Method for rapidly screening Cucurbitaceae new RIP gene

A technology of Cucurbitaceae and genes, applied in the field of ribosomal inactivation protein gene screening, can solve the problems of heavy workload and inability to screen new RIP genes, etc., and achieve rapid results

Inactive Publication Date: 2004-03-17
FUJIAN AGRI & FORESTRY UNIV +1
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  • Application Information

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Problems solved by technology

The previous method of the present invention all is to isolate RIP first, and then clones its gene, and this method often has a large workload, and often cannot screen out new RIP genes after a large amount of separation work; therefore, there is no such method so far. An effective and rapid method for screening new RIP genes

Method used

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Embodiment Construction

[0011] Taking the rapid screening of Cucurbitaceae RIP gene as an example, the steps are as follows:

[0012] a. Primer design

[0013] After a systematic comparison of the known RIP amino acid sequences of Cucurbitaceae, a degenerate primer pair LY1 / LY2 was designed according to the highly conserved amino acid sequences of the upstream and downstream and their codon preferences. The sequence is as follows:

[0014] LY1,5'-GT[A / G]AC[G / C / A]AA[C / T]GT[C / T]TA[T / C]ITIATGGG-3'

[0015] LY2,5'-[C / G / T]GCIA[A / T]I[A / T][G / A]IAT[T / C]TG[T / C]TTGGAIAG-3'

[0016] b. Extraction of total DNA

[0017] Reagent preparation:

[0018] Reagent A: CTAB extract containing 2% CTAB, 100mM Tris-HCl pH8.0, 1.4M NaCl, 20mM EDTA pH8.0, 1% β-mercaptoethanol

[0019] Reagent B: phenol: chloroform: isoamyl alcohol = 25:24:1

[0020] Reagent C: Chloroform: Isoamyl Alcohol (24:1)

[0021] Reagent D: the CTAB precipitation solution contains 5g CTAB per L, 0.04M NaCl

[0022] Reagent E: TE buffer containing...

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Abstract

The fast cucurbitaceae RIP gene screening method includes designing degenerate primer pair LY1 / LY2 based on highly conservative amino acid sequence and its codon bias in the upstream and downstream of known cucurbitaceae RIP, Touchdown-PCR amplification with total DNA as template, direct PCR product clonal sequencing of cucurbitaceae plant with un-reported RIP gene to obtain new RIP gene; and fast distinguishing known and unknown RIP genes of cucurbitaceae plant with reported RIP gene according to the length polymorphism of limiting segment in RIP gene LY1 / LY2 segments. Compared with available method of first separating RIP and then cloning gene, the method of the present invention has the features of being fast and accurate, and may be used widely in RIP gene screening.

Description

Technical field [0001] The invention belongs to the field of biopesticide and biomedicine, and relates to a screening method for ribosome-inactivating protein (RIP) gene. Background technique [0002] Ribosome-inactivating protein is favored by medical scientists and pesticide researchers because of its medical value such as anti-tumor and anti-virus, as well as agricultural value such as anti-plant virus and pathogenic fungus. So far, at least 50 gene sequences of type I RIP and 15 type II RIP have been determined. However, people's understanding of RIP is still in its infancy, especially the understanding of its physiological function has not made great progress. RIP is a protein with a variety of enzymatic activities and diverse biological functions. Different RIPs have their own unique functions, and their potential activities are still being discovered [8] . The research on the RIP gene family and its expression regulation in plants is helpful to understand its physi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 林奇英林毅吴祖建谢联辉
Owner FUJIAN AGRI & FORESTRY UNIV
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