Endosperm specific expression promoter

A molecular and sequence listing technology, applied in the field of endosperm-specific expression promoters, can solve the problems of increasing the metabolic burden of plants, being toxic, and detrimental to the normal growth of plants.

Inactive Publication Date: 2014-07-23
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, plant constitutive promoters widely used in genetic engineering have gradually exposed some problems. The genes driven by such promoters are expressed to varying degrees in different tissues and organs of plants, and in most cases, people do not want exogenous The gene is widely expressed in the whole plant and the whole growth period of the transgenic plant, because on the one hand, it will increase the metabolic burden of the plant, on the other hand, some foreign proteins are not necessary or even toxic to the plant, which is not conducive to the normal growth of the plant (Kasuga M, Liu Q, Miura S, et al. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol, 1999, 17:287-291.)

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1, the cloning of promoter sequence

[0054] 1. Wheat Genomic DNA Extraction

[0055] Wheat varieties tested: Yunmai 33, Zhongguochun, Jimai 20 and Atlas66.

[0056] After the seeds of the four tested wheat varieties germinated for 7-10 days, fresh leaves were ground into powder in liquid nitrogen, and genomic DNA was extracted by CTAB method.

[0057] CTAB extraction buffer: 0.1M Tris-HCl (pH8.0), 20mM EDTA (pH8.0), 1.4M NaCl, 2% (w / v) CTAB, 2% (w / v) PVP40 and 3% (v / v) β-mercaptoethanol (add just before use).

[0058] Extraction steps:

[0059] 1. Take an appropriate amount of material (5g) and grind it fully in liquid nitrogen, transfer the ground powder into a pre-cooled 50ml centrifuge tube, add 20ml CTAB extraction buffer, vortex quickly, and lyse in a 65°C water bath for 45 minutes ;

[0060] 2. Add an equal volume of chloroform:isoamyl alcohol (24:1, v / v), invert and mix thoroughly, centrifuge at 8000g at room temperature for 20 minutes, transfer...

Embodiment 2

[0079] Embodiment 2, transient expression experiment detects promoter activity

[0080] 1. Construction of transient expression vector

[0081] The pAHC25 vector was incompletely digested with EcoR I, and the 6800bp fragment was selected from the product to recover, and the recovered fragment was added to DNA ligase for self-ligation. The ligation system: T4 DNA ligase 1 μl; 2×T4 DNA ligase buffer 2.5 μl; recovered fragment (6800bp) 1.5 μl. Transformation after ligation is transformed into a usable vector, denoted as vector A, which carries the GUS gene, and its plasmid map is as follows: image 3 Shown in A.

[0082] The carrier A was completely digested with BamH I and Hind III. It can be clearly seen from the vector map that the fragment sizes after digestion should be 2000bp and 4800bp, and the results of restriction electrophoresis showed that the sizes of the two fragments were just in line with it ( image 3 Middle B), indicating that the vector A was constructed suc...

Embodiment 3

[0117] Embodiment 3, promoter activity analysis result in transgenic rice

[0118] 1. Construction of transgenic vector

[0119] Obtain Pro-1Bx7 with embodiment 1 respectively OE Promoter sequence (sequence 1), Pro-1Bx7 promoter sequence (sequence 2) and Pro-1Bx13 promoter sequence (sequence 3) as templates, through primers 1Bx-PF2 and 1Bx-PR2 (see step 1 of Example 2 for the sequence) Perform PCR amplification.

[0120] The reaction system and reaction procedure are carried out with reference to Step 2 of Example 1.

[0121] After the reaction, each PCR product was double-digested with BamH I and Hind III, recovered from the gel, and connected to the large backbone fragment of the pCAMBIA1391Z vector (CAMBIA, Canberra, Australia) that had undergone the same double digestion to obtain a recombinant plasmid. Ligation system: T4 DNA ligase 1 μl; 2×T4 DNA ligase buffer 2.5 μl; pCAMBIA1391Z vector backbone fragment 1 μl; PCR product after digestion 0.5 μl.

[0122] The recombi...

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Abstract

The invention discloses an endosperm specific expression promoter. The endosperm specific expression promoter is any one of (1) a DNA (deoxyribonucleic acid) molecule as shown in a sequence l of a sequence table, (2) a DNA molecule which is hybridized with a definitive DNA sequence under a strict condition and has a promoter function, and (3) a DNA molecule which has more than 90% of homology with the DNA sequence defined in (1) or (2) and has a promoter function. The experiment proves that the endosperm specific expression promoter provided by the invention can be used for driving the specific expression of a target gene in plant endosperm. The invention provides a useful core component for the study on expression, adjustment and control of plant genes in grains and improvement of crops by using a genetic engineering method. The endosperm specific expression promoter has important significance for cultivation of new crop varieties through specific improvement and transformation of crop seeds.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to an endosperm-specific expression promoter. Background technique [0002] Among the multi-level levels of gene expression regulation, transcriptional regulation is a very cost-effective regulation method for plants. Various regulatory factors combine with promoters to turn on or turn off gene expression to achieve the purpose of regulation. The promoter is the most important regulation method in gene transcription. Because genes are controlled by different regulatory factors at different levels, this control mechanism not only determines the level of gene expression, but also determines the temporal and spatial order of gene expression. occupy a very important position. Promoters are important components of genetic engineering expression vectors, and the study of promoters is of great significance for the regulation mechanism of gene expression and the improvement of crop production tra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/84C12N1/21A01H5/00C12N15/11
Inventor 庞斌双耿玉珂李甜郝晨阳张学勇
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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