Expression cassette and vector for recovering male fertility of rice OsCYP704B2 mutant as well as detection method and application thereof

An oscyp704b2 and expression cassette technology, applied in the field of plant molecular biology, can solve the problems of poor quality, difficult to identify genotypes of mutation sites, disease susceptibility, etc.

Inactive Publication Date: 2020-08-21
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The three-line breeding method has problems such as disease susceptibility, poor quality, and low breeding efficiency, and due to the constraints of the restoration relationship, 95% of rice resources cannot be used for hybrid breeding
Finally, the larger the transformed fragment, the lower the transformation efficiency
Therefore, Li et al. used the rice original genome sequence up to 3.8kb (excluding the flanking vector sequence) as a restoration gene fragment, which has a higher possibility of genetic

Method used

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  • Expression cassette and vector for recovering male fertility of rice OsCYP704B2 mutant as well as detection method and application thereof
  • Expression cassette and vector for recovering male fertility of rice OsCYP704B2 mutant as well as detection method and application thereof
  • Expression cassette and vector for recovering male fertility of rice OsCYP704B2 mutant as well as detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Acquisition of pOsMs26 promoter

[0078] Through the analysis of the Nipponbare and 93-11 genome sequence, it was found that there was a 1112 bp DNA sequence between the start codon of the OsCYP704B2 (ie OsMs26) gene and the stop codon of the upstream predicted gene. According to the analysis of the promoter element prediction website New PLACE (https: / / www.dna.affrc.go.jp / PLACE / ?action=newplace), this region has a high density of anther-specific promoter element POLLEN1LELAT52 sequence AGAAA. DNA fragments of different lengths and containing different numbers of POLLEN1LELAT52 elements were intercepted for promoter activity identification. The results showed that although some of these intercepted fragments contained the sequence of the anther-specific promoter element POLLEN1LELAT52, they did not have a promoter Activity or promoter activity is low. After layer-by-layer screening and comparison of promoter activities, it is finally determined that the -712~-...

Embodiment 2

[0079] Example 2 Construction of expression cassette and vector containing pOsMs26 promoter and OsCYP704B2 gene

[0080] According to the screening results in Example 1, the expression of the OsCYP704B2 complementary gene was driven by the pOsMs26 promoter whose sequence is shown in SEQ ID NO.1, and the ability of the promoter to drive the complementary gene to restore anther abortion was tested.

[0081] According to the OsCYP704B2 (OsMs26) gene coding region and its upstream and downstream sequences of the indica rice variety 93-11 disclosed on the Gramene website, the sequence of the expression cassette containing the pOsMs26 promoter and OsCYP704B2 gene shown in SEQ ID NO.1 is designed, and the Its sequence is optimized, and by adding or eliminating the recognition sites of certain endonucleases, it is convenient for subsequent operations such as digestion and transfer of expression cassettes, as follows:

[0082] (1) Connect the pOsMs26 promoter whose sequence is shown in...

Embodiment 3

[0091] Example 3 cyp704b2-3 was backcrossed to Zhonghua 11 rice plants

[0092] In previous studies, the inventor obtained two OsCYP704B2 mutants of indica rice 93-11 background, namely cyp704b2-2 and cyp704b2-3 (which were disclosed in Chinese patents CN104894144A and CN105002191A respectively), and both mutants showed It is male sterile without pollen. This example adopts the method of backcross breeding of mutant genes described in Example 9 of patent CN105002191A, using the cyp704b2-3 mutant as the mutant gene donor and Zhonghua 11 as the parent of the backcross recipient, through two generations of backcross, from One cyp704b2-3-wild type heterozygous strain whose Zhonghua 11 background was more than 92% was selected from 55 BC2 individuals, and its self-seeds were used to identify homozygous mutants and mutant-wild type heterozygotes.

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Abstract

The invention relates to the field of plant molecular biology, in particular to an expression cassette and a vector for recovering male fertility of rice OsCYP704B2 mutant as well as a detection method and application of the expression cassette and the vector. The invention provides a plant anther promoter as shown in SEQ ID NO.1 and the rice OsCYP704B2 gene expression cassette which is driven bythe promoter and is subjected to sequence optimization. The anther promoter provided by the invention can drive genes to be efficiently expressed in plant anther; according to the rice OsCYP704B2 geneexpression cassette provided by the invention, high-level expression of the OsCYP704B2 gene in anther under the driving of the anther promoter can be realized; the rice male sterility caused by OsCYP704B2 function deletion is completely recovered, and the rice male sterility promoter has high genetic stability and can be used for recovering rice male fertility or preparing a rice male genic malesterility maintainer line and a sterile line.

Description

technical field [0001] The invention relates to the field of plant molecular biology, in particular to an expression cassette for restoring the male fertility of rice OsCYP704B2 mutants, a detection method and application thereof. Background technique [0002] Rice is an important food crop, and hybrid rice has played an important role in increasing food production. Hybrid rice is the selection of two rice parents with different genetic backgrounds and complementary traits to produce the first-generation hybrids with heterosis for rice production. Hybrid rice has obvious heterosis phenomenon, which can increase yield by 30% compared with conventional rice. Hybrid rice can be divided into three-line hybrid rice and two-line hybrid rice. Three-line hybrid rice (three-line hybrid rice) means that the production of this hybrid rice requires three rice lines: rice cytoplasmic male sterile line, rice cytoplasmic male sterile line and rice cytoplasmic male sterile restoration Ti...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/46
CPCC12N15/8233C12N15/8287C12N2830/008
Inventor 李新鹏陈磊李燕群陈江淑陈思兰安保光龙湍吴永忠黄培劲
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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