Plant leaf specific expression promoter and application thereof

A plant leaf and promoter technology, applied in applications, plant peptides, plant products, etc., can solve the problems of plant metabolic imbalance, death, hindering the normal growth and development of plants, etc., to achieve broad application prospects, relieve concerns, and improve photosynthesis characteristics Effect

Inactive Publication Date: 2018-03-20
XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main purpose of the present invention is to solve the existing plant genetic engineering technology, due to the use of constitutive promoters to make foreign genes overexpress heterologous proteins or metabolites in transgenic plants, so that the original metabolism of plants is unbalanced, hindering plant normal growth and development, and even death

Method used

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  • Plant leaf specific expression promoter and application thereof
  • Plant leaf specific expression promoter and application thereof
  • Plant leaf specific expression promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Extracting Upland Cotton Coker 312 Genomic DNA

[0050] The seeds of upland cotton Coker 312 were sown in a nutrient pot filled with vermiculite mixed with nutrient soil, and placed in an RXZ intelligent artificial climate incubator for cultivation. After the seeds germinated, they were cultured in Hoagland nutrient solution, at 28°C during the day, 18°C ​​at night, 16h / d light, light intensity 30001x, and relative humidity 75; the young leaves of cotton seedlings that had grown for 2 weeks were taken, and a small amount of cotton genomic DNA was extracted according to the improved CTAB method , measure the absorbance value of DNA at 260nm and 280nm with a UV spectrophotometer, calculate the DNA concentration and purity, and the resulting DNA concentration is OD 260 / OD 280 = 1.865, the purity was 850 ng / μl.

Embodiment 2

[0051] Example 2 Cloning of the 5' flanking sequence of the Ghlsp promoter of the cotton Ghi.10424 gene.

[0052]The 5' flanking sequence of the cotton Ghlsp promoter was cloned by Genome walking method, and the specific operation steps were referred to the instructions of Genome Walking Kit from TaKaRa Company, with slight changes according to specific conditions. Using the upland cotton gene expression profile chip as a reference, a leaf-specifically expressed gene Ghi.10424 was preliminarily screened out, and the gene fragment size was as follows figure 1 shown. The expressed sequence tag ESTs sequences were compared by Blast on NCBI, and the UNIGENE database was searched for clustering and electronic splicing of similar sequences to obtain a complete cDNA sequence. According to the largest open reading frame (ORF) of the gene, primers 10424-F and 10424-R (Table 1) were designed to amplify the full length of the gene.

[0053] Using genomic DNA as a template, use AP1, AP2...

Embodiment 3

[0055] The acquisition of embodiment 3 cotton GhLsp promoter

[0056] Specific primers were designed according to the sequence obtained by Genome walking method: GhLsp-F, GhLsp-R. Using upland cotton Coker 312 genomic DNA as a template, using GhLsp-F and GhLsp-R as primers to amplify the target gene, and through 1% agarose gel electrophoresis detection, the results show that there is a single specific band at about 2 kb ( figure 2 ), which is consistent with the expected theoretical value. The PCR product was recovered with a DNA gel recovery kit, and the purified product was connected to the pMD18-T vector, transformed into E.coliDH5α competent cells, screened with ampicillin and blue and white spots, and the recombinant plasmid was selected for PCR and enzyme digestion identification ( image 3 ) were all positive clones were sent to Shanghai Sangon for sequencing. The analysis of the sequencing results showed that the length of the fragment was 2kb, and the known sequenc...

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Abstract

The present invention relates to a plant leaf specific expression promoter and application thereof. An upstream promoter of a highly-active cotton leaf specific expression gene screened by gene microarray data and RT-PCR is cloned on the basis of upland cotton Goker Coker 312 genomic DNA as a template to obtain a DNA fragment of about 2kb at the upstream of a translation initiation site. Pant expression vector pGhlsp::GUS for the promoter to drive GUS is constructed, arabidopsis thaliana is transformed by agrobacterium floral dip method, the arabidopsis thaliana is observed by GUS histochemical staining, results show that the gene is mainly specifically expressed in leaves, and hardly expressed in roots and stems, and the plant leaf specific expression promoter is a new leaf specific expression promoter.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a cotton leaf-specific expression promoter Ghlsp promoter and its application in transgenic plants. Background technique [0002] Cotton belongs to the Malvaceae family and is one of the most important fiber crops in the world. It is also an important economic crop in my country and plays an important role in the national economy and people's lives. At present, the use of traditional breeding, improved cultivation and other methods to increase cotton production has fallen into a bottleneck. Therefore, using genetic engineering to obtain high-quality, high-yield and stable-yield cotton is an urgent task in our country. During the growth and development of cotton, gene expression is regulated and controlled by various physical and chemical factors, and the required gene products need to be expressed in a timely, quantitative and localized manner according to its own needs and environme...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82C12N5/10A01H5/00A01H6/60
CPCC07K14/415C12N15/8225
Inventor 司爱君
Owner XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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