EjAG gene of loquat, protein encoded by EjAG gene and application

A loquat and protein technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem that there is no AG homologous gene research report and other problems, reduce the number of petals and the number of rounds, have a good application prospect, and restore stamens and pistils. effect of structure

Active Publication Date: 2019-08-02
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, loquat is an important garden ornamental tree species in my country, and there is no research report on its AG homologous gene.

Method used

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  • EjAG gene of loquat, protein encoded by EjAG gene and application
  • EjAG gene of loquat, protein encoded by EjAG gene and application
  • EjAG gene of loquat, protein encoded by EjAG gene and application

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Experimental program
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Effect test

Embodiment 1

[0023] Cloning of embodiment 1 loquat EjAG gene cDNA sequence

[0024] (1) Extraction of loquat flower bud total RNA

[0025] Collect fresh loquat flower buds at the dew-white stage, put them into cryopreservation tubes, freeze them quickly in liquid nitrogen, and put them in a -80°C ultra-low temperature freezer for later use. Use the RNA extraction kit to extract total RNA: Take 0.2 mg of the material from the -80°C refrigerator, put it into a mortar with 2 mL of lysate and 200 μL of PLANTaid, and grind thoroughly; transfer the grinding liquid into a 2 mL EP tube, and centrifuge at 13,000 rpm After 10 minutes, take 500 μL of the supernatant and transfer it to a new 1.5 mL centrifuge tube; add 250 μL of absolute alcohol to the supernatant, and mix by pipetting; add the above liquid to the adsorption column, and put the adsorption column into the collection tube; Add 500 μL of washing solution to the column, centrifuge at 13,000 rpm for 2 minutes, and discard the waste in the...

Embodiment 2

[0036] The tissue specificity of embodiment 2 loquat EjAG gene expression

[0037] Total RNA was extracted from loquat young leaves, sepals, petals, stamens and pistils, and after removing trace amounts of DNA in the total RNA, it was reverse transcribed into cDNA as a template. According to the full-length cDNA sequence of loquat EjAG gene, oligo 6.0 software was used to design real-time fluorescent quantitative primers qEjAGF:5'-CAATCGTCAAGTGACCTTCTGCA-3' and qEjAGR:5'-TTTCACTATCTGCGCACGCAGTT-3'. Under the premise of PCR-specific amplification, real-time fluorescence quantitative PCR was carried out.

[0038] The loquat actin gene was used as an internal reference gene, and the primers were qEjactinF: 5'-AATGGAACTGGAATGGTCAAGGC-3' and qEjactinR: 5'-TGCCAGATCTTCTCCATGTCATCCCA-3'. Amplification was performed using a three-step method with three biological replicates per reaction. The PCR reaction program was as follows: pre-denaturation at 94°C for 3 minutes; 40 cycles of 94...

Embodiment 3

[0040] The expression vector pBI121-EjAG construction of embodiment 3 loquat EjAG gene

[0041] Use PCR to amplify primers that introduce restriction sites: upstream primer EjAG-F5'-ATCCCGAAAGCTT TCTAGA ATGG-3' (introducing XbaI restriction site), downstream primer EjAG-R: 5'-CAAGCA CCCGGG TTAAACTAGTT-3' (introduction of SmaI restriction site).

[0042] Using the reverse-transcribed cDNA of loquat flower bud total RNA as a template, the high-fidelity enzyme EX-taq was used for PCR reaction. PCR amplification program: 94°C for 5min; 94°C for 40s, 56°C for 40s, 72°C for 40s, for 30 cycles; 72°C for 10min. After the reaction, 1% agarose gel electrophoresis was performed, and the gel was cut and recovered. The recovered PCR product was ligated with the pMD19-T vector, transformed into Escherichia coli competent cells, single clones were picked, and then sent for sequencing. After the sequence analysis of the introduced enzyme cutting site is correct, extract the plasmid, use...

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Abstract

The invention relates to the field of plant molecular biology, in particular to an EjAG gene determined by stamen and pistil characteristics of loquat, protein encoded by the EjAG gene and application. The whole length of the cDNA (complementary deoxyribonucleic acid) sequence of the EjAG gene is shown in SEQ ID No.1; the amino acid sequence of the protein encoded by the EjAG gene is shown in SEQID No.2. The EjAG gene is only expressed in the stamen and pistil of the loquat, but not expressed in the leaves, sepal and petals. An expression carrier pBI121-EjAg is guided into an arabidopsis ag-1mutant by an agrobacterium-mediated bud infection method; when the transgenic arabidopsis ag-1 mutant flowers, the number of petals and the number of rings are reduced, and the stamen and pistil arerestored. The EjAG gene has the advantages that the theoretical foundation is laid for the modification of plant flower organs by the EjAG gene of the loquat, and the technical scheme for the modification of plant flower organs by the EjAG gene of the loquat is provided; the EjAG gene of the loquat is over-expressed in the arabidopsis ag-1 mutant, so that the phenotype of double flower is restoredinto the normal phenotype; the application prospect is good.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and specifically relates to a loquat EjAG gene, its encoded protein and application. Background technique [0002] Loquat (Eriobotrya japonica) is a subtropical evergreen species of Rosaceae Loquat. Its inflorescence is a terminal conical mixed inflorescence consisting of a main axis and 5 to 10 branches. The number of flowers per flower spike varies with the variety and the nutritional status of the flower spike branches, generally 70-100 flowers, and as many as 150-200 flowers. Loquat flower differentiation has unique characteristics: the differentiation time begins in summer and autumn, and the flowering period is longer from differentiation to flowering. The stamens of loquat flower are generally more than 20 pieces, the pistil style is shorter than the stamen filaments, there are 5 styles, and the base is the ovary with 5 ventricles. The formation and development of loquat stamens an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/02
CPCC07K14/415C12N15/827
Inventor 景丹龙梁国鲁陈薇薇郭启高夏燕吴頔王淑明党江波
Owner SOUTHWEST UNIVERSITY
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