Rice callus-specific promoter and application thereof

A callus and promoter technology, applied in the field of plant genetic engineering, to improve screening efficiency, reduce safety concerns, and improve expression

Inactive Publication Date: 2011-09-14
WUHAN HEALTHGEN BIOTECHNOLOGY CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different species have different preferences for the use of codons. The usage rates of the first and second bases of

Method used

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  • Rice callus-specific promoter and application thereof
  • Rice callus-specific promoter and application thereof
  • Rice callus-specific promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] [Example 1] Cloning of Rice Callus Specific Promoter (OsCSP)

[0026] 1. Extraction of rice TP309 genomic DNA

[0027] A) Take 0.1 gram of young leaves and place them in a mortar, add 0.6ml of CTAB extract, and grind the leaves evenly;

[0028] B) Warm bath in a water bath at 65°C for 30 minutes, during which gently shake 2-3 times, take out after warm bath and cool to room temperature naturally;

[0029] C) After adding 0.6ml of extract solution (chloroform: isoamyl alcohol = 24:1), shake gently for 5 minutes, and centrifuge (10000rpm) for 5 minutes;

[0030] D) Draw the supernatant into another 1.5ml centrifuge tube, add 75 μL of 3M sodium acetate and 1ml of pre-cooled absolute ethanol;

[0031] E) Centrifuge (12000rpm) at room temperature for 10 minutes, discard the supernatant, add 1ml of 70% ethanol, centrifuge to wash the precipitate, pour off the ethanol, and dry the DNA;

[0032] F) After the DNA is fully dried, add 200 μl of sterile ddH2O to dissolve the DNA, ...

Embodiment 2

[0042] [Example 2] Construction of a vector containing the OsCSP promoter

[0043] 1. Construction of OsCSP-stuff-nos vector

[0044] The amplified fragment of the OsCSP promoter was double-digested with HindIII / NaeI to recover the product and the pBI221 plasmid. The promoter after enzyme digestion was recovered by precipitation, inserted into the 4.8kb pBI221 vector recovered after enzyme digestion, and the correct transformant was screened after transformation of DH10B. The obtained vector OsCSP-stuff-nos number is pOsPMP209.

[0045] 2. Construction of OsCSP-gus-nos vector

[0046] Using pCAMBIA1301 as a template, the GUS gene was amplified. The forward primer is 5'-cggtgccggcATGGTAGATCTGAGGG-3' (SEQ ID NO.6), the reverse primer is 5'-ccgctcgagTCACACGTGGT GGTG-3' (SEQ ID NO.7), and the amplification temperature is 60°C. The product was recovered by digesting the PCR gel with NaeI / XhoI, and the digested product was precipitated and recovered, inserted into the pOsPMP209 ...

Embodiment 3

[0053] [Example 3] Agrobacterium-mediated transformation

[0054] 1. Culture of callus

[0055] Take mature rice seeds, soak them with 20% sodium hypochlorite (adding 1-2 drops of Tween 20) for 30-40 minutes on the ultra-clean bench after shelling, and rinse them with sterile water for 4-5 times. Blot the water with sterile filter paper, put it on the callus induction medium, and culture it in the dark at 26°C for 4 weeks, pick the bright yellow, dense, relatively dry callus and transfer it to the subculture medium, and culture it in the dark at 26°C for 2 weeks. week.

[0056] 2. Agrobacterium transformation

[0057] The Agrobacterium strains of pCAMBIA1300(35S-hpt-nos), pOsPMP122(JH-OsCSP-hpt-nos), pOsPMP522(JH-OsCSP-opt hpt-nos) and pOsPMP43(OsCSP-gus-nos) plasmids were respectively streaked The bacteria were inoculated on YEB solid medium containing 50mg / L kanamycin, and cultured in the dark at 28°C for 2 days. Transfer the Agrobacterium to the suspension medium respec...

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Abstract

The invention provides a callus-specific promoter cloned from rice, an expression vector containing the promoter and a selective marker gene optimized by a rice codon, and a transformant. A nucleotide sequence of the promoter is shown in SEQ ID NO.1; and the promoter has the expression specificity of calli, and has higher activity in the calli. The expression of the selective marker gene (a hygromycin B phosphotransferase gene with a sequence shown in SEQ ID No.2) optimized by the codon in a normal rice plant is controlled by using the promoter, so that the transgenic rice can be efficiently screened, and the problem of the safety of the selective marker gene to the biological environment can be completely solved; therefore, the biological safety of the transgenic rice is improved, and the selection efficiency is improved at the same time.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a rice callus-specific promoter and application thereof. Background technique [0002] Screening marker genes are used as an effective means to screen positive transformants after plant genetic transformation, but at present, constitutive promoters are used to mediate the expression of marker genes, among which the most widely used in plant genetic transformation is the tobacco mosaic virus. CaMV35S promoter (Xu Chunbo, Wang Yong, Li Xingyou, Zhao Haixia, construction of CBF4 gene plant expression vector under the regulation of two promoters, Biotechnology, 2010), followed by Ubiquitin promoter (Garbarino, J.E., T.Oosumi, and W.R. Belknap, Isolation of a polyubiquitin promoter and its expression in transgenic potato plants.Plant Physiol, 1995), although these promoters have been widely used in plant genetic transformation, but due to constitutive expression, the...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00
Inventor 杨代常宁婷婷汪相宏
Owner WUHAN HEALTHGEN BIOTECHNOLOGY CORP
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