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Banana SSUII gene, cloning method, expression vector and application

A technology of gene expression and cloning method, which is applied in the field of regulation of banana carotenoid synthesis, and can solve problems such as lack of carotenoid content

Pending Publication Date: 2022-07-15
TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Banana (Musa spp.), a perennial large-scale evergreen monocotyledonous herbaceous plant belonging to the Musa family Musa, is one of the four major fruits in the world and is also the main food crop in many developing countries. It has high economic value and nutritional value, but the common Carotenoid content in banana varieties is relatively scarce

Method used

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  • Banana SSUII gene, cloning method, expression vector and application
  • Banana SSUII gene, cloning method, expression vector and application
  • Banana SSUII gene, cloning method, expression vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Cloning method of banana gene

[0045] 1. Extraction of total RNA:

[0046] (1) Dispense 1 mL of CTAB extract into 2 mL centrifuge tubes and preheat at 65°C;

[0047] (2) Weigh 0.2 g of the material, grind it in liquid nitrogen, and immediately transfer it to a centrifuge tube containing CTAB extract, vortex to mix, and incubate at 65°C for 2 to 3 minutes;

[0048] (3) Immediately add an equal volume of chloroform / isoamyl alcohol (24:1), vortex to mix, and centrifuge at 10,000 rpm for 15 min at room temperature;

[0049] (4) Aspirate the supernatant into a new centrifuge tube and repeat step (3);

[0050] (5) Take the supernatant into a new centrifuge tube, add 1 / 3 volume of 8M LiCl, and precipitate overnight at 4°C;

[0051] (6) 4 ℃, 10000rpm centrifugation for 30min;

[0052] (7) discard the supernatant, wash the precipitate with 70% ethanol, and then wash with absolute ethanol;

[0053] (8) Dissolve the precipitate in 100 μl STE (preheated at 65°C), and...

Embodiment 2

[0064] Example 2: Real-time fluorescence quantitative RT-PCR expression detection of banana SSUII gene in different genotypes of banana

[0065] RNA was extracted from AAA, BB and TTT banana leaves, and cDNA was reverse transcribed. The cDNA templates of each genotype were analyzed by real-time quantitative PCR using real-time fluorescence quantitative PCR. Specific primers were designed according to the principle of qPCR primer design, and the internal reference gene was banana Musa actin. The primer sequences are as follows:

[0066]

[0067] Using SYBR Green I as a fluorescent dye, the relative expression levels of SSUII gene in different genotypes of bananas were determined by qPCR, using 2 -ΔΔCt The relative expression of SSUII gene in each sample was calculated by the method. React in QuantStudio TM 3 Real-time PCR instrument (Thermo Fisher Scientific). The reaction system was 20 μL (template 2 μL, forward and reverse primers 0.4 μL each, 50×ROX Reference Dye2 dye...

Embodiment 3

[0068] Embodiment 3: Construction of banana SSUII gene plant expression vector pBI121 1. Design primers according to the nucleotide sequence of the isolated banana SSUII gene:

[0069] Forward primer:

[0070] 5'-CGGGGGACGAGCTCGGTACCCCCAATCGTCTTCTTTATCG-3'

[0071] Reverse primer:

[0072] 5'-ACCATGGTGTCGACTCTAGAACTCAGCCATCAGTCAACCT-3'

[0073] The polymerase chain reaction was performed using the 5'Race cDNA reverse transcribed from total RNA as a template.

[0074] 2. Take 5 μl of PCR product and ligate it with pEASY-Blunt vector. The operation steps are carried out according to the instructions of Trans's product pEASY-BluntCloningKit. Escherichia coli DH5α strain was then transformed, and the surface was coated with isopropylthio-β-D-galactoside (IPTG) and 5-bromo-4-chloro-3-indole-β-D-galactoside (X-gal) ) on LB plates containing ampicillin (100 μg / ml) overnight. Pick white colonies and grow overnight in LB liquid medium. Plasmid DNA was extracted by alkaline method...

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Abstract

The banana SSU-II gene is obtained by extracting total RNA (Ribonucleic Acid) of banana 'Musa spp.', synthesizing cDNA (Complementary Deoxyribonucleic Acid) through reverse transcription, designing a specific primer, obtaining full-length cDNA of the SSU-II gene by utilizing a PCR (Polymerase Chain Reaction) method, connecting the full-length cDNA with a pEASY-Blunt vector, transforming an escherichia coli DH5alpha competent cell and selecting positive clone, the full length of the cDNA of the gene is Mt21507 which is 1309bp, and the cDNA comprises an upstream sequence in front of an initiation codon and a poly-A tail. The expression levels of the SSUII genes of bananas with different genotypes are measured by utilizing real-time fluorescent quantitative PCR (Polymerase Chain Reaction), and the expression quantity of the SSUII genes in TTT type Karat bananas is proved to be higher than that of AAA type and BB type bananas. The SSUII gene is connected to a plant expression vector pBI121 to construct a new plant expression vector. The gene can influence the accumulation of the carotenoid content of banana fruits, and the SSUII gene has important theoretical and practical significance for researching the molecular mechanism of high carotenoid content of bananas, transforming low carotenoid bananas and providing a theoretical basis for high carotenoid content breeding of bananas.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the cloning of banana SSUII gene, analysis of expression characteristics, construction of an expression vector, and regulation of banana carotenoid synthesis. Background technique [0002] Carotenoids are ubiquitous in all higher plants. Because they are the precursors of vitamin A and antioxidants that can strongly scavenge singlet oxygen, they have become one of the most essential components for human nutrition and health. They are called provitamin A. Medical research has proved that carotenoids play an important role in protecting human health by quenching free radicals, enhancing human immunity, preventing cardiovascular diseases, and preventing cancer. When the intake of carotenoids in the human body is not up to the standard, it will cause corresponding diseases, such as vitamin A deficiency (VAD) and micronutrient deficiencies (MNDs). The World Health Organization (WHO) estima...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/82C12N15/10
CPCC12N9/1085C12N15/825C12N15/1003C12Y205/01029C12Q2527/125
Inventor 徐立王加宾李志英王小冰李春燕
Owner TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI
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