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A key gene for peanut vitamin E synthesis and its application

A key gene and vitamin technology, applied in the key gene and application field of peanut vitamin E synthesis, can solve the problems of nutrient destruction, peculiar smell, prone to rancidity, etc., and achieve the effect of extending shelf life, improving content and activity

Inactive Publication Date: 2017-07-07
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Peanut oil is one of the main edible oils for Chinese residents, but the content of unsaturated fatty acids in peanut oil is high, and it is prone to rancidity under the action of air, light, high temperature and metal ions, the nutrients are destroyed, and there are peculiar smells

Method used

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  • A key gene for peanut vitamin E synthesis and its application
  • A key gene for peanut vitamin E synthesis and its application
  • A key gene for peanut vitamin E synthesis and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] [Example 1] Cloning by electronic cloning technology AhCYP707A gene open reading frame

[0021]According to the candidate gene fragments obtained from the transcriptome data of peanut 454 sequencing, primers AhHPPD-F (5'-TCCACGAGTTCGCTGAGTTC-3') and AhHPPD-R (5'-CAAGTGCTGCAACCCTGCACCTTCGTTG -3') were designed to verify the mixing of Minhua 6 tissues Whether there is the gene fragment in the full-length cDNA library, and then combined with LibF-45 (5'-GATTCTGTGGATAACCGTATTACCGCCTTACGCGTGTAAAACGAC-3') and LibR-39 (5'-ACCAGGATCTCCTAGGGAAACAGCTATGACCATGTTCAC-3') primers designed according to the library vector, RACE reaction is carried out. RACE reaction conditions: 5′ RACE reaction with AhHPPD-R and LibF-45 primers, PCR amplification conditions: 94°C for 5min; 94°C for 30s, 70°C for 2min, 10 cycles; 94°C for 30s, 66°C for 30s, 72°C 2min at ℃, 25 cycles; 10min at 72℃. Use AhHPPD-F and LibR-39 as primers for 3′ RACE reaction, PCR amplification conditions: 94°C 5min; 94°C ...

Embodiment 2

[0024] [Example 2] Extraction of Total RNA from Peanut Embryos in Different Tissues and Different Development Stages

[0025] Taking Minhua No.6, an excellent peanut variety bred by Fujian Agriculture and Forestry University, as the material, 8 tissues of roots, stems, leaves, flowers, fruit needles, pericarp, seed coats, and embryos of different developmental stages were collected, as well as the different developmental stages. Embryos are fresh embryos taken 20 days, 40 days, and 60 days after the fruit needles were buried in the soil, and stored at -80°C for later use. Preheat 1.7mL CTAB extract (65°C) and 80µl mercaptoethanol to 65°C; add the ground material, vortex for 2min, incubate at 65°C for 15-30min, invert and mix 3-4 times, add 1 / 10 Volume of absolute ethanol, 1 / 10 volume of 3MKAc, equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), invert and mix at room temperature for 10 min. Centrifuge at 12000r / min for 15min at 4°C; take the supernatant, add an equal...

Embodiment 3

[0026] [Example 3] qRT-PCR analysis AhHPPD expression pattern

[0027] Total RNA was extracted from different peanut tissues and embryos at different developmental stages by the above-mentioned modified CTAB method, and 1 μg was reverse-transcribed with PrimeScript™ Reverse Transcriptase reverse transcriptase (purchased from TAKARA Company). After diluting the single-stranded cDNA 10 times, take 2 µL as a template, and operate according to the SYBR® Premix Ex Taq™ kit protocol of TAKARA Company, using Mastercylcer ep realplex (Eppendorf) with specific primers AhHPPD_RT_F (5'-AGAAGAACTGCATAATGGGATTCGGTC-3'); AhHPPD_RT_R ( 5'-GAAAGGAGCATTGCCCAAGTGACTGTG-3'); Ahactin F (5'-GAGGAGAAGCAGAAGCAAGTTG-3'), Ahactin R (5'-AGACAGCATATCGGCACTCATC-3') for qRT-PCR. The expression of the gene in the sample was analyzed according to the ΔΔCT method. Analysis by qRT-PCR AhHPPD See Figure 4 for the relative expression levels of mRNA in each peanut tissue and three stages of embryo developme...

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Abstract

The invention relates to a peanut vitamin E synthesis key gene and its application. Our laboratory designs primers based on analysis results of peanut transcriptome data, and adopts improved RACE technology to clone and isolate the cDNA sequence of peanut HPPD gene. The overexpression vector p35S::AhHPPD was constructed, and the model plant tobacco was transformed. The content of α-tocopherol in the transgenic tobacco and wild-type tobacco was analyzed by RP-HPLC. The results showed that the content of α-tocopherol in the transgenic tobacco increased by 2-3 times. It shows that AhHPPD cloned from peanut can encode functional p-hydroxyphenylpyruvate dioxygenase; the activity of AhHPPD can improve the important role of tocopherol such as α-tocopherol synthesis. This will help increase the content of vitamin E in peanuts, prolong the storage period and shelf life of oils and fats, and is of great significance.

Description

technical field [0001] The invention relates to a peanut vitamin E synthesis key gene and its application, belonging to the field of plant genetic engineering. Background technique [0002] Vitamin E (Vitamin E) is a kind of fat-soluble antioxidant that is only synthesized in photosynthetic organisms and plays an important role in animals and plants. Vitamin E can enhance the ability of plants to resist adversity stress, improve the stability of oil and prolong the storage period of oil seeds. It can also maintain the normal metabolism of the human body, reduce the occurrence of cancer, inhibit cardiovascular and cerebrovascular diseases, and improve the body's immunity. Vitamin E is also widely used in the fields of food, medicine, health care products and cosmetics. [0003] Vitamin E consists of a polar aromatic head and a non-polar isoprenoid side chain tail. According to the saturation of the side chain, it is divided into two types: tocopherol and tocotrienol. The po...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/84A01H5/00
Inventor 庄伟建陈华张冲邓烨蔡铁城张福婷
Owner FUJIAN AGRI & FORESTRY UNIV
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