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Rice constitutive strong promoter and application thereof

A strong promoter, constitutive technology, applied in the field of biotechnology and genetic engineering, to achieve the effect of good application prospects

Active Publication Date: 2021-05-07
BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The gene corresponding to LOC_Os02g45770 is OsMADS6. This gene is mainly expressed strongly in the early stage of floral meristem, and its transcription can be detected in palea, lodicule, carpel, integument of ovule and receptacle This (Li et al., 2010; Duan et al., 2012), and the constitutive strong promoter element at the 3' end of the gene has not been reported

Method used

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  • Rice constitutive strong promoter and application thereof
  • Rice constitutive strong promoter and application thereof
  • Rice constitutive strong promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016]Example 1: A total of 1 promoter full length segment.

[0017]Extraction of Japan Qing Rice Genomic DNA: The fresh leaves were extracted with CTAB using CTAB method using CTAB method, and the refrigerator was placed in the refrigerator to prepare.

[0018]The LOC_OS02G45770 genome gene is extracted on the biological information website (http: / / rice.plantbiology.msu.edu) to obtain the gene 3 'end reverse region (figure 1 Gene sequence, total of 1676 bp length, design 1 pair specific primer (see Table 2), PCR amplification condition is: 94 ° C for 5 min; 98 ° C 30S, 58.5 ° C 30S, 72 ° C 2 min, 35 cycles; 72 ° C 10 min . PCR reaction system: 10 * Kod Buffer 2 ul, 10 mmdNTP 1.5 ul, 10 mm primer PRF 2 ul, 10 mm primer Pr 2 ul, genome DNA 2 ul, KOD POLYMERASE 1 ul, the rest of the double steamed to 50 ul.

[0019]Table 1 Analysis of the core components of full length promoter

[0020]

[0021]Table 2 Primer sequences for the design of the present invention

[0022]

[0023]The PCR product TA was cloned ...

Embodiment 2

[0025]Example 2: RT-PCR analysis of transgenic rice and analysis of GUS staining

[0026]The above-mentioned constructed carrier PCAMBIA1301-Nubi5 transforms japonica (Japan Qing) callus as genetic transformation receptor materials, and uses Agrobacterium to promote rice embedum seed callus to achieve genetic transformation, screened, differentiation and rooting of resistant callus. The steps of growth positive plants, the specific steps are as follows:

[0027]1. Exhibit: Take the seed embryo of 10-14 days, peeling, peeling, 3 min with 75% anhydrous ethanol, then disinfected with 3% sodium hypochlorite solution for 10 min, sterile water was cleaned 3-4 times, After the filter paper is sued, then the embryo is picked, inoculated on N6 + 2.0 mg / l2, 4-D medium, and 7-15 d at 27 ° C.

[0028]2. Pick 1-2 mm pale yellow, dense callus use as Agrobacterium transformation.

[0029]3. Agrobacteria Infest: From the Picking Agrobacterium, Picking Single Silitter in the 2D Single Silitter has fallen in Y...

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Abstract

The invention relates to a rice constitutive strong promoter and application thereof. Specifically, the invention provides a promoter Nubi5 nucleotide sequence capable of being expressed in each tissue of a plant and a preparation and use method. The invention discloses that a 3' end reverse sequence of a plant gene has a strong constitutive promoter functional element for the first time. The invention also relates to an expression cassette containing the promoter, a recombinant expression vector and a method for obtaining a corresponding transgenic plant. The Nubi5 promoter disclosed by the invention shows very strong activity in organs such as roots, stems, leaves, flowers and seeds of the plant, and has a very good application prospect in the field of plant transgenosis.

Description

Technical field[0001]The invention belongs to the technical field of biotechnology and genetic engineering. Specifically, a rice constitutive force and its application thereof are specifically involved.Background technique[0002]With the research and development of genetically modified technology, the promoter is applied in many different species in many agriculture. The success of transgenic plant R & D is mainly dependent on several factors, including optimizing cell culture conditions, selecting a suitable transformation method, and use the appropriate promoter to become a critical factor in transformation of transgenic plants (MCELROY ETAL., 1990). Previously, broccoli flower leaf virusCamv35s The constituent promoter has been widely used in the plant transformation system of bonchi and monocotonic leaves (Odell et al., 1985; ZHANG et al., 1988), of course, it also has a lower vitality in the monocratic plant (Peterhans et al. Al., 1990). The other two currently have a wide range...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8223
Inventor 颜静宛林智敏苏军王锋
Owner BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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