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Flanking sequences of transgenic insect-resistant herbicide-resistant maize CM8101 exogenous insertion fragment and application thereof

A technology of transgenic corn and exogenous insertion, which is applied in the field of plant biology and can solve unrealistic problems

Inactive Publication Date: 2019-03-29
INST OF CROP SCI CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, with the acceleration of the migration of rural labor to cities, the scale and mechanization of corn planting has become a trend, which makes the traditional manual weeding method unrealistic

Method used

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  • Flanking sequences of transgenic insect-resistant herbicide-resistant maize CM8101 exogenous insertion fragment and application thereof
  • Flanking sequences of transgenic insect-resistant herbicide-resistant maize CM8101 exogenous insertion fragment and application thereof
  • Flanking sequences of transgenic insect-resistant herbicide-resistant maize CM8101 exogenous insertion fragment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Integration of the target gene in the genome of insect-resistant and herbicide-resistant transgenic maize CM8101

[0036] 1. Southern blot hybridization detection technology

[0037] (1) Agarose electrophoresis: Take a certain amount of DNA sample to be tested, and digest 10-20 μg with an appropriate restriction endonuclease; after digestion, perform electrophoresis on agarose gel; after electrophoresis, transfer to membrane.

[0038] (2) Blot transfer: cut the gel and mark it (excision in the lower left corner) for easy positioning, then place the gel in a container, depurinate it with 0.25M HCl for 10 min; soak the gel in an appropriate amount of denaturing solution, Place it at room temperature for 30 minutes to denature it, and shake gently without interruption; cut the filter paper and nylon membrane to the same size as the glue, and immerse the nylon membrane in the transfer buffer for 30 minutes to balance; spread layer by layer, leaving no air bubbles ...

Embodiment 2

[0054] Example 2 The establishment of exogenous insertion sequence analysis and specific PCR detection method

[0055] The flanking genome sequence of the inserted fragment and the sequences inserted into the maize genome near the 5' end and 3' end were obtained by optimized Tail-PCR method, and the transformant-specific PCR detection method was determined according to the flanking sequence.

[0056] 1. Tail-PCR captures flanking sequences

[0057] According to the hiTAIL-PCR method reported by Liu Yaoguang et al., the specific primers for hiTAIL-PCR were designed according to the sequence of the 5' end and 3' end of the vector. The nucleotide sequence and the length of the amplified fragment are shown in Table 3. See Table 4 for arbitrary degenerate primers AD1-n (n is 1 to 4), AC-1 and AD6 used in hiTAIL-PCR.

[0058] Table 3 specific primers

[0059]

[0060] Table 4 Arbitrary degenerate primers AD1-n, AC1 and AD6

[0061]

[0062] The PCR reaction system of each s...

Embodiment 3

[0085] Example 3 Quantitative detection of target protein in different tissues in different periods

[0086] 1. ELISA quantitative detection technology

[0087] Each material was sampled in different periods and different tissues, and 3 strains were taken from each tissue. Take 0.1g and simply cut into 4-8 segments. Put it into liquid nitrogen for quick freezing, and put each material into a sealed bag, and put them collectively into -80 for storage. The Cry1Ab / Ac kit produced by Agdia Company in the United States was used. The analysis method was operated according to the specific kit instructions. The analysis results were based on the expressed gene protein as a percentage of the sample tissue weight ng protein / g weight and the soluble protein percentage as a mg protein / g weight per gram. Heavy representation. Different tissue samples were taken from different stages of field growth, and 3 plants were taken from each tissue for detection.

[0088] Weigh 0.1 g of the tis...

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Abstract

The invention belongs to the plant biotechnical field, and in particular, relates to flanking sequences of a transgenic insect-resistant herbicide-resistant maize CM8101 exogenous insertion fragment and an application thereof. The transgenic maize CM8101 provided by the invention is identified to have an insertion site by Southern blot. The flanking sequences of 5' end and 3' end of the exogenousinsertion fragment of the insertion site are shown in SEQ ID No.1 and SEQ ID No.2. The invention provides primer pairs for detecting the flanking sequences, wherein the primer pairs have nucleotide sequences shown in SEQ ID No.3-4 and SEQ ID No.5-6 respectively. The identification of the flanking sequences of the transgenic insect-resistant and herbicide-resistant maize CM8101 exogenous insertionfragment is suitable for the detection of the transgenic maize CM8101 including parents, hybrid F1 and offspring, and plants, tissues, seeds and products.

Description

technical field [0001] The present invention relates to the field of plant biotechnology, in particular, to a transgenic insect-resistant and herbicide-resistant maize CM8101 exogenous insertion fragment side sequence and a specific primer pair for detecting the transgenic maize, and the present invention also relates to the use of the primer pair A method and a kit for detecting transgenic maize CM8101 parents or hybrid offspring and products thereof. Background technique [0002] Corn is an important grain, feed and industrial raw material crop, and plays an important role in national food security and national economic development. In recent years, my country's corn planting area has remained at more than 500 million mu, mainly distributed in more than 20 provinces, municipalities and autonomous regions across the country. Due to changes in farming system and natural climate, the Asian corn borer has seriously affected the yield, total yield and quality of corn, and has ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6895
CPCC12Q1/6895C12Q2600/13
Inventor 李新海翁建峰雷开荣杨小艳谢树章张德贵鲁鑫张爽李明顺郝转芳雍洪军韩洁楠
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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