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Specific molecular marker of pbelf3b gene in pear and its application

A gene and genome technology, applied in the field of molecular markers of pear flower bud differentiation and development, N-d type and D+d type, can solve problems such as undefined functional domains

Active Publication Date: 2020-08-04
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ELF3 protein is relatively conserved in pear, apple, tomato, soybean, Arabidopsis, rice and maize, but there is no clear functional domain

Method used

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  • Specific molecular marker of pbelf3b gene in pear and its application
  • Specific molecular marker of pbelf3b gene in pear and its application
  • Specific molecular marker of pbelf3b gene in pear and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: InDel sequence identification of sand pear and Xinjiang pear PbELF3b gene intron region

[0056]According to the white pear genome database of the Pear Engineering Technology Center of Nanjing Agricultural University and the resequencing data of different pear varieties, the full-length DNA sequences of the PbELF3b genes of Sand pear and Xinjiang pear were obtained by homologous alignment. Use MUSCLE (https: / / www.ebi.ac.uk / Tools / msa / muscle / ) to compare and analyze the sequences of the two species, sequence information such as SEQ ID NO: 1 and sequence information such as SEQ ID NO: 2 shown. The comparison results showed that there were 3 synonymous mutations in the exons, 2 base mutations and a long InDel sequence in the intron. The specific performance is that there is a 58bp fragment deletion in Xinjiang pear relative to sand pear sequence ( figure 1 ). Therefore, the PbELF3b gene of sand pear was named as N type, and the PbELF3b gene of Xinjiang pear as...

Embodiment 2

[0058] Embodiment 2: Cloning and vector construction of sand pear and Xinjiang pear PbELF3b gene

[0059] The sequence of the vector and the PbELF3b gene was selected according to the requirements of the recombinase to construct the vector, primers were designed with Primer premier5.0, and the full-length PCR was amplified using the DNA corresponding to two representative pear varieties as templates. The detailed steps are as follows:

[0060] Research materials The representative variety of sand pear 'Cuiguan' and the representative variety of Xinjiang pear 'Korla Xiangli' were planted in the Jiangpu Pear Germplasm Resource Center of the School of Horticulture, Nanjing Agricultural University. The trees are about 10 years old. All healthy pear leaves were selected, 500 mg samples were randomly weighed, immediately frozen in liquid nitrogen, DNA samples were extracted using a plant genomic DNA extraction kit (purchased from Chengdu Fuji Biotechnology, China), and the operation...

Embodiment 3

[0063] Example 3: Functional Identification of PbELF3b Gene in Sand Pear and Xinjiang Pear

[0064] The correctly sequenced recombinant plasmids were named pCAMBIA1300-35S:PbELF3b-N-GFP and pCAMBIA1300-35S:PbELF3b-D-GFP, respectively, and referred to as PbELF3b-N and PbELF3b-D. Extract the corresponding recombinant plasmid. The recombinant plasmids PbELF3b-N and PbELF3b-D were introduced into Agrobacterium GV3101 by freeze-thaw method to obtain positive Agrobacterium strains.

[0065] Agrobacterium infects Arabidopsis thaliana using the classic flower dipping method, the specific steps are as follows:

[0066] First, the positive Agrobacterium strains stored in the ultra-low temperature refrigerator (-80°C) were streaked on the plate medium of LB supplemented with kanamycin 100mg / L and rifampicin 50mg / L for activation, and cultured at 28°C Cultivate the box upside down for 1-2 days, pick a single clone in good growth state and shake it in 100ml LB liquid medium supplemented ...

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Abstract

The invention discloses a specific molecular marker of a pear PbELF3b gene and an application of the specific molecular marker. Clonal PbELF3b allele N type and D type and a modified N-d type and D+dtype are separated from pyrus pyrifolia and pyrus sinkiangensis, respectively; the D type misses a d fragment of 58 bp in size relative to the N type, and deletion of the fragment can lead to strongerdelayed flowering activity of the PbELF3b gene; an InDel sequence-specific marker primers SEQ ID NO:4 and SEQ ID NO:5 designed based on intron deletion fragments can be used as molecular markers to identify the differentiation and development situations of pear flower buds, so that the breeding efficiency can be improved; and different types of the PbELF3b can be used for regulation and control of flowering time of other plant species by using a genetic engineering process.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a molecular marker for the differentiation and development of pear flower buds and an application thereof. The molecular marker of the present invention is derived from the PbELF3b gene. The invention discloses PbELF3b allele N type and D type isolated and cloned from sand pear and Xinjiang pear respectively, and transformed N-d type and D+d type. Compared with the N type, the D type lacks a d segment with a size of 58 bp. The deletion of this fragment can lead to stronger delayed flowering activity of PbELF3b gene. Background technique [0002] China is the center of origin of the genus Pyrus and one of the centers of diversity of cultivated pears in the world. Pear trees are widely distributed in China, with large cultivation areas and high yields. In recent years, on the basis of the traditional main varieties, excellent varieties represented ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/20C12Q1/6895C12N15/11
CPCC07K14/415C12N15/827C12Q1/6895C12Q2600/13C12Q2600/156
Inventor 王鹏刘哲谢智华程梦雨吴巨友张绍铃齐开杰
Owner NANJING AGRICULTURAL UNIVERSITY
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