Thaumatin-like protein gene PpTLP from pyrus pyrifolia nakai with antifungal activity and application

A sweet protein-like, anti-fungal technology, applied in the fields of molecular biology and genetic engineering, can solve the problems of endangering human and animal health, high chemical pesticide residues, long cycle, etc., achieve broad market application prospects, reduce the use of disease-resistant agents, and breed short cycle effect

Inactive Publication Date: 2010-06-16
KUNMING UNIV OF SCI & TECH
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Problems solved by technology

Traditional disease control mainly relies on chemical pesticides and cultivating resistant varieties. Although some results have been achieved, there are serious drawbacks
Conventional breeding methods are long-term, time-consuming and labor-intensive, high residues of chemical pesticides, easy to pollute the environment, endanger human and animal health and other shortcomings limit the large-scale application of traditional disease control methods

Method used

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  • Thaumatin-like protein gene PpTLP from pyrus pyrifolia nakai with antifungal activity and application
  • Thaumatin-like protein gene PpTLP from pyrus pyrifolia nakai with antifungal activity and application
  • Thaumatin-like protein gene PpTLP from pyrus pyrifolia nakai with antifungal activity and application

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Embodiment

[0023] 1) PpTLP full-length gene cloning and sequence analysis

[0024] Grind the red peel of the torched pear into powder with liquid nitrogen, transfer it to a centrifuge tube, and extract total RNA by the guanidine isothiocyanate method. Use reverse transcriptase M-MLV (Tiangen) to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process are as follows: Take 5 μg Total RNA, add 50ng oligo(dT)15, 2μL dNTP (2.5mM each) in sequence ,ddH 2 O (RNase-free) to a reaction volume of 13.5 μL; after mixing, heat denaturation at 70°C for 5 min, then rapidly cool on ice for 5 min, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200 U), 1 μL M- MLV (200U), 1 μL DTT (0.1M), mix well and centrifuge for a short time, incubate at 42°C for 1.5h, take it out and heat at 95°C for 5min to terminate the reaction. After the first strand of cDNA was synthesized, it was stored at -20°C for future use.

[0025] Using the synthesized first-stra...

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Abstract

The invention relates to a thaumatin-like protein gene PpTLP from pyrus pyrifolia nakai with antifungal activity and application. The gene PpTLP has the base sequence shown in SEQID and coded thaumatin-like proteins. The invention verifies that the gene PpTLP has the function of improving the antifungal ability of the plants through correlation technique of functional genomics. When the antifungal gene PpTLP is constructed on a plant expression vector and is transformed into tobacco to be overexpressed, the transgenic tobacco has high antifungal activity. The protein from the transgenic tobacco expressing PpTLP has obvious inhibitory effects on various fungi such as sclerotinia sclerotiorum, phytophthora parasitica var. nicotianae, phomopsis sp. and the like.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering, in particular to a PpTLP gene PpTLP with antifungal activity and its application. Background technique [0002] In agricultural production, plant diseases are a very difficult problem, especially fungal diseases, which account for more than 80% of the total plant diseases. Among various diseases, fungal diseases have the most symptoms, which can appear in various parts of plants. Traditional disease control mainly relies on chemical pesticides and cultivating resistant varieties. Although some results have been achieved, there are serious drawbacks. The disadvantages of conventional breeding methods such as long cycle, time-consuming and laborious, high residues of chemical pesticides, easy pollution to the environment, and harm to human and animal health limit the large-scale application of traditional disease control methods. [0003] With the establishment and develo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/84A01P3/00
Inventor 刘迪秋李文娴葛锋王光勇王继磊田荣欢方松刚
Owner KUNMING UNIV OF SCI & TECH
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