Peach auxin primary response factor PpIAA1 gene and application thereof

A technology of response factor and auxin, applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Pending Publication Date: 2019-06-14
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

in the peach fruit PpIAA1 Genetic research has rarely been reported

Method used

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  • Peach auxin primary response factor PpIAA1 gene and application thereof
  • Peach auxin primary response factor PpIAA1 gene and application thereof
  • Peach auxin primary response factor PpIAA1 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Peach PpIAA1 Isolation and identification of genes

[0046] RNA was extracted from peach pulp with a plant polysaccharide polyphenol kit (DP441, Tiangen, Beijing), and single-stranded cDNA was obtained through a reverse transcription kit (Tiangen, Beijing). The single-stranded cDNA was used as a template and the following sequences were used as primers, Obtained by PCR PpIAA1 The full-length sequence of the gene, as shown in SEQ ID NO: 1, the electropherogram of the PCR amplification product is shown in figure 1 . PCR amplification program: pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 59°C for 30s, extension at 72°C for 30s, 30 cycles; PCR amplification system: upstream primer 0.5ul (10uM / ml), downstream primer 0.5ul ( 10uM / ml), DNA 1ul (20ng / ul), PCR Mix 25ul, the most useful ddH 2 O fill up to 50ul. The primer sequences are:

[0047] Forward IAA1f: 5'-ATGAACATGCCACCGGATGCTG-3', as shown in SEQ ID NO:3;

[0048] Revers...

Embodiment 2

[0052] Example 2 Peach PpIAA1 Gene Expression Analysis

[0053] Detection by qRT-PCR PpIAA1 The expression of genes at different stages of CN13 peach ripening. select Actin ( ppa007242m ) as an internal reference gene, with 2 -ΔΔCt Formula to calculate relative gene expression, qRT-PCR amplification program: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30s, annealing at 60°C for 30s, extension at 72°C for 30s, 45 cycles; qRT-PCR amplification system: upstream primer 0.3ul (10uM / ml), downstream primer 0.3ul (10uM / ml), cDNA 1ul (20ng / ul), Mix 7.5ul, the most useful ddH 2 O fill up to 15ul. Quantitative analysis primer sequence is:

[0054] Forward RTIAA1f: 5'-GGCTGTTGGGATAGCTCCAA-3', as shown in SEQ ID NO:9;

[0055] Reverse RTIAA1r: 5'-GCTTGATCAGTACCATTCATTTCATT-3', shown in SEQ ID NO:10.

[0056] The results show, PpIAA1 The gene was expressed in different ripening stages of CN13 peach fruit, and at the same time PpIAA1 The gene increased rapid...

Embodiment 3

[0057] Example 3 PpIAA1 Gene function identification test

[0058] for research PpIAA1 Whether the gene is involved in the auxin signaling pathway during the peach ripening process was analyzed and identified by transgenic tomato.

[0059] 3.1 Screening of transgenic tomato positive strains

[0060]The tomato transformation method was optimized with reference to Sun et al (2006), Bee Lynn Chew and Yu Pan (University of Nottingham). Micro-Tom tomato seeds were surface-sterilized with 75% alcohol, rinsed with sterile water three times, and then disinfected with 10% sodium hypochlorite for 1 h. After taking it out, wash it 6 times in sterile water and dry it on filter paper. Seeds were sown in 50% MS medium containing 0.8% agar at pH 5.9. Plants were kept in 14 h / 10 h light and dark, 25°C, 80% relative humidity, 250 μmol m -2 the s -1 Cultivate in a tissue culture room with high light intensity.

[0061] To construct the overexpression vector PpIAA1-pK2GW7, use PCR to am...

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Abstract

The invention relates to a peach auxin primary response factor gene PpIAA1 and application thereof. The PpIAA1 gene is cloned from peach fruits, and the PpIAA1 gene is further stably transformed intoMicro-Tom tomatoes for functional verification. The results show that compared with wild tomatoes, root branches of PpIAA1 transgenic strain plants are decreased, the leaf color is obviously green, fruits are ripened in advance and the shelf life is shortened. And the mature period of the tomato fruits of the PpIAA1 transgenic strain is advanced by 6-8 days. The expression of the PpIAA1 is significantly inhibited by 1-MCP treatment of 'middle nectarine No. 13'. The expression of the PpIAA1 is significantly promoted by NAA or ethylene treatment of 'middle nectarine No. 16', indicating that thegene plays an important role in the ripening and softening process of peach fruits. The screened PpIAA1 gene can be helpful for researching the response mechanism that auxin and ethylene together promote the ripening of peach fruits in the ripening period of the peach fruits.

Description

technical field [0001] The invention belongs to the field of auxin response factor gene and its application, in particular to peach auxin primary response factor PpIAA1 Genes and their applications. Background technique [0002] Peach[ Prunus persica (L). Batsch] fruit is generally solute type (melting flesh, MF), which belongs to the respiratory climacteric type fruit, which softens rapidly after the ethylene jump and reaches commercial maturity, while the hard type peach fruit ripening stage (stonyhard, SH) does not Ethylene jumps to the peak, and the fruit does not soften. Tatsuki et al. (Tatsuki et al., 2013) compared solute-type and hard-type peach fruits at maturity and found that there were differences not only in the amount of ethylene released, but also in the accumulation of auxin and abscisic acid. The lower level of ethylene synthesis in peach may be due to the lower level of its auxin (IAA). Treatment of hard peach fruits with the growth regulator 1-Naphth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/06A01H5/12A01H5/08A01H6/74A01H6/82
Inventor 曾文芳王志强王小贝牛良潘磊崔国朝王雁邓丽
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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