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Loquat flower organ development related transcription factor EjPI protein and coding gene and application thereof

A technology of loquat and protein, applied in the fields of application, genetic engineering, plant gene improvement, etc., to achieve good application prospects and improve ornamental effect

Active Publication Date: 2019-10-25
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the regulation of flower organ development by loquat PI homologous genes

Method used

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  • Loquat flower organ development related transcription factor EjPI protein and coding gene and application thereof
  • Loquat flower organ development related transcription factor EjPI protein and coding gene and application thereof
  • Loquat flower organ development related transcription factor EjPI protein and coding gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0024] Cloning of embodiment 1 loquat EjPI gene cDNA sequence

[0025] Extraction of Total RNA from Loquat Flower Buds

[0026] Collect fresh loquat flower buds with a length of about 1.0 cm, quickly put them into cryopreservation tubes, freeze them in liquid nitrogen for 3 hours, and put them in a -80°C ultra-low temperature freezer for later use. Use the RNA extraction kit to extract the total RNA of flower buds: take out the flower bud materials from the -80°C ultra-low temperature refrigerator, put them into a mortar that has been frozen in advance and add 2 mL of RLT lysate and 200 μL of PLANTaid, and grind them thoroughly at room temperature; Transfer the grinding solution to a 2mL eppendorf centrifuge tube, centrifuge at 13000rpm for 15min, absorb 500μL of the supernatant, and transfer it to a new 2mL centrifuge tube; add 250μL of absolute alcohol to the supernatant, mix well by pipetting, and then add it to the adsorption column , and put the adsorption column into th...

Embodiment 2

[0037] Semi-quantitative RT-PCR expression analysis of embodiment 2 loquat EjPI gene

[0038] Total RNA was extracted from loquat sepals, petals, filaments, anthers, and pistils, and after removing trace amounts of DNA in the total RNA, it was reverse-transcribed into cDNA. According to loquat cDNA as a template, oligo 6.0 software was used to design semi-quantitative RT-PCR primers RTEjPIF: 5'-TGCTAAGCATGAGAACCTCAGCAATGA-3' and RTEjPIR: 5'-CAGCTGCCTCTGATGATACCCAT-3'. Use PCR to test its specificity, and semi-quantitative RT-PCR experiments can only be carried out under the premise of ensuring PCR-specific amplification. The loquat actin gene was used as an internal reference gene, and the primers were RTEjactinF: 5'-AATGGAACTGGAATGGTCAAGGC-3' and RTEjactinR: 5'-TGCCAGATCTTCTCCATGTCATCCCA-3', and PCR amplification was performed, with 3 biological replicates for each reaction. The PCR reaction program was: pre-denaturation at 94°C for 5min; 25 cycles at 94°C for 30s, 55°C for ...

Embodiment 3

[0039] The plant transgenic vector pBI121-EjPI construction of embodiment 3 loquat EjPI gene

[0040] PCR amplification was used to introduce restriction sites at both ends of the CDS region of the loquat EjPI gene. Using the reverse transcribed cDNA of loquat flower bud total RNA as a template, TEjPI-F: 5′- TCTAGA ATGGGGAGGGGTAAGATTGAGATCA-3′(introduce XbaⅠ restriction site) and TEjPI-R: 5′- CCCGGG TTAGATTCTCTCCTGGAGATTTGG-3' (introduced SmaI restriction site) was used as primer, and EX-taq enzyme was used for PCR amplification. PCR reaction program: 94°C for 4min; 30 cycles of 94°C for 45s, 57°C for 45s, and 72°C for 45s; 72°C for 10min. After the reaction, the PCR products were subjected to 1% agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit. The recovered PCR product was connected to the pMD19-T vector, and transformed into E. coli competent cells. After single clones were picked, they were sent for sequencing. According to the sequenci...

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Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to a loquat flower organ development related transcription factor gene EjPI and application thereof. The full length of the cDNA sequence of EjPI gene is shown in SEQ ID NO. 1, and the amino acid sequence of the coded protein is shown in SEQ ID NO. 2. The EjPI gene of the invention is expressed only in petals,filaments and anthers, but not in sepals and pistils. The EjPI gene overexpression vector is transformed into wild-type arabidopsis and pi-1 mutant by agrobacterium-mediated inflorescence staining. The results show that overexpression of the EjPI gene in wild-type arabidopsis could make the sepals of arabidopsis valvulae and open; and the overexpression of the EjPI gene in the arabidopsis pi-1 mutant can restore petals and stamens. The transgenic plant material obtained by using the EjPI gene overexpression vector can enable the wild-type angiosperms to achieve sepal petalization and can be used for breeding double petal flowers; and the plant pi mutant restores petals and stamens, and a reproduction process is completed effectively. The gene can be used for plant double flower transformation so as to improve ornamental value and restore stamen organs for cross breeding, and has good application prospect.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and specifically relates to a loquat EjPI protein, its coding gene and application. Background technique [0002] Loquat (Eriobotrya japonica) is an important evergreen fruit tree belonging to Rosaceae Malinae, which is widely distributed. Flowering is one of the most important events in the loquat life cycle. Different from the flower development pattern of most fruit trees, loquat flower organs begin to differentiate and develop in summer, and bloom in autumn and early winter. During the flower development of loquat, its apical meristem continuously grows into a panicle, and then differentiates into floral organs, and then the development of floral organs is regulated by a complex network of environmental and endogenous genes. Therefore, the research on floral organ development of loquat will help to better understand the floral organ development process of Rosaceae. [0003] During th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/02A01H6/20
CPCC07K14/415C12N15/827
Inventor 夏燕梁国鲁陈薇薇石敏景丹龙郭启高吴頔王淑明
Owner SOUTHWEST UNIV
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