EjFRI gene for delaying loquat flowering time and encoded protein and application thereof

A gene and loquat technology, which is applied to the loquat EjFRI protein and its encoding gene and application fields, can solve the problems of ungene sequence accuracy verification, functional research and reporting, etc., and achieves delayed flowering time, delayed result time, and good application prospects. Effect

Inactive Publication Date: 2020-04-28
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, other angiosperms have only predicted FRI homologous genes, and have not verified the accuracy of these gene sequences, and have not yet studied and reported their functions

Method used

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  • EjFRI gene for delaying loquat flowering time and encoded protein and application thereof
  • EjFRI gene for delaying loquat flowering time and encoded protein and application thereof
  • EjFRI gene for delaying loquat flowering time and encoded protein and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0023] Extraction of Total RNA from Loquat Flower Buds

[0024] Fresh flower buds with a length of about 0.5 cm in the differentiation stage of loquat were collected, quickly sampled, 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 use. Extract total RNA from loquat flower buds using an RNA extraction kit: take out the collected flower buds from the -80°C ultra-low temperature freezer, put them into a pre-frozen mortar with 1 mL of RLT lysate and 100 μL of PLANTaid, at room temperature, Grind thoroughly; 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, pipette to mix After uniformity, add to the adsorption column, and then put the adsorpti...

Embodiment 2

[0035] Example 2 The subcellular localization analysis of loquat EjFRI gene

[0036] Use the software Oligo7 to analyze the restriction site of the ORF sequence of the EjFRI gene, and design the restriction site primers at both ends, LEjFRI-BamHI: 5'- GGATCC ATGGGAAAGAAGAAGAAGAGAGT-3'; LEjFR-SalI: 5'- GTC GAC AAATGCCATTCGACCAGCAAGC-3'. Using the correctly sequenced pMD18-EjFRI plasmid as a template for amplification, the ORF sequence of the EjFRI gene containing BamHI and SalI restriction sites was obtained. The target gene and the transformed vector pCAMBIA1300 plasmid were respectively extracted, and double-digested with restriction endonucleases BamHI and SalI, and recovered after agarose gel electrophoresis. Use T 4 DNA ligase connects the double-digested target gene EjFRI and 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 sequ...

Embodiment 3

[0039] Real-time fluorescent quantitative PCR analysis of embodiment 3 loquat EjFRI gene

[0040] The total RNA of loquat stems, leaves, leaf buds, flower buds and flowers were extracted respectively, and after the trace amount of DNA in the total RNA was removed, it was reverse transcribed into cDNA. Based on loquat cDNA as a template, oligo 7.0 software was used to design real-time fluorescent quantitative PCR primers qEjFRIF:5'-GCAGCACCAGAAAGCCAAGCA-3' and qEjFRIR:5'-GTTGACTCCCTGCCATCCTTCG-3'. The loquat actin gene was used as an internal reference gene, and the primers were qRTEjactinF: 5'-AATGGAACTGGAATGGTCAAGGC-3' and qRTEjactinR: 5'-TGCCAGATCTTCTCCATGTCATCCCA-3', and its specificity was detected by PCR. Real-time fluorescence quantitative PCR experiments can be carried out, and each reaction is set up with 3 biological replicates. The PCR reaction program is: 94°C pre-denaturation for 5min; 94°C for 20s, 56°C for 20s, 72°C for 20s, 41 cycles, and then, collect the melt...

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Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to an EjFRI gene for delaying loquat flowering time and application of the EjFRI gene. The full length of a sequence of a coding region of EjFRI gene cDNA is shown as SEQ ID No.1, and the amino acid sequence of the protein coded by the EjFRI gene cDNA is shown as SEQ ID No.2. The EjFRI gene disclosed by the invention is transiently expressed in tobacco leaves and is positioned in a cell nucleus, which indicates that the protein encoded by the gene belongs to a typical transcription factor. The expression quantity of the gene in loquat leaf buds is the highest, and the expression quantity of the gene in flower buds is low. An EjFRI gene overexpression vector is transferred into wild-type arabidopsis thaliana for overexpression through an inflorescence dip dyeing method. Results show that the flowering time of the arabidopsis thaliana can be significantly delayed by overexpression of the EjFRI gene inthe wild-type arabidopsis thaliana. A transgenic plant material obtained by using the EjFRI gene overexpression vector can obviously delay the flowering time of the plant and further delay the fruiting time of the plant, can be used for directional breeding of late flowering and late 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 EjFRI protein, its coding gene and application. Background technique [0002] Loquat is an important evergreen fruit tree in subtropical and tropical regions, and plays an important role in my country's economic production. In loquat production, the shelf life of its fruit is short, which has become an important factor restricting the development of loquat industry. Currently, loquat fruits are available on the market in November in Panzhihua, Sichuan, around March in Guangzhou, and in April and May in Chongqing. The current survey shows that the flowering period of different loquats is variable and longer, which provides a basis for the breeding of loquat fruit varieties at different maturity stages. Therefore, it is of great significance to carry out loquat flowering regulation research, and then guide the production practice of late-maturing loquat ...

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 景丹龙陈薇薇王鹏梁国鲁夏燕郭启高何桥党江波
Owner SOUTHWEST UNIV
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