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Identification and applications of plant pulvinus specific expression promoter ProCol1

A technology for plant expression vectors and plant leaves, applied to plant leaf pillow-specific expression promoters, plant expression vectors, the application of the above-mentioned promoters in plant transgenic engineering, can solve the problem of worrying about the safety of transgenic plants, and it is difficult to achieve orientation Improve plant traits, waste of plant growth resources, etc., to achieve the effect of improving crop agricultural traits, increasing plant density, and increasing photosynthetic efficiency

Inactive Publication Date: 2014-04-02
RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In plant transgenic engineering, most of them use constitutive promoters. Since such promoters can promote gene expression in all tissues, they are persistent and do not show spatiotemporal specificity, so it is difficult to achieve the purpose of directional transformation of plant traits. , and the constitutive promoter is continuously expressed in time and space, which also causes a waste of plant growth resources
In terms of biological transgenic safety, the high expression level, strong priming effect, indiscriminate selection of biological species and tissues, and the unsafe source of constitutive promoters make transgenic organisms using such promoters prone to gene drift. , The source of the excessive expression of exogenous genes, making people very worried about the safety of transgenic plants

Method used

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  • Identification and applications of plant pulvinus specific expression promoter ProCol1
  • Identification and applications of plant pulvinus specific expression promoter ProCol1
  • Identification and applications of plant pulvinus specific expression promoter ProCol1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, containing enzyme cutting site ProCol1 Acquisition of the promoter

[0063] Step 1. Design of primers

[0064] According to the whole genome sequence of rice variety Nipponbare (Oryza sativa L cv. Nipponbare) provided by NCBI, the amplification primers were designed according to the upstream 2100bp sequence of the rice gene (LOC_Os08g07760.1), and the primers were designed according to the selected vector and the characteristics of the target gene In this example, the rice binary expression vector pCAMBIA1381 (from CAMBIA, a publicly available vector, preserved by the Rice Group of the Supervision, Inspection and Testing Center for Components of GMO Products, Ministry of Agriculture, Anhui Academy of Agricultural Sciences) was used as an example in this example. Because of the GUS gene, the specifically designed primers are: the forward primer has an EcoRI restriction site at the 5' end (GAATTC), and the reverse primer has a SalI restriction site at t...

Embodiment 2

[0072] Embodiment 2, the construction of plant expression vector and the transformation of Agrobacterium

[0073] Plasmids were extracted from the TA clone obtained in Example 1, double-digested with EcoRI and SalI, and the promoter was recovered ProCol1 fragment. Recover the EcoRI and SalI fragments of pCAMBIA1381 at the same time, and connect the above two fragments with T4 ligase (purchased from TaKaRa Company) to obtain the promoter ProCol1 and gus Gene fusion plant expression vector pCAMBIA1381- ProCol1 ( figure 1 ), using the freeze-thaw method to transfer the expression vector into Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) EHA105 (preserved by the Rice Group of the Supervision, Inspection and Testing Center for GMO Product Components, Ministry of Agriculture, Anhui Academy of Agricultural Sciences), extract the positive plasmid, perform enzyme digestion verification with EcoRI and SalI, and clone it for use.

[0074] Contrast: turn CaM...

Embodiment 3

[0075] Embodiment 3, utilize promoter ProCol1 Driving GUS reporter gene expression in rice

[0076] Step 1: Agrobacterium-mediated genetic transformation of rice

[0077] After removing the glumes of mature seeds, soak the seeds in 70% alcohol for 1 min, pour off the alcohol, and soak the seeds in 50% sodium hypochlorite (the concentration of available chlorine in the original solution is greater than 4%) containing 1 drop of Tween 20 for 40 min (150 r / min). Then wash the seeds with sterilized water until the solution is clear without the taste of sodium hypochlorite, and soak the seeds in sterilized water overnight. The embryos were peeled off along the aleurone layer of the seeds with a scalpel, and the embryos were inoculated on callus induction medium. After 11 days of dark culture at 30°C, the callus was separated from the endosperm and germ, and the primary callus that was in a good debudding state and vigorously divided was pre-cultured for 3-5 days and then use...

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Abstract

The invention discloses identification and applications of a plant pulvinus specific expression promoter ProCol1, relates to the field of plant gene engineering, in particular relates to a rice pulvinus specific expression promoter including a plant expression carrier of the promoter and a converter, and also relates to applications of the promoter in plant transgenic engineering. A plant expression carrier containing the promoter DNA (Deoxyribose Nucleic Acid) sequence and a GUS (glucuronidase) gene is subjected to a genetic transformation method of agrobacterium tumefaciens mediated transformation by the inventor to obtain transgenic plants; through staining identification, the GUS gene only has specific expression at the rice pulvinus part, thereby speculating that the promoter provided by the invention can drive outer source genes to be specifically expressed in plant pulvinus; and the plant pulvinus specific expression promoter ProCol1 can be applied to directional improvement of crop agricultural characteristics, can improve the crop yield, can culture efficient and high-safety transgenic novel varieties of plants, is convenient to use, and is environment-friendly.

Description

technical field [0001] The present invention relates to the field of plant genetic engineering, in particular to a plant leaf pillow-specific expression promoter, a plant expression vector containing the promoter, and a transformant of the promoter. The present invention also relates to the use of the above-mentioned promoter in plant transgenic engineering Applications. Background technique [0002] A promoter is a DNA sequence that RNA polymerase can recognize and bind to, thereby initiating gene transcription, usually located upstream of the gene. A typical promoter includes CAAT-box and TATA-box, which are the recognition and binding sites of DNA-dependent RNA polymerase, and are generally located dozens of bases upstream of the transcription start site, upstream of the core promoter There are usually some special DNA sequences, namely cis-acting elements, which transcription factors bind to activate or inhibit the transcription of genes. Once RNA polymerase is located...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/84C12N1/21C12N5/10A01H5/00
Inventor 杨剑波魏鹏程李娟张银萍李浩宋丰顺倪大虎李莉杨亚春
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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