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Constitutive promoter PSCBV-CHN1 from sugarcane baculovirus and application thereof

A PSCBV-CHN1, promoter sequence technology, applied in the field of plant genetic engineering and plant genetic breeding, can solve the problems of narrow genetic background, laborious, limited genotype, etc.

Pending Publication Date: 2022-06-03
广东省科学院南繁种业研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The initial interspecific hybridization provided high-sugar and high-resistance varieties for establishing the basis of modern sugarcane hybrid breeding. However, due to the limited genotypes used in the noble breeding process, modern sugarcane cultivars generally have a narrow genetic background, which has become a limitation for sugarcane breeding. Major barriers to further increases in yield and resistance to biotic / abiotic stresses
The traditional sugarcane hybrid breeding method has been widely used in the genetic improvement of sugarcane varieties, but this method is time-consuming and laborious.

Method used

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  • Constitutive promoter PSCBV-CHN1 from sugarcane baculovirus and application thereof
  • Constitutive promoter PSCBV-CHN1 from sugarcane baculovirus and application thereof
  • Constitutive promoter PSCBV-CHN1 from sugarcane baculovirus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Cloning of SCBV-CHN1 Promoter Nucleotide Sequence

[0048] 1-1 Experimental Materials

[0049] Leaf samples of sugarcane cultivar ROC27 infected with SCBV-CHN1 were collected from Fujian, China. The leaves were collected from sugarcane + 1 leaf in the sugarcane field (the highest visible hypertrophy with leaves), brought back to the laboratory, cleaned and disinfected with 75% alcohol, placed in a ziplock bag, and stored in an ultra-low temperature refrigerator at -80°C.

[0050] 1-2 Extraction of total DNA from sugarcane leaves

[0051] The extraction method of total DNA from sugarcane leaves adopts the modified CTAB method (Sun et al., 2016). The absorbance and concentration of total DNA were measured by NanoVue ultra-micro spectrophotometer (GE Healthcare) protein nucleic acid analyzer, and the integrity of total DNA was detected by electrophoresis.

[0052] 1-3 SCBV-CHN1 Genome Cloning

[0053] According to the two SCBV genome sequences published in th...

Embodiment 2

[0056] Example 2 P SCBV-CHN1 Construction of promoter plant recombinant expression vector

[0057] 2-1 Construction of EYFP expression vector

[0058] Preparation of plant expression vector backbone: using fast restriction endonucleases XhoI and NcoI (Fermentas, USA) to CaMV 35S : The EYFP vector was digested, and the 25 μL double-enzyme digestion reaction system contained 2.5 μL 10×FastDigest Buffer, 0.5 μL XhoI, 0.5 μL NcoI and 1 μg target plasmid. After 30 min in a water bath at 37°C, electrophoresis was performed on a 1% agarose gel to recover a large fragment, ie, the target vector backbone, and stored in a -20°C refrigerator for later use.

[0059] PCR amplification: Design the amplification promoter P by the seamless cloning primer design tool (http: / / 123.56.75.195 / ) SCBV-CHN1 Sequences of primers IF-EYFP-CHN1-F: 5'-CGGGCCCCCCCTCGAGAAGAACCAACTCTGCTATGTGCATG-3' (SEQ ID NO: 6) and IF-EYFP-CHN1-R: 5'-CCCTTGCTCACCATGGCAAAGAGCTCAAATGATCAGCTG-3' (SEQ ID NO: 7). pass The...

Embodiment 3

[0063] Example 3 P SCBV-CHN1 : Transient expression of EYFP plasmid in onion epidermal cells, Arabidopsis protoplasts and sugarcane young leaf tissue

[0064] 3-1 Preparation of onion scale epidermis and plasmid transformation

[0065] Wash the onion with sterile water, soak it in 75% alcohol for 1 min; remove the top and base of the onion and cut the two outer layers of onion scales; cut the onion in half four times, take the outer three layers, tear off the inner skin of the onion scales, and cut into about 2cm 2 Square sheet, place its smooth side up and inner surface down in MS osmotic medium (4.4g / L MS medium base salt (with vitamins), 36.4g / L mannitol, 0.6mg / L 2,4 -D, 6g / L agar powder, pH 5.8~6.2). The osmotic medium on which the onion scale epidermis was placed was placed in a dark place and incubated at 28°C for 4 h for use. put P SCBV-CHN1 : The EYFP plasmid is coated in tungsten powder (1.1 μm), the specific operation steps refer to Gao et al. (2013), the bombar...

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Abstract

The invention provides a plant constitutive promoter from sugarcane baculovirus and application of the plant constitutive promoter. The promoter has a nucleotide sequence as shown in SEQ ID NO: 1. By detecting the expression conditions of exogenous EYFP and GUS genes in transgenic onion, transgenic sugarcane and transgenic arabidopsis thaliana, it is proved that the promoter is a constitutive promoter and can promote efficient expression of exogenous genes in plant roots, stems and leaves. Therefore, the promoter can be used for preparing transgenic plants and carrying out plant transgenic breeding, can be used as an element for constructing a plant recombinant expression vector, and has a wide application prospect in the aspects of changing plant resistance characters and plant transgenic breeding.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering and plant genetic breeding, and in particular relates to a constitutive promoter P from sugarcane baculovirus SCBV-CHN1 and applications. Background technique [0002] Plant genetic engineering technology is to introduce exogenous genes into recipient plant cells and make them express correctly and effectively, so as to achieve the purpose of changing plant resistance traits or rapidly cultivating new plant varieties. Plant genetic engineering technology has great potential in cultivating high-quality new plant varieties. However, this process is restricted by a variety of influencing factors, and the promoter sequence is the key to the effective expression of foreign genes. [0003] Due to the sessile nature of plants, external biotic and abiotic stresses can induce various immune responses in plants. The transcription of resistance-related genes and the expression of later pro...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/82C12N15/84A01H5/00A01H6/20A01H6/56A01H6/46
CPCC07K14/005C12N15/8222C12N15/8212C12N2710/14022Y02A40/146
Inventor 王竹青王勤南孙生仁常海龙吴建涛王建强陈俊吕
Owner 广东省科学院南繁种业研究所
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