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Corn nutritive organ specificity promoter and application thereof

A vegetative organ and promoter technology, applied in the field of plant genetic engineering, can solve the problem of non-expression of reproductive organs, and achieve the effect of strong stage, high sensitivity and good commercial value

Inactive Publication Date: 2015-12-09
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is a lack of a promoter in maize that can regulate the expression of the target gene in the vegetative organs, but not in the reproductive organs

Method used

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  • Corn nutritive organ specificity promoter and application thereof
  • Corn nutritive organ specificity promoter and application thereof
  • Corn nutritive organ specificity promoter and application thereof

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Experimental program
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preparation example Construction

[0031] A method for preparing a transgenic plant containing the maize vegetative organ-specific expression promoter of the present invention comprises the following steps:

[0032] (a) introducing the promoter according to claim 1 or the plant expression vector according to claim 3 into rice tissue cells;

[0033] (b) cultivating the rice tissue cells under conditions that promote plant growth to obtain transgenic plants.

[0034] A total of 18 transgenic plants were obtained by Agrobacterium-mediated transformation of rice, and 10 positive plants were detected by PCR. GUS histochemical staining was performed on different tissue parts of the positive plants. GUS histochemical staining confirmed that the PZmCCR promoter was a vegetative organ-specific expression promoter, and pCAM-PZmCCR was a vegetative organ-specific expression vector.

[0035] figure 1 PZmCCR promoter molecular detection results. A is the result of PCR; B is the result of double enzyme digestion.

[003...

Embodiment 1

[0042] Cloning of Maize Vegetative Organ Specific Promoter PZmCCR

[0043] According to the full-length sequence of the PZmCCR promoter published on the NCBI website, primers for PCR amplification of this fragment were designed, the upstream primer was added with the AAGCTT restriction site of HindIII, and the downstream primer was added with the CCATGG restriction site of NcoI.

[0044] The primer sequences are as follows:

[0045] Primer 1 (upstream primer): 5'- CCCAAGCTT CAGGCTGTGGGACACCTCCATTCTA-3'

[0046] Primer 2 (downstream primer): 5'- CATGCCATGG GTGCTCCTCTGCTCTAGCTCTT-3'

[0047] Using the corn B73 genomic DNA extracted from Quanshijin Company’s plant genome kit as a template, PCR amplification was performed with primers 1 and 2. The PCR reaction system is shown in Table 1:

[0048] Table 1

[0049]

[0050] The PCR reaction conditions were: pre-denaturation: 94°C for 5min; denaturation: 94°C for 30s; annealing: 62°C for 30s; extension: 72°C for 2min, 27 cycl...

Embodiment 2

[0053] Establishment of pZmCCR gene promoter expression vector

[0054] The small fragment PZmCCR fragment that has been connected to pEASYT1CloningVector and the large fragment CaMV35S promoter on the Agrobacterium binary vector pCAMBIA1301 were double-enzymatically digested with HindⅢ and NcoI enzymes, and 20uL of the system was digested in a water bath at 37°C for 3h As shown in table 2:

[0055] Table 2

[0056]

[0057] Ligate the above size fragments with T4 DNA ligase, and ligate at 25°C for 2-12 hours. The ligation system is shown in Table 3:

[0058] table 3

[0059]

[0060] Take 4-6 μL of the constructed vector pCAM-PZmCCR plasmid and gently inject it into 200 μL LEHA105 Agrobacterium competent cells, ice bath for 5 min, liquid nitrogen quick freezing for 1 min, and 37 °C water bath for 5 min, add 200 μL YEP liquid medium, 28 °C, Pre-express at 220rpm for 4-5h; centrifuge at 10000rpm for 30s, discard the supernatant, add 100μL YEP liquid medium, resuspend t...

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Abstract

The invention discloses a corn nutritive organ specificity promoter and application thereof. The corn nutritive organ expression promoter can be expressed in corn nutritive organs and has the nucleotide sequence shown in SEQ ID NO.1. A plant expression vector with the corn nutritive organ expression promoter is provided. The plant expression vector is established by replacing a CaMV35S promoter on a plant binary expression vector pCAMBIA1301 with the corn nutritive organ expression promoter. The corn nutritive organ specificity promoter is only expressed in nutritive organs of transgenic rice (Zhonghua 11) instead of seeds and can have good application prospects in corn cultivation on the aspects of insect resistance, disease resistance and the like of transgene.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a maize vegetative organ-specific promoter and application thereof. Background technique [0002] Promoters are important cis-acting elements that regulate the transcription and expression of genes. According to the different transcription modes of promoters, they can be divided into three types: constitutive, inducible and tissue-specific promoters. The choice of which promoter to drive gene expression is a hot spot in current genetic engineering research. Tissue-specific promoters, also known as organ-specific promoters, are one of the types of promoters. Driven by such promoters, gene expression is often only highly expressed in certain specific organs and tissue parts, which can effectively avoid Unnecessary waste of plant nutrition and adverse effects on plants, and exhibit characteristics such as growth regulation. [0003] Different types of organ-specific promot...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/84A01H5/00
Inventor 项艳吴胜男吴敏董庆孔晶晶张敏杨建平
Owner ANHUI AGRICULTURAL UNIVERSITY
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