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EjAGL17 protein for regulating and controlling eriobotrya japonica flowering time as well as coding gene and application of gene

A technology of loquat and protein, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problem that the flowering time has not been reported, and achieve the effect of promoting early flowering, good application prospects, and promoting early fruiting

Inactive Publication Date: 2020-01-10
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the loquat AGAMOUS-Like 17 (EjAGL17) gene regulating its flower development and flowering time.

Method used

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  • EjAGL17 protein for regulating and controlling eriobotrya japonica flowering time as well as coding gene and application of gene
  • EjAGL17 protein for regulating and controlling eriobotrya japonica flowering time as well as coding gene and application of gene
  • EjAGL17 protein for regulating and controlling eriobotrya japonica flowering time as well as coding gene and application of gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Cloning of embodiment 1 loquat EjAGL17 gene cDNA sequence

[0023] Extraction of Total RNA from Loquat Flower Buds

[0024] The flower buds of the loquat differentiation stage with a fresh length of about 1.0 cm were collected, quickly put into cryopreservation tubes, put into liquid nitrogen for quick freezing for 2 hours, and then put into -80°C ultra-low temperature freezer for later use. Use the RNA extraction kit to extract the total RNA of loquat flower buds: take out the collected flower buds from the -80°C ultra-low temperature freezer, put them into a pre-frozen mortar with 1 mL RLT lysate and 100 μL PLAN Taid, and grind them thoroughly at room temperature ;Transfer the above grinding solution to a 1.5mL eppendorf centrifuge tube, centrifuge at 13000rpm for 10min, absorb 500μL supernatant, and transfer to a new 1.5mL centrifuge tube; add 250μL absolute alcohol to the supernatant, and mix well by pipetting , put it into the adsorption column, then put the adsor...

Embodiment 2

[0035] Example 2 Analysis of subcellular localization of loquat EjAGL17 gene

[0036] The ORF sequence of the EjAGL17 gene was analyzed using the software Oligo7, and primers for the restriction sites at both ends were designed. EjAGL17-SacI: 5'-cgagctcATGGGGAGAGGAAAGATTGTGAT-3'; The pMD18-EjAGL17 plasmid with correct sequencing was used as a template to amplify, and the ORF sequence of the EjAGL17 gene containing SacI and BamHI restriction sites was obtained. The target gene and the transformed vector pCAMBIA1300 plasmid were respectively extracted, and double-digested with restriction endonucleases SacI and BamHI respectively, and recovered after agarose gel electrophoresis. Use T 4 DNA ligase connects the target gene EjAGL17 after double digestion with the modified pCAMBIA1300 vector, and transfers the recombinant vector into Escherichia coli competent cells, and then performs bacterial liquid PCR and double digestion verification before sequencing to ensure that the targ...

Embodiment 3

[0039] Example 3 Real-time fluorescent quantitative PCR expression analysis of loquat EjAGL17 gene

[0040] The flowering process of loquat includes: (A) Physiological differentiation period of flower buds, (B) Morphological differentiation period of flower buds, (C) Main axis differentiation period of inflorescence, (D) Branch axis differentiation stage of inflorescence, (E) Rapid growth of lateral branches of inflorescence Elongation stage, (F) floret differentiation stage, (G) flower bud dew stage, (H) full flower stage and (I) flower fade stage. The total RNA of the nine developmental stages of loquat was extracted respectively, and after removing the trace amount of DNA in the total RNA, it was reverse transcribed into cDNA. Based on loquat cDNA as a template, oligo 6.0 software was used to design real-time fluorescent quantitative PCR primers qRTEjAGL17F:5'-TTGACGAGCAGGCAAGTGAC-3' and qRTEjAGL17R:5'-TAGCATAATCGTAAAGCCTG-3'. The loquat actin gene was used as an internal ...

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Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to an EjAGL17 gene related to eriobotrya japonica flowering time regulation and control and an application of the EjAGL17 gene. The full length of the sequence of a coding region of cDNA of the EjAGL17 gene is shown as SEQ ID No.1, and the amino acid sequence of the protein coded by the EjAGL17 gene is shown asSEQ ID No.2. The EjAGL17 gene disclosed by the invention is transiently expressed in tobacco leaf cells and is positioned at a cell nucleus, so that the protein coded by the gene has typical transcription factor characteristics; meanwhile, the expression of the gene has significant difference in different stages of eriobotrya japonica flower development; the EjAGL17 gene overexpression vector istransferred into wild type arabidopsis thaliana through an agrobacterium mediated inflorescence dip dyeing method, and results show that overexpression of the EjAGL17 gene in the wild type arabidopsisthaliana can promote flowering time of the arabidopsis thaliana by about 14 days; and a transgenic plant material obtained by using the EjAGL17 gene overexpression vector can obviously advance the flowering phase of a plant and further promote the fruiting time to be advanced, can be used for directional breeding of early blossoming and early maturing varieties of the plant, and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and specifically relates to a loquat EjAGL17 protein, its coding gene and application. Background technique [0002] Loquat (Eriobotrya japonica) is a plant of the genus Loquat in the Rosaceae family. It is an important evergreen fruit tree in southern my country; its fruit matures in the off-season of fruit in late spring and early summer, and the flesh is fresh and juicy. to consumers. Significantly different from most other plants in the Rosaceae family, loquat flower buds begin to differentiate in summer and usually bloom in late autumn to early winter. Its flower development goes through the flower bud morphological differentiation stage, inflorescence spindle and branch axis differentiation stage, and lateral branch axis rapid elongation stage to form a panicle; then floret differentiation stage and flowering stage appear on the branch axis. Therefore, the research on the molecular r...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/02A01H6/20
CPCC07K14/415C12N15/827
Inventor 景丹龙梁国鲁夏燕刘新亚占峰薛宝贵郭启高王淑明
Owner SOUTHWEST UNIVERSITY
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