Nitrogen supply recovery specific induced expression promoter Y2 after paddy rice nitrogen deficiency, and application thereof

A promoter and specific technology, applied in the field of plant genetic engineering, can solve the problems of single plant biological yield and grain yield decline

Inactive Publication Date: 2014-06-18
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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  • Nitrogen supply recovery specific induced expression promoter Y2 after paddy rice nitrogen deficiency, and application thereof
  • Nitrogen supply recovery specific induced expression promoter Y2 after paddy rice nitrogen deficiency, and application thereof
  • Nitrogen supply recovery specific induced expression promoter Y2 after paddy rice nitrogen deficiency, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Determination of the regulatory mode of the Y2 gene promoter and acquisition of the sequence

[0028] In order to discover the promoters of nitrogen deficiency or high nitrogen supply induced gene expression in rice, and to provide new promoter materials for genetic engineering to improve nitrogen uptake in rice, the applicant designed figure 1 In the technical route, 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).

[0029] 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 (hydropo...

Embodiment 2

[0030] Embodiment 2: Construction of promoter expression transformation vector

[0031] According to the known promoter Y2A full-length sequence (2266bp, see sequence listing SEQ ID NO: 1) and its truncated promoter segment: Y2B (see sequence listing SEQ ID NO: 2), Y2C (see sequence listing SEQ ID NO: 3) Three pairs of primers were designed (see Table 1 for the sequences of the primer pairs), and the total DNA of the rice variety Zhonghua 11 was used as a template to amplify to obtain the full-length sequence (2266bp) of the promoter Y2A and the truncated Y2B (1056bp) and Y2C (519bp). When amplifying these three fragments, the same restriction endonuclease site BamHI was added to the 5' ends of the three pairs of primers (as shown in Table 1-1), so the amplified fragments can be used for restriction nucleic acid Endonuclease BamHI was cut, and then connected to the promoter vector DX2181b (the vector DX2181b was obtained by the researchers of the State Key Laboratory of Crop ...

Embodiment 3

[0038] Example 3: Rice transformation experiments after the full-length Y2 promoter and its truncated promoter fragments were fused to GUS

[0039] After the full-length and truncated fragments of the Y2 promoter are respectively connected to the vector DX2181b, the transformed rice positive plants are obtained by using the method of Agrobacterium-mediated transgenesis. The specific steps of transformation are as follows:

[0040] The obtained correctly cloned plasmids (DX2181b-Y2A, DX2181b-Y2B and DX2181b-Y2C) were introduced into the rice variety Zhonghua 11 through the rice genetic transformation system mediated by Agrobacterium (EHA105 provided by CAMBIA Laboratory, Australia). Cultivate, infect, co-cultivate, screen hygromycin-resistant calli, differentiate, take root, train seedlings and transplant to obtain transformed plants. The Agrobacterium-mediated genetic transformation system of rice (subspecies japonica) mainly adopts the further optimized method based on the re...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and specifically relates to a nitrogen supply recovery specific induced expression promoter Y2 after paddy rice nitrogen deficiency, and an application thereof. With a chip technology, promoter Y2 downstream gene nitrogen supply induced expression information is obtained. With different paddy rice varieties, Y2 downstream gene nitrogen supply induced expression mode is verified. With a PCR method, the promoter Y2 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 nitrogen supply recovery specific induced expression promoter after nitrogen deficiency. A functional section is at AT6 upstream 519bp.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering. In particular, it relates to a promoter Y2 that restores nitrogen supply specific induction expression after nitrogen deficiency in rice 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 the 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/84A01H5/00
Inventor 练兴明杨猛张星
Owner HUAZHONG AGRI UNIV
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