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Paddy rice nitrogen deficiency specific induced expression promoter Y5 and application thereof

A technology for inducing expression and promoters, which is applied in the field of plant genetic engineering and can solve the problems of single plant biological yield and grain yield decline

Inactive Publication Date: 2013-08-21
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

GS is involved in NH4 + The main enzyme for the assimilative synthesis of glutamine (Gln), Cai et al. (2009) used the 35S promoter to overexpress rice glutamine synthetase genes GS1; 1, GS1; 2 and E. coli glutamine in rice variety Zhonghua 11 Synthetase gene glnA, GS activity, total nitrogen content, amino acid content, soluble protein, etc. have all been significantly increased, but the biological yield per plant and grain yield have decreased significantly

Method used

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  • Paddy rice nitrogen deficiency specific induced expression promoter Y5 and application thereof
  • Paddy rice nitrogen deficiency specific induced expression promoter Y5 and application thereof
  • Paddy rice nitrogen deficiency specific induced expression promoter Y5 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Determination of the regulatory mode of the promoter Y5 and acquisition of the sequence

[0029] In order to discover the promoters of nitrogen deficiency-induced gene expression in rice and provide new promoter materials for genetic engineering to improve nitrogen uptake in rice, we designed the attached figure 1 In the technical roadmap, two well-known and commonly used conventional rice varieties were selected as experimental materials: Nipponbare and Zhenshan 97. Microarray technology is used to select new genes that have a significant response to nitrogen stress. This technology can be used to quantitatively detect the expression levels of a large number of genes at different times (Yang Rong et al., 1999).

[0030] The seeds of Nipponbare and Zhenshan 97 were soaked at 37°C for 3 days, accelerated for 2 days, seedlings emerged in sand culture for one week, and transplanted to rice full nutrient solution hydroponics (hydroponic nutrient solution composit...

Embodiment 2

[0031] Embodiment 2: Construction of promoter expression transformation vector

[0032] According to the known full-length promoter sequence of gene Y5 (see sequence listing SEQ ID NO: 1) and its truncated sequence Y5B (see sequence listing SEQ ID NO: 2), Y5C (see sequence listing SEQ ID NO: 3), Y5D (see the sequence table described in SEQ ID NO: 4) design primers (see Table 1 for the primer sequence), and use the total DNA of the rice variety Zhonghua 11 as a template to amplify to obtain the full-length sequence of the promoter Y5A respectively (1305bp) and truncated Y2B (1062bp), Y2C (784bp) and Y5D (546bp). When amplifying these four fragments, the same restriction endonuclease site HindIII (as shown in Table 1-1) was added to the 5' ends of the four pairs of primers, so the amplified fragments can be obtained by restriction The endonuclease HindIII was digested, and then connected to the promoter vector DX2181b (the vector DX2181b was transformed by the researchers of th...

Embodiment 3

[0038] Example 3: Rice transformation experiment after the full-length Y5 promoter and its truncated promoter fragment were fused to GUS

[0039] After connecting the full length of the Y5 promoter and its truncated promoter fragments to the vector DX2181b, the transformed rice positive plants were obtained by using the method of Agrobacterium-mediated transgenesis. The specific transformation steps are as follows:

[0040] The obtained correctly cloned plasmids (DX2181b-Y5A, DX2181b-Y5B, DX2181b-Y5C and DX2181b-Y5D) were introduced into the rice variety Hua 11 through the rice genetic transformation system mediated by Agrobacterium (EHA105 provided by CAMBIA Laboratory, Australia) In , transformed plants were obtained through precultivation, infection, co-cultivation, selection of hygromycin-resistant calli, differentiation, rooting, seedling training and transplanting. The Agrobacterium-mediated genetic transformation system of rice (subspecies japonica) mainly adopts the fu...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and specifically relates to a paddy rice nitrogen deficiency specific induced expression promoter Y5 (also known as Y5A) and an application thereof. With a chip technology, promoter Y5 (Y5A) downstream gene nitrogen supply induced expression information is obtained. With different paddy rice varieties, Y5A downstream gene nitrogen supply induced expression mode is verified. With a PCR method, the promoter Y5A full length and a 5'-terminal truncated fragment thereof are amplified, and are connected to a promoter vector DX2181b, such that a promoter fusion GUS expression vector is obtained. The constructed vector is used in genetic transformation upon a paddy rice variety Zhonghua11. The regulation of the promoter upon GUS gene is verified in a transformation positive plant. Through Realtime expression level verification and GUS protein activity detection, it is further determined that the promoter is a paddy rice nitrogen deficiency specific induced expression promoter. A functional section is at ATG upstream 546bp.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. In particular, it relates to a rice nitrogen-deficiency-specific induction promoter Y5 and its application. Background technique [0002] At present, transgenic research mainly uses constitutive promoters to drive the expression of target genes, the most typical is the CaMV35S promoter isolated from cauliflower mosaic virus (Hirt et al., 1990; Battraw et al., 1990), and some plant sources in recent years promoters, such as the promoter of actin gene Actin1 cloned from rice and the ubiquitin gene promoter cloned from maize (Schledzewski et al., 1994). However, the constitutive expression of exogenous genes will often cause unnecessary waste of resources, and the accumulation of a large number of heterologous proteins will also break the original metabolic balance of plants and hinder the normal growth of plants (Nie Lina et al., 2008; Rai et al., 2009). Kasuga et al. (1999) fo...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/84A01H5/00
Inventor 练兴明张星杨猛
Owner HUAZHONG AGRI UNIV
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