in the regulation of many physiological processes in plants. Since the actual function of the ZmPHD11 gene in maize has no published research data, in order to study the relationship between this gene and the male reproductive development of maize, the present invention firstly uses qRT-PCR to analyze The expression pattern of this gene in different stages of maize anther development. Specific steps are as follows:

[0036] 1. Sampling of corn anthers a...

Embodiment 2

[0042] Example 2 Function of Maize ZmPHD11 (Zm00001d013416) Gene and Creation of Maize Male Sterile Lines Using CRISPR / Cas9 Method

[0043] In order to clarify the function of maize ZmPHD11 (Zm00001d013416) in maize, the present invention uses the CRISPR / Cas9 gene editing method to mutate the Zm00001d013416 gene sequence and knock out the function of the gene in maize. The present invention selects the corn hybrid Hi II as the recipient material for gene editing. In the present invention, the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 of the conserved region of the gene are respectively selected as the target region for CRISPR / Cas9 gene editing.

[0044] 1. Construction of CRISPR / Cas9 gene editing vector of ZmPHD11

[0045] The gene editing vector of the present invention is pBUE411-MT1T2-Cas9, the base vector of the vector is pBUE411-Cas9, the intermediate vector is pCBCmT1T2, and gRNA is provided. In the present invention, the MT-sgRNA is obtained by designing the targ...

Embodiment 3

[0071] Example 3 Phenotype analysis of phd11 sterile line

[0072] F2 generation seeds were obtained after selfing of the F1 generation plants identified in the above example 2 that do not contain the Cas9 gene, and one of each of the three phd11 mutation types (ZmPHD11-Cas9-1, ZmPHD11-Cas9-2 and ZmPHD11-Cas9-3) was obtained Self-crossed single panicles were sown in rows, and the phenotypes were investigated at the mature stage. Among the three F2 lines, the ratio of fertile plants to sterile plants is consistent with 3:1 segregation, which further indicates that the sterility trait of the phd11 sterile line is controlled by a single recessive gene, and then the stable non-transgenic obtained for the F2 generation Detailed phenotypic comparison of the Phd11 CMS line with wild type.

[0073] 1. Observation of vigor of tassels, anthers and pollen

[0074] In terms of vegetative growth and ear development, the plants of the phd11 sterile lines (ZmPHD11-Cas9-1, ZmPHD11-Cas9-2, a...

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Abstract

The invention discloses male sterility gene ZmPHD11 It has the nucleotide sequence shown in SEQ ID NO.1, and the protein encoded by the gene has the amino acid sequence shown in SEQ ID NO.2. The present invention uses CRISPR / Cas9 gene editing technology to mutate the gene in wild-type maize, which can cause abnormal anther development and pollen abortion, leading to complete male sterility, and found that ZmPHD11 Regulatory functions of genes on male reproductive development in maize. Through offspring screening, a sterile line without transgenic components can be obtained, and a stable maize male sterile line can be created, which is of great significance for maize male fertility control and hybrid seed production. The present invention also aims at obtaining phd11 Functional molecular markers were designed for male sterile mutants, which have important application value in maize male sterile line breeding, sterile hybrid seed production and molecular marker-assisted selection.

Description

technical field [0001] The invention belongs to the field of plant biotechnology breeding, and in particular relates to the male sterile gene ZmPHD11 and its application in creating maize male sterile lines. Background technique [0002] Corn is the largest food crop in my country, with an annual planting area of ​​more than 550 million mu. The healthy development of the corn seed industry has great strategic significance for ensuring national food security. At the same time, the corn seed industry is also the seed industry with the largest market value, the highest degree of commercialization, and the highest technological content in the world, and is a strategic location for global seed industry competition. Compared with the international leading level, my country's corn seed industry still has a huge gap in terms of technological innovation and industrial models. First, due to factors such as the lack of fundamental breakthroughs in the basic research on maize male ster...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/113C12N15/11C12Q1/6895C07K14/415A01H1/02A01H1/04
Inventor 李金萍侯全璨谢科柳双双龙艳魏珣冷燕吴锁伟安学丽万向元
Owner BEIJING SHOU JIA LI HUA SCI TECH CO LTD