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Multivalent transgenic insect-resistant rice genotype identification primer pair and method

A genotype and gene technology, applied in the field of primer pairs for identifying genotypes of multivalent transgenic insect-resistant rice, can solve the problems of inability to distinguish monovalent and multivalent in insect-resistant phenotype identification, and speed up the breeding process of insect-resistant transgenic rice, etc. Achieve the effect of obvious band size distinction, clear band, and good amplification results

Active Publication Date: 2019-07-05
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the above problems, the object of the present invention is to target the transgenic rice TT51-1 (transfer cry1Ab / cry1Ac fusion gene) that has obtained the transgenic safety certificate, and the donor material T2A-1 (transcry2A transgenic gene) that is widely used in transgenic breeding research. gene) and T1C-19 (transcry1C gene), a series of multiplex primers were designed to detect the genotypes of monovalent, bivalent and trivalent transgenic Bt genes in rice breeding materials, so as to confirm the homozygosity of the Bt gene as soon as possible Or heterozygous state, solve the problem that the identification of insect-resistant phenotypes cannot distinguish between monovalent and multivalent, and can speed up the process of breeding insect-resistant transgenic rice

Method used

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  • Multivalent transgenic insect-resistant rice genotype identification primer pair and method
  • Multivalent transgenic insect-resistant rice genotype identification primer pair and method
  • Multivalent transgenic insect-resistant rice genotype identification primer pair and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] [Example 1] Material preparation

[0055] Transgenic Bt gene homozygous lines TT51(cry1Ab / 1Ac), T2A-1(cry2A), T1C-19(cry1C) were provided by the State Key Laboratory of Crop Genetic Improvement / National Plant Gene Research Center (Wuhan), Huazhong Agricultural University. Non-transgenic rice 9311, R988, and Chaotai B are preserved by the State Key Laboratory of Hybrid Rice (Wuhan University).

[0056] 9311 was crossed with TT51, R988 with T2A-1, Chaotai B with T1C-19, and the F1 obtained was self-crossed to obtain F2 for screening different genotype plants of the monovalent Bt transgene; Hybridization, screening for different genotypes of the bivalent Bt transgene in the F2 offspring of the cross; screening out the bivalent homozygous cry1Ab / 1Ac(+ / +)cry2A(+ / +) genotype rice single plant was crossed with T1C-19 (cry1C), and different genotypes of the trivalent Bt transgene were screened in the hybrid F2 progeny.

Embodiment 2

[0057] [Example 2] Primer Design

[0058] The foreign fragment insertion sequence of TT51 (Genebank: EU880444.1) and the rice genome sequence on both sides of the insertion site, part of the foreign fragment sequence and insertion site of T2A-1 (Genbank: HQ161063.1) were obtained from the NCBI database The rice genome sequence on the left, the partial exogenous fragment sequence of T1C-19 (Genbank: HQ161062.1) and the rice genome sequence on the left of the insertion site. Such as figure 1 As shown, L+R and I+R combinations were designed according to the TT51 insertion sequence and the genome sequences on both sides to detect genomic fragments and insertion events, respectively. According to the T2A-1 and T1C-19 insertion sequence and the left genome sequence, L+I primer combination was designed to detect the insertion event, and R primers were designed by selecting a genome fragment of about 1 kb on the right side of the insertion site, and the genome fragment was detected w...

Embodiment 3

[0062] [Example 3] DNA extraction

[0063] Cut rice leaves about 1-2 cm long and put them in a 2 mL round-bottomed centrifuge tube, and add steel balls and 40 μL 0.25 mol / L NaOH solution, put them in a QIAGEN Tissuelyser sample maker to grind and homogenate, and then add 160 μL 0.25 mol / L NaOH solution. 05 mol / L (pH=8.0) Tris-HCl, vortexed to mix and shake well, centrifuged at 12000r / min, and took the supernatant.

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Abstract

The invention discloses a multivalent transgenic insect-resistant rice genotype identification primer pair and method. A series of multiplex primers are designed aiming at transgenic rice TT51-1 (trans-cry1Ab / cry1Ac fusion gene), T2A-1 (trans-cry2A gene) and T1C-19 (trans-cry1C gene) and used for detecting genotypes of univalent, divalent and trivalent trans-Bt genes in rice breeding material so as to confirm Bt gene homozygous or heterozygous states as soon as possible, and the problem of failure in distinguishing of univalence and multivalence in insect-resistant phenotype identification issolved. According to experiments, a detection method is high in specificity, expensive or high-time-consumption detection means are avoided, and an insect-resistant transgenic rice breeding process can be accelerated.

Description

technical field [0001] The invention belongs to the technical field of agricultural bioengineering, and specifically relates to a primer pair and a method for identifying multivalent transgenic insect-resistant rice genotypes, which are used for identification and assistance of rice seed resources containing cry1Ab / cry1Ac fusion genes, cry2A genes and cry1C genes breeding. Background technique [0002] Rice Lepidoptera pests are one of the important factors affecting the high and stable yield of rice. The rice leaf roller, rice stem borer, and rice stem borer can cause losses of 5% to 10% of the total rice production in my country, and their main damages are white leaves, dead seedlings, and white ears. At present, no rice seed resources resistant to rice Lepidoptera pests have been found in rice. Therefore, in the past, the control of borers was mainly based on the application of pesticides, but there were also problems such as poor control effects, pesticide residue pollu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/16
Inventor 阳菁方欣怡王井章李阳生
Owner WUHAN UNIV
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