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Inducible promoter separated from leaf mustard

A promoter and sequence technology, applied in the field of genetic engineering, can solve problems such as weak expression activity, increased plant metabolic burden, and toxicity

Inactive Publication Date: 2006-09-13
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

In most cases, people do not want exogenous genes to be widely expressed in the entire plant and throughout the growth period of transgenic plants, because on the one hand, it will increase the metabolic burden of the plants, on the other hand, some exogenous proteins are not essential or even toxic to plants, so it is not necessary It is beneficial to the normal growth of plants; in addition, in some plant organs, the expression activity of constitutive promoters is not strong (Ren Maozhi, etc., a promoter function analysis of a dominantly expressed gene in cotton reproductive organs, Chinese Science Series C Life Science, 2005, 35( 1): 22-28)

Method used

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  • Inducible promoter separated from leaf mustard
  • Inducible promoter separated from leaf mustard

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Isolation of the BjCHI1 gene promoter (BjC-p) from the mustard genome by linker PCR

[0030] 1.1 Preparation of mustard genomic DNA

[0031] For mustard greens (Brassica juncea) planted indoors for one month, take about 3 grams of young leaves, refer to the method of McCouch et al. (McCouch S.P., et al., Molecular mapping of rice chromosome, Theor Appl Genet, 1988, 76: 815-829) Genomic DNA was extracted by SDS method. The extracted genomic DNA of mustard was detected by agarose gel electrophoresis, and the concentration was measured by an ultraviolet spectrophotometer and stored at -20°C.

[0032] 1.2 Enzyme digestion and adapter ligation of mustard genomic DNA

[0033] Referring to the method of Siebert et al. (Siebert P.D., et al., An improved PCR method for walking inuncloned genomic DNA, Nucleic Acids Research, 1995, 23 (6): 1087-1088), take 2 micrograms of mustard genomic DNA, and use restriction The endonucleases EcoRV, Dra I and Sma I were completely...

Embodiment 2

[0038] Example 2 Verification of promoter function

[0039] It is carried out by connecting the promoter with the reporter gene, and then transforming it into plants to detect the expression of the reporter gene.

[0040] 2.1 Construction of recombinant expression vector pBICHP0

[0041] pBICHPO was constructed by replacing the 35S promoter sequence upstream of the GUS gene in the expression vector pBI121 with the BjC-p promoter (92nd to 1378th nucleotides shown in SEQ ID NO: 1) sequence. The plant binary expression vector pBI121 as expression vector contains neomycin phosphotransferase gene (Npt II) and β-glucuronidase (GUS) gene as reporter gene. Obtain the BjC-p promoter sequence (92nd to 1378th nucleotides shown in SEQ ID NO: 1) from Brassica juncea genome by PCR method, the used PCR primer pair:

[0042] KjXeB11: 5'-TAA AGC TTT CTC AAA TAT GAG GTT TTA CTA TTC AT-3' (SEQ ID NO: 8)

[0043] KjWXF7: 5'-TAG GAT CCG TTT CTC TGA GCT GTA TGG TTG-3' (SEQ ID NO: 9)

[0044] Sinc...

Embodiment 3

[0051] The promoter activity analysis of embodiment 3 truncated

[0052] Researchers believe that there is an active region in the above-mentioned full-length promoter, so the full-length promoter shown by SEQ ID NO: 1 (nucleotides 92 to 1378 shown in SEQ ID NO: 1) has been truncated, The corresponding truncated promoters were obtained to analyze their activity.

[0053] 3.1 Construction of recombinant expression vector

[0054] Using the pBICHP0 plasmid as a template, PCR amplification was performed using the corresponding primer pairs to obtain three truncated promoter sequences, and the following expression vectors were constructed:

[0055] 1) The 302nd to 1378th nucleotides shown in SEQ ID NO: 1 are used as a promoter, amplified by primers KjXeB1 and KjWXF7, and the constructed vector is called pBICHP1;

[0056] 2) The 456th to 1378th nucleotides shown in SEQ ID NO: 1 are used as a promoter, amplified by primers KjXeB2 and KjWXF7, and the constructed vector is called pB...

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Abstract

The invention discloses a MeJA inducement promoter separated from mustard that could construct recombination expression carrier using the connecting of the external source gene. When the external gene is anti-insect gene, the transformed plant would be gained.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a plant promoter. In particular, it concerns a damaged, methyl jasmonate (MeJA)-inducible promoter isolated from Brassica juncea. Background technique [0002] Because plants cannot move autonomously, they can only adapt to the environment passively. After long-term evolution, plants have established a set of protection mechanisms to resist unfavorable factors such as pests and diseases, such as forming physical barriers such as thick cell walls; and quickly start the expression of some genes to resist the attacks of diseases and pests , such as chitinase gene, protease inhibitor gene, and disease process protein gene. It has been found that hormones in plants, such as methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA), etc., change significantly when pests and diseases occur. It is speculated that these hormones play a role in the process of plant resistance to p...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82
Inventor 赵开军吴雪峰谢恩倍王春连
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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