Gene switch system for directional expression of insect-resistant and/or herbicide-resistant genes of corn as well as construction method and application of gene switch system

A gene switch and herbicide-tolerant technology, applied in the field of genetic engineering, can solve the problems that hinder the promotion and commercial planting of genetically modified crops, and achieve the effects of eliminating food safety concerns, great application value, and wide application prospects

Pending Publication Date: 2021-04-13
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use safety of genetically modified food crops has always been a hot topic of public controversy, and it is also an important reason why genetically modified food crops have not been fully recognized or widely commercialized so far in

Method used

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  • Gene switch system for directional expression of insect-resistant and/or herbicide-resistant genes of corn as well as construction method and application of gene switch system
  • Gene switch system for directional expression of insect-resistant and/or herbicide-resistant genes of corn as well as construction method and application of gene switch system
  • Gene switch system for directional expression of insect-resistant and/or herbicide-resistant genes of corn as well as construction method and application of gene switch system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction of "key" and "lock" expression vectors for directional expression in maize

[0054] Based on the plant expression vector pCOMBIA 3300 (purchased from Sigma Biotechnology Company), the maize green tissue promoter Zm1rbcS and the site-specific recombinase gene Cre were cloned, and the maize green tissue-specific promoter Zm1rbcS, nuclear localization gene Krp2, Point-specific recombinase gene Cre and terminator NosT to construct the maize "key" vector pKEY(pZm1rbcS-Krp2-Cre-NosT)( figure 1 A), kanamycin is the selection marker gene, HindⅢ and SmaI are restriction sites, and the "key" pKEY overexpression vector is constructed by homologous recombination. At the same time, using the plant expression vector pCOMBIA3300 as the basic vector, the maize endogenous constitutive ZmUbi, the insect resistance gene Cry1Ab / 1Ac, the insect resistance gene Vip3ArLr1, and the herbicide tolerance gene RePAT were cloned, and the maize "lock" gene vector pLockBt(pZmUb...

Embodiment 2

[0055] Embodiment 2 Maize acceptor genetic transformation

[0056] Transformants were obtained by infecting maize immature embryos with Agrobacterium-mediated method. The specific process is: transfer the constructed maize gene directional expression "key" vector pKEY and maize gene expression "lock" vectors pLockBt, pLockVip, pLockReP to Agrobacterium EHA105, transfer, ice bath (10min), liquid nitrogen quick freezing ( 5min), 37°C water bath (5min), ice bath (5min), activation (adding YEP liquid 28°C shaking culture for 2-3h), plating culture (28°C, 48-72h), single-clonal bacterial inspection and other steps to obtain positive clones. The confirmed positive single clones were inoculated in liquid YEP medium containing kanamycin and rifamycin, and cultured in a shaker at 28°C at 200r / min overnight for 12-16h. Use a spectrophotometer to measure the OD value of the bacterial solution at a wavelength of 600nm. When the OD value reaches 0.2-0.4, divide it into sterile centrifuge...

Embodiment 3

[0057] Embodiment 3 Transgenic positive plants are obtained

[0058] Take genetic transformation to obtain maize plant leaves, for T 0 The target gene of transgenic plants was detected by PCR, and a total of 233 positive plants were obtained (Table 1). In order to verify whether the maize directional expression regulatory gene Cre, insect resistance gene Cry1Ab / 1Ac, insect resistance gene Vip3ArLr1 and herbicide tolerance gene RePAT are really recombined into the maize genomic DNA, the genomic DNA of the leaves of the transgenic plants of the T0 generation was extracted for test strips and PCR detected by the T 0 Molecular detection of transgenic plants showed that the target gene had been integrated into the maize genome ( image 3 ).

[0059] Table 1 T obtained by PCR detection 0 Transgenic positive plants

[0060]

[0061] T will be obtained 0 The seeds of transgenic positive plants were planted in the field to obtain transgenic plants T 1 generation, through the ...

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Abstract

The invention discloses a gene switch system for directional expression of insect-resistant and/or herbicide-resistant genes of corn as well as a construction method and application of the gene switch system. The gene switch system comprises a locking element containing the insect-resistant and/or herbicide-resistant genes, and a nucleotide sequence which opens the locking element as shown in SEQ ID NO.1 and enables the locking element to be expressed in specific tissue, wherein the nucleotide sequence shown as SEQ ID NO.1 is a site-specific recombinase gene Cre. An endogenous green tissue specific promoter of the corn is utilized, a site specific recombination system and related regulation elements are combined, a corn gene directional expression 'gene switch ' technical platform is constructed, precise expression of exogenous genes is achieved, and powerful technical support is provided for cultivation of high-quality, safe and efficient new transgenic crop strains.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gene switch system for directional expression of corn insect-resistant and / or herbicide-tolerant genes, its construction method and application. Background technique [0002] Corn is the largest food crop integrating grain, economy, feed and energy, and plays a pivotal role in my country's national economy and agricultural production. Ensuring grain production and green safety is a long-term strategic task in my country, and the occurrence of pests and diseases directly threatens corn grain production and safety (Bu Huahu et al., 2019). With the development of biological sciences, the development of genetic engineering technology provides a reference for solving this problem, and disease-resistant transgenic corn is an important measure to effectively resist the threat of pests and diseases (Wang Xueli et al., 2020). The use safety of genetically modifi...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N5/10A01H5/00A01H5/10A01H6/46A01H1/02
CPCA01H1/02C07K14/32C07K14/325C07K14/415C12N9/12C12N9/14C12N15/8223C12N15/8277C12N15/8286C12N2830/008C12Y207/11001
Inventor 袁广胜何慧陈浩沈亚欧潘光堂马浪浪高世斌兰海史家豪杨焱王智颖邹超英
Owner SICHUAN AGRI UNIV
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