Exogenous inserting fragment flanking sequence of transgenic corn BBHTL8-1 and application of exogenous inserting fragment flanking sequence of transgenic corn BBHTL8-1

A technology of transgenic corn and flanking sequences, applied in the field of plant biology

Inactive Publication Date: 2020-04-03
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Using one insecticidal protein alone can easily lead

Method used

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  • Exogenous inserting fragment flanking sequence of transgenic corn BBHTL8-1 and application of exogenous inserting fragment flanking sequence of transgenic corn BBHTL8-1
  • Exogenous inserting fragment flanking sequence of transgenic corn BBHTL8-1 and application of exogenous inserting fragment flanking sequence of transgenic corn BBHTL8-1
  • Exogenous inserting fragment flanking sequence of transgenic corn BBHTL8-1 and application of exogenous inserting fragment flanking sequence of transgenic corn BBHTL8-1

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The acquisition of embodiment 1 transgenic material

[0027] According to the codon usage frequency pairs in maize Cry1Ab, Cry3Bb and cp4epsps The gene is codon-optimized, and the optimized cp4epsps, Cry1Ab and Cry3Bb genes and from maize BYZGR and ZmT The gene is constructed into a plant expression vector. The vector backbone comes from the pCAMBIA1300 vector sequence. The vector size is 25221 bp. For the vector map, see figure 1 , where RB to LB contains Cry1Ab, ZmTMT, ZmHPT, Cry3Bb, and cp4epsps Five expression cassettes (Table 1-Table 5).

[0028] Table 1 Cry1Ab expression cassette

[0029] name size Features E35S 619 bp Cauliflower mosaic virus 35S promoter (double CaMV35S), containing repeat enhancer regions. Act2 intron 591 bp The first intron of rice actin gene Act2 stabilizes the expression of Cry1Ab. Cry1Ab

2451 bp from Bacillus thuringiensis cry1Ab Gene, expressing Cry1Ab protein, against Lepidopte...

Embodiment 2

[0042] Example 2 Detection of exogenous genes in BBHTL8-1 transformation materials

[0043] The "Plant Genomic DNA Extraction Kit" of Quanshijin Company was used to extract BBHTL8-1 genomic DNA, and the Cry1Ab , Cry3Bb and cp4epsps Genes are specifically amplified. Primer sequences are listed in Table 6.

[0044] Table 6 Information of primers for exogenous gene detection in BBHTL8-1 transformation materials

[0045]

[0046] The PCR (polymerase chain reaction) reaction system is as follows:

[0047] 2xTSINGKE Master Mix 10 µl

[0048] Forward primer (10 µM) 0.4 µl

[0049] Reverse primer (10 µM) 0.4 µl

[0050] wxya 2 O 8.2 µl

[0051] DNA template 1.0 µl

[0052] The PCR reaction procedure is as follows:

[0053] 94°C for 3 minutes

[0054] 94°C 30s

[0055] 58℃ 30 s 35 cycles

[0056] 72°C 30s

[0057] 72°C for 10 minutes

[0058] After the reaction, the PCR products were subjected to agarose gel electrophoresis, and the PCR electrophoresis results wer...

Embodiment 3

[0059] Example 3 Side sequence analysis and specific PCR detection of BBHTL8-1

[0060] In the previous experiment, the inventor carried out PCR detection of exogenous gene on BBHTL8-1 backcrossed offspring for three consecutive generations (BC4F1, BC5F1 and BC6F1), and the PCR results showed that the exogenous gene was stably inherited in the offspring of transgenic plants , the segregation ratio of transgenic plants and non-transgenic plants conforms to the Mendelian law of single-copy gene, which is a single-copy insertion.

[0061] The inventors used the whole gene resequencing method and PCR technology to detect the integration of the exogenous target gene in the BBHTL8-1 genome. The obtained sequencing results were combined with bioinformatics analysis, and it was found that the exogenous insertion sequence was integrated on the maize chromosome. The exogenous gene of BBHTL8-1 was inserted between Chr4:180575645—180577000 in maize genome.

[0062] The inventor determin...

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Abstract

The invention discloses an exogenous inserting fragment flanking sequence of transgenic corn BBHTL8-1 and an application of the exogenous inserting fragment flanking sequence of the transgenic corn BBHTL8-1. The nucleotide sequences of the flanking sequence are respectively shown as a sequence table SEQID NO:1 and a sequence table SEQID NO:2. The invention further provides a primer pair for detecting a 3' end flanking sequence. The identification of the exogenous inserting fragment flanking sequence of the transgenic pest-resistant herbicide-resistant quality improvement corn BBHTL8-1 disclosed by the invention are suitable for corn BBHTL8-1 including parents, hybrid and descendant relevant materials, and includes detection of plants, tissue, seeds and relevant products.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and in particular relates to a side sequence of a transgenic insect-resistant and herbicide-resistant maize BBHTL8-1 exogenous insert fragment and application thereof. Background technique [0002] Corn is an important food crop in my country, and it is also an important feed and industrial raw material, which plays an important role in ensuring national food security and national economic development. The corn borer and the two-spot firefly beetle cause great losses to the corn production in my country every year. Weeds also cause serious harm to my country's corn production. Generally, they cause a 10-30% reduction in production, and can reach more than 50% in serious cases. With the increase in labor costs, relying on manual weeding will greatly increase production costs. In order to control pests and reduce the occurrence of weeds, farmers often use a large amount of highly toxic chemical ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6895
CPCC12Q1/6895C12Q2600/13C12Q2600/158
Inventor 王磊邹俊杰张兰徐妙云郑红艳
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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