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Promoter Y8A for specific induced expression by recovering nitrogen supply after nitrogen deficiency of rice and application thereof

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

Inactive Publication Date: 2015-06-17
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 Synthase gene glnA, GS activity, total nitrogen content, amino acid content, soluble protein, etc. have been significantly increased, but the biological yield per plant and grain yield have been significantly decreased

Method used

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  • Promoter Y8A for specific induced expression by recovering nitrogen supply after nitrogen deficiency of rice and application thereof
  • Promoter Y8A for specific induced expression by recovering nitrogen supply after nitrogen deficiency of rice and application thereof
  • Promoter Y8A for specific induced expression by recovering nitrogen supply after nitrogen deficiency of rice and application thereof

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Experimental program
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Effect test

Embodiment 1

[0026] Example 1: Determination of the regulatory mode of the Y8 gene promoter and acquisition of the sequence

[0027] 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. Apply microarray technology to select new genes that have obvious responses 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).

[0028] Soak the seeds of Nipponbare and Zhenshan 97 at 37°C for 3 days, accelerate germination for 2 days, and seedlings emerge in sand culture for one week, and transplant the seedlings to rice full nutrient solution hydropon...

Embodiment 2

[0029] Embodiment 2: Construction of promoter expression transformation vector

[0030] The present invention uses the total DNA of rice variety Zhonghua 11 as a template to amplify the full-length sequence of the Y8A promoter through the PCR method. At the same time, the full-length sequence of the promoter was truncated to 982 bp upstream of the ATG according to the 5'-end truncation mode, named: Y8B (see SEQ ID NO: 1 in the sequence listing), and the truncated fragment was obtained by PCR. The details are as follows: According to the known full-length sequence of the promoter Y8A (1936bp, see SEQ ID NO: 1 in the sequence listing) and its truncated promoter segment Y8B (see SEQ ID NO: 2 in the sequence listing), design two pairs of primers (The sequence of the primer pair is shown in Table 1). Using the total DNA of rice variety Zhonghua 11 as a template, the full-length sequence of promoter Y8A (1936bp) and the truncated Y8B (982bp) were amplified respectively. When amplif...

Embodiment 3

[0036] Example 3: Rice transformation experiments after the full-length promoter Y8A and its truncated promoter fragments are fused to GUS

[0037] After connecting the full-length promoter Y8A and the truncated fragment Y8B 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:

[0038] The obtained correctly cloned plasmids (DX2181b-Y8A, DX2181b-Y8B) were introduced into the rice variety Zhonghua 11 through the rice genetic transformation system mediated by Agrobacterium (EHA105 provided by CAMBIA Laboratory, Australia), and pre-cultivated, infected , co-cultivation, selection of hygromycin-resistant calli, differentiation, rooting, training and transplanting of seedlings to obtain transformed plants. The Agrobacterium-mediated genetic transformation system of rice (subspecies japonica) mainly adopts the further optimized method based on the report...

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Abstract

The invention belongs to the field of a plant genetic engineering technology and specifically relates to a promoter Y8A for specific induced expression by recovering nitrogen supply after nitrogen deficiency of rice. By a chip technology, nitrogen supply induced expression information of downstream gene of the promoter Y8A is obtained, and a nitrogen supply induced expression pattern of the downstream gene of the promoter Y8A is verified through different rice varieties. Through a PCR method, overall length of the promoter Y8A and its 5' end truncated fragment Y8B are amplified and linked to a vector DX2181b so as to construct a promoter-fused GUS expression vector. Then, the constructed vector is transformed into the rice variety Zhonghua 11. In positive transformed plants, regulation of GUS gene expression by the promoter is verified. Through expression quantity verification of Realtime, it is further confirmed that Y8A and Y8B belong to the promoters for specific induced expression by recovering nitrogen supply after nitrogen deficiency and a functional fragment of the promoter is at the position of ATG upstream 982bp. The invention provides a new promoter resource for rice.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering. In particular, it relates to a promoter Y8A that specifically induces the expression of nitrogen supply 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 often causes 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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IPC IPC(8): C12N15/113C12N15/82A01H5/00
Inventor 练兴明杨猛张星
Owner HUAZHONG AGRI UNIV
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