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Tea tree polygalacturonase inhibitor protein gene CamPGIP and application thereof

A polygalactose and inhibitory protein technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of reducing the quality of tea products, affecting photosynthesis, and declining.

Active Publication Date: 2014-01-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Leaf diseases of tea trees seriously affect photosynthesis and cause tea yield and quality decline

Method used

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  • Tea tree polygalacturonase inhibitor protein gene CamPGIP and application thereof
  • Tea tree polygalacturonase inhibitor protein gene CamPGIP and application thereof
  • Tea tree polygalacturonase inhibitor protein gene CamPGIP and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Isolation and cloning of tea tree polygalacturonase inhibitory protein gene CamPGIP

[0034] 1) Isolation of tea tree polygalacturonase inhibitory protein gene CamPGIP by suppression subtractive hybridization (SSH)

[0035] The pathogen-infected leaves of the Fuding Dabai tea tree variety in the tea germplasm resource nursery and the healthy leaves of the same variety with the same maturity were collected, and after being fully ground with liquid nitrogen, the total RNA of the leaves of the two groups of tea trees was extracted by the TRIzol method (Invitrogen Life Technologies) , respectively take the above two groups of total RNA with OligotexTM-dT30 mRNA Purification Kit (Bao Biological [Dalian] Co., Ltd.) to purify mRNA, using PCR-Slelect TM cDNA Subtraction Kit (Clontech Laboratories, Lnc., Mountain View, USA) was used for suppression subtractive hybridization. Take 2 μg of purified mRNA from the above two groups, and use SMARTScribe reverse transcri...

Embodiment 2

[0042] Example 2: Correlation Analysis of Relative Expression Intensity of Polygalacturonase Inhibitor Protein Gene CamPGIP Gene in Tea Tree Leaf and Disease

[0043] 1) CamPGIP gene expression in tea tree diseased leaves and healthy control leaves:

[0044] According to the tea plant polygalacturonase inhibitory protein gene CamPGIP sequence obtained above, use Primer Premier5 software (Premier Inc.) to design expression primers (sequences shown in SEQ ID No: 15 and SEQ ID No: 16), design The product size is 229bp. Simultaneously design reference gene tea tree actin (Actin) gene (http: / / www.ncbi.nlm.nih.gov / nuccore / FJ355923) expression primers (sequences such as SEQ ID No: 17 and SEQ ID No: 18), The designed product size is 419bp.

[0045] "Anthracnose" diseased leaves of "Longjing 43" and "Shuigu" varieties in the tea germplasm resource nursery and healthy control leaves of the same maturity, and "cloud leaf blight" of "Jiukeng" and "Zhenong 25" varieties "Disease...

Embodiment 3

[0053] Example 3: Acquisition and identification of tea tree polygalacturonase inhibitory protein gene CamPGIP tobacco

[0054] 1) RT-PCR amplifies the CamPGIP gene and the amplified fragment is connected to the T-vector

[0055] Primers were designed according to the sequence of the open reading frame (from the 31st to 1023rd base at the 5' end) of the gene shown in SEQ ID No: 1, and Nde I and Sac1 restriction sites were introduced into the primers. The upstream and downstream primers were as follows: Shown in SEQ ID No:19 and SEQ ID No:20, wherein AA is a protective base, the underlined part in SEQ ID No:19 is the Nde I restriction site introduced, and the underlined part in SEQ ID No:20 is The introduced Sac1 restriction site. Total RNA was extracted from shoots of Fuding Dabai tea tree variety with TRIzol reagent, and the first strand of cDNA was synthesized using TaKaRa PrimeScript II1 st Strand cDNA Synthesis Kit (Bao Biological [Dalian] Co., Ltd.). The PCR re...

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Abstract

The invention belongs to the field of tea tree gene engineering, and specifically relates to a tea tree polygalacturonase inhibitor protein gene CamPGIP and an encoded protein thereof, and also relates to application of controlling tea tree diseases by inducing and regulating the expression intensity of polygalacturonase inhibitor protein gene CamPGIP. Specifically, the invention discloses a tea tree polygalacturonase inhibitor protein gene CamPGIP, which is a nucleotide sequence shown as SEQ ID No:1. A protein encoded by the gene CamPGIP is a nucleotide sequence shown as SEQ ID No:2. The gene CamPGIP is used to construct transgenic plants and helps improve the resistance to diseases (particularly referring to anthracnose or marble leaf blight) of the transgenic plants shown.

Description

technical field [0001] The invention belongs to the field of tea tree genetic engineering, and in particular relates to a tea tree leaf polygalacturonase inhibitory protein gene CamPGIP and its encoded protein, and also relates to the method of inducing and regulating the expression intensity of polygalacturonase inhibitory protein gene CamPGIP in Application in tea plant disease defense. Background technique [0002] Leaf diseases of tea trees seriously affect photosynthesis and result in reduced yield and quality of tea leaves. Fungi are the main pathogens of tea leaf diseases. Fungal diseases go through the invasion period, latent period, and onset period from the invasion of the fungus until the disease symptoms appear. The invasion period is the stage from the germs contacting the leaves of the tea tree to the establishment of a nutritional or parasitic relationship between the two; the process from the establishment of the parasitic relationship to the appearance of o...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415A01H5/00
Inventor 陆建良刘阳范方媛甘泉郑新强梁月荣
Owner ZHEJIANG UNIV
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