Early anther specific promotor and application thereof

A technology in the promoter and sequence list, applied in the field of plant genetic engineering, can solve the problems of destruction, phytotoxic environment, fertility restoration and maintenance of sterility in cumbersome methods, etc.

Active Publication Date: 2017-03-08
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the utilization of heterosis, the existing methods of artificially creating male sterility to restore fertility and maintain sterility are relatively cumbersome, while chemical male-killing methods are likely to poison plants or cause environmental damage

Method used

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  • Early anther specific promotor and application thereof
  • Early anther specific promotor and application thereof
  • Early anther specific promotor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, the cloning of LOC_Os05g42240 gene promoter fragment EASpro:

[0020] The length of 3kb upstream of the start codon ATG site of the rice LOC_Os05g42240 gene was selected as a candidate promoter region, and the genomic DNA of the rice variety Nipponbare was used as a template to amplify using primers F1 and R1 (see below). Subsequently, the amplified PCR product was constructed into the pENTR intermediate vector by TOPO reaction, and then carried out LR reaction with the pHGWFS7 final vector to construct the analysis vector EASpro-GUS with EASpro promoter fused to GUS. Among them, TOPO enzyme and LR enzyme were purchased from Invitrogen, and the pHGWFS7 vector was purchased from Ghent University.

[0021] F1: 5'-CACCGAATTCGCTGCGTCTAATTTCCGG-3' (SEQ ID NO: 2)

[0022] R1: 5'-GGCGGAAGAAGCCGAGAGGTTAGTG-3' (SEQ ID NO: 3)

Embodiment 2

[0023] Embodiment 2, the acquisition of transgenic plants

[0024] (1) Rice callus induction:

[0025]Select plump Nipponbare rice seeds, peel off the seed coat, sterilize and wash, and evenly inject them into the sterilized MS solid medium with 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) , 32 ℃ continuous light for 5 days to induce callus formation.

[0026] (2) Agrobacterium transformation:

[0027] At the same time, the pHGWFS7 vector carrying the EASpro promoter fragment was transformed into Agrobacterium EHA105 competent cells by heat shock method, coated with solid LB medium with 50 μg / L spectinomycin, cultured in the dark at 28°C for 2 days, and screened for positive clones . The obtained positive clones were cultured in the dark at 28° C. for 3 days in solid AB medium containing 50 mg / L spectinomycin for rice transformation.

[0028] (3) Rice callus transformation:

[0029] Take the callus with good growth condition, soak in the bacterial solution for 2 minutes,...

Embodiment 3

[0034] Embodiment 3, the activity analysis of EASpro promoter in anther tissue

[0035] Take the florets of positive transgenic rice at different reproductive stages, and treat them with pre-cooled 90% acetone for 20 minutes; discard the acetone, add 50 mM phosphate buffer (pH 7.2) to rinse twice; absorb and discard the phosphate buffer, and add GUS staining solution (50mM phosphate buffer solution with pH 7.2, potassium ferricyanide 2mM, potassium ferrocyanide 2mM, X-gluc 1mM) appropriate amount, so that the plant material is completely submerged in the staining solution, and vacuumized for 1hr; Stain in the box. After the color development of the plant material is completed, discard the GUS staining solution, add 1mL of 70% ethanol and mix gently to decolorize. Observe and photograph under a stereomicroscope.

[0036] The results showed that in the early stage of stamen primordium differentiation, when the stamen primordium was just raised and the length of the entire flore...

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Abstract

The invention discloses an early anther specific promotor and an application thereof. The promoter is the promoter of a rice gene LOC_Os05g42240; the promoter can be used for controlling the specific expression of a target gene in such cells as anther primordium, tapetum and microspore cells in an anther early development stage of a plant; and the promoter is applicable to researches in the aspects of plant breeding and gene function analysis.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a high-efficiency promoter sequence specifically expressed in the early development stage of rice anthers and the application of the promoter in plant transgenesis. Background technique [0002] Rice (Oryza sativa L.) is a monocot model crop for the study of plant gene function, and it is also one of the most important food crops in the world. However, with the increase of population and the decrease of arable land and a series of problems, how to effectively increase the yield of rice at the unit level has become a long-term research goal in rice breeding. At present, the application of heterosis can not only improve the ability of crops to resist diseases and insect pests, but also effectively improve crop quality and increase crop yield. [0003] In the utilization of heterosis, the methods of restoring the fertility and maintaining the sterility...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
CPCC07K14/415C12N15/8231
Inventor 秦跟基杨琰郭冬姝
Owner PEKING UNIV
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