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Maize transformation event and specificity identification method and application thereof

A technology for transforming events and identification methods, which is applied in the cultivation of transgenic crop varieties and the field of plant molecular biology, and can solve problems affecting the expression of endogenous genes

Pending Publication Date: 2016-11-30
HANGZHOU RUIFENG BIOTECH LIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, the insertion of exogenous genes may also affect the expression of endogenous genes

Method used

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  • Maize transformation event and specificity identification method and application thereof
  • Maize transformation event and specificity identification method and application thereof
  • Maize transformation event and specificity identification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Obtaining conversion events

[0064] (1) Acquisition of plasmid vectors containing foreign genes

[0065] The carrier map that the present invention is used for corn transformation is asfigure 1 As shown, the nucleotide sequence of the transformation plasmid vector is SEQ ID NO.13, and the names and positions of specific vector components are shown in Table 1. Wherein the nucleotide sequence of the T-DNA gene is shown in SEQ ID NO:2. SEQ ID NO: 2 contains a complete glyphosate-resistant expression cassette and an insect-resistant expression cassette, specifically composed of the following parts, the glyphosate-resistant expression cassette: a complex formed by the 35S promoter derived from CaMV and the corn Polyubiqutin-1 promoter Promoter (nucleotide sequence is shown in SEQ ID NO.9), this composite promoter drives the 5' end to be linked with the anti-glyphosate gene G10evo (EPSPS) (nucleotide sequence is SEQ ID NO.9) of a section of coding AHAS gene chlor...

Embodiment 2

[0078] Example 2: Screening of Transformation Events

[0079] 240 independent transformants of insect-resistant and glyphosate-resistant maize were obtained through Agrobacterium mediated (Example 1). According to carrier sequence (SEQID NO.13 design primer, screen with PCR method to contain anti-glyphosate gene G10eve (nucleotide sequence is shown in SEQID NO.5) and insect-resistant gene cry1Ab-cry2Aj (nucleotide sequence is SEQ ID NO.5) NO.4), and the maize transformant without the vector backbone sequence, and carried out the preliminary test of insect resistance and glyphosate resistance performance in the greenhouse: by spraying 0.4wt% glyphosate, the glyphosate resistance Transformants with poor sex were inoculated with corn borer on the remaining transformants, and the transformants without corn borer damage were screened, and a total of 15 transformants with transformation events numbered "Double Antibody 12-1" ~ "Double Antibody 12-15" were obtained. Maize transforma...

Embodiment 3

[0104] Example 3: Identification of the flanking sequence of the insertion site of the foreign gene

[0105] Use the TAIL-PCR (Thermal asymmetric interlaced PCR) method reported by Liu et al. (Liu, Plant Journal1995, 8(3): 457-463) for the excellent transformation events "Double Antibody 12-5" and "Double Antibody 12-15". The sequence of the region flanking the insertion point of the source transgenic DNA was determined. In this method, three nested specific primers are combined with degenerate primers to carry out continuous PCR amplification, and different annealing temperatures are used to selectively amplify the target fragment. The amplified DNA fragments were cloned, sequenced and compared with the online maize genome database (http: / / www.maizegdb.org).

[0106] Sequence and compare the DNA fragment of the left flank region of the T-DNA of the transformation event "Double Antibody 12-5", and the obtained sequence is SEQ ID NO.1, wherein the sequence between nucleotides ...

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Abstract

The invention discloses a maize transformation event and a specificity identification method and application thereof. According to the transformation event, a nucleotide sequence as shown in SEQ ID NO.1 is the left flanking region of an exogenous gene; a nucleotide sequence as shown in SEQ ID NO.3 is the right flanking region of an exogenous gene or a nucleotide sequence as shown in SEQ ID NO.14 is the left flanking region of an exogenous gene, and a nucleotide sequence as shown in SEQ ID NO.15 is the right flanking region of an exogenous gene. By the maize transformation event, specificity introduction of the exogenous gene into maize lines can be realized, and the receptor maize is endowed with insect resistance and glyphosate resistance. The insect resistant and glyphosate resistant gene can be stably inherited in the receptor maize. Expression of the insect resistant and glyphosate resistant gene will not produce adverse effects on agronomic trait of the receptor maize. A detection method can provide molecular marker for breeding by the transformation event so as to enhance breeding efficiency.

Description

(1) Technical field [0001] The invention belongs to the field of plant molecular biology, in particular to the field of breeding transgenic crop varieties. The invention relates to the transformation events "Double Antibody 12-5" and "Double Antibody 12-15" of insect-resistant and glyphosate-resistant corn and a specific detection method thereof. (2) Background technology [0002] Maize (Zea mays) is an important crop and a major food source in many parts of the world. With the development of plant genetic engineering, genetic modification by introducing foreign genes has become one of the main means of genetic breeding. Both insect and herbicide resistance are highly desirable agronomic traits in maize production. Insect-resistant transgenic corn can greatly reduce the use of chemical pesticides, thereby reducing production costs and reducing the pollution of pesticides to the environment and crop products. Herbicide-resistant corn can greatly reduce the agricultural lab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N5/10A01H5/00A01H1/02
CPCA01H1/02C07K14/325C07K2319/00C12N9/1092C12N15/8275C12N15/8286C12Q1/6895C12Y205/01019C12N5/14C12N15/11C12N15/82C12Q1/68
Inventor 沈志成林朝阳张先文徐晓丽
Owner HANGZHOU RUIFENG BIOTECH LIMITED
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