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A promoter regulating gene expression in non-secreting glandular trichomes and its application

A non-secretory glandular trichome and gene regulation technology, applied in the field of plant biology, can solve the problems of normal plant growth burden, excessive consumption of cell material and energy, time and space regulation of target genes, etc.

Active Publication Date: 2017-11-10
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the current genetic engineering research of Artemisia annua, constitutive promoters are often used, such as the cauliflower mosaic virus 35S promoter (CaMV35S), which can drive the expression of foreign genes in all tissues and organs, and will cause excessive consumption. The substance and energy in the cell, and the expression of the target gene cannot be regulated in time and space, which is likely to cause a certain burden and harm to the normal growth of plants

Method used

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  • A promoter regulating gene expression in non-secreting glandular trichomes and its application
  • A promoter regulating gene expression in non-secreting glandular trichomes and its application
  • A promoter regulating gene expression in non-secreting glandular trichomes and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Cloning of the Genome Sequence of the Promoter from the Artemisia annua Genome

[0031] 1. Cultivate aseptic vaccines of Artemisia annua:

[0032] Artemisia annua seeds were soaked in ethanol with a volume fraction of 75% for 1 min, then soaked in 20% (w / v) NaClO for 20 min, rinsed with sterile water for 3 to 4 times, blotted the surface moisture with sterile absorbent paper, and inoculated Cultivate on hormone-free MS solid medium at 25°C and 16h / 8h (sunlight / darkness) under light, after 14 days you can obtain sterile A. annua seedlings;

[0033] 2. Genomic DNA extraction

[0034] Put a leaf of Artemisia annua (1cm 2 About the size, take it in an ice box), add 2 steel balls. Add 300 μL TPS buffer (operate in a fume hood, TPS contains mercaptoethanol), shake at 55-60 Hz for 100 seconds. Then add 300 μL TPS buffer (fume hood). Water bath at 65°C for 1h (shake every 20min), and the time can be extended appropriately, up to 1.5h. Cool to room temperature and centrifu...

Embodiment 2

[0050] Analyze the cis-acting elements of the AaTPS7 gene promoter to determine the type of AaTPS7 gene promoter

[0051] The length of the AaTPS7 gene promoter sequence obtained in the present invention is 1167bp. In order to find the cis-acting elements on the promoter, the promoter of AaTPS7 gene was analyzed with Plantcare (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). The analysis found that besides TATA box and CAAT box, there are many cis-acting elements: CGTCA-motif, HSE, MBS, MRE, TC-rich repeats, WUN-motif, etc. on the promoter of polycloning.

[0052] The cis-acting elements in the promoter sequence are shown in Table 4:

[0053] Table 4: Analysis of regulatory elements in the promoter sequence

[0054]

[0055] In Table 4, the positions of -980, -907, -678 and -130 refer to the corresponding positions in the sequence listed in Example 1.

[0056] CGTCA-motif is a methyl jasmonate response element; HES responds to heat stress; MBS and MRE are M...

Embodiment 3

[0058] Connect the obtained promoter into the pCAMBIA-1391z vector and fuse the GUS reporter gene

[0059] In order to study the expression of the gene promoter in different plant parts, the promoter proTPS7 of the AaTPS7 gene was connected to the pCAMBIA-1391z vector and fused with the GUS reporter gene. site, the NcoI restriction site was introduced into the reverse primer, and the primer sequence is shown in the table below:

[0060] Table 5 PCR primers constructed by pCAMBIA1391z-proTPS7 vector

[0061]

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Abstract

The invention discloses a promoter for regulating gene expression in non-secretion glandular trichomes. The nucleotide sequence of the promoter is shown in SEQ ID No: 1. The invention also provides a preparation method of the promoter, a vector and an expression box containing the promoter. The promoter provided by the present invention can regulate the expression of the target gene in time and space, so as to further study the occurrence and development of non-secretory glandular hairs and the metabolic regulation of secondary metabolites therein, and can be used in the production of metabolite genes Application in engineering breeding. Therefore, the present invention is of great significance to genetic engineering breeding utilizing plant glandular hair tissue to express and produce metabolites.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to a terpene synthase gene promoter (proTPS7) specifically expressed in plant non-secretory glandular hairs and its application in transgenic plants. Background technique [0002] Artemisia annua (Artemisia annua L.) is an annual herb of the genus Artemisia in the Asteraceae family. The plant has a strong volatile aroma. The essential oil of Artemisia annua L. extracted from the glandular hairs of Artemisia annua contains a large number of terpenoids with pharmacological or biocidal activity. and flavonoids. There are two kinds of glandular hairs on the leaf surface of Artemisia annua: glandular secretorytrichome (GST) and non-secretory glandular hairs (T shape trichome, TST), two kinds of glandular hairs are important for plant growth, development, defense and pollen dispersal etc. play a vital role. Artemisinin is a sesquiterpene lactone compound containing a peroxide bridge s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/84A01H5/00
Inventor 唐克轩陈俏沈乾付雪晴石璞孙小芬颜廷祥
Owner SHANGHAI JIAOTONG UNIV