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A method for isolating and regulating genes of peanut embryo development

A separation method and embryo development technology, applied in the direction of DNA preparation, DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of unclear molecular mechanism and achieve the effect of simple method

Inactive Publication Date: 2016-11-30
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the molecular mechanism of peanut embryo development, especially the molecular mechanism of peanut embryo abortion caused by calcium deficiency has not yet been clarified

Method used

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  • A method for isolating and regulating genes of peanut embryo development
  • A method for isolating and regulating genes of peanut embryo development
  • A method for isolating and regulating genes of peanut embryo development

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] [Example 1] Extraction of Total RNA in Early Embryos of Peanut Deficiency and Sufficiency Calcium

[0023] The fine peanut variety Minhua No. 6 bred by Fujian Agriculture and Forestry University was used as the material, and a typical calcium-deficient peanut empty pod producing area was selected in Pingtan County, Fujian Province. The exchangeable calcium content in the soil was 49.54 ug / kg. For calcium deficiency treatment, on this basis, 75kg of lime is applied per mu, and the exchangeable calcium content in the soil reaches 129.61 mg / kg (the exchangeable calcium content in the soil that causes calcium deficiency in peanuts is 100mg / kg), which is used as sufficient calcium. deal with. Under the conditions of calcium deficiency and sufficient calcium, the young fruits of peanuts on the 6th, 9th, and 15th days after the needles were inserted into the soil were removed, and the immature embryos were quickly thrown into liquid nitrogen and stored at -80°C for later use. ...

Embodiment 2

[0024] [Example 2] Construction of suppression subtractive hybridization library

[0025] The suppression subtractive hybridization library of peanut early embryos under calcium deficiency and sufficient conditions was constructed according to the PCR-SelectTM cDNASubtraction Kit (BD Biosciences-Clontech, Palo Alto, CA). When the early peanut embryos under the calcium deficiency condition were the test group, the peanut early embryos under the sufficient calcium condition were the driving group; on the contrary, when the peanut early embryos under the calcium deficiency condition were the detection group, the early peanut embryos under the calcium deficiency condition were the driving group. Take 1 μg of mRNA from the detection group and the driver group respectively and carry out reverse transcription to synthesize cDNA according to the M-MLV RTase cDNA Synthesis Kit (Takara, Japan). Rsa Ⅰ Digest at 37°C for 1.5 h, then ligate linker 1 (5'-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCC...

Embodiment 3

[0026] [Example 3] Calcium Deficiency Induced Early Peanut Embryo Suppression Subtractive Hybridization Library Screening

[0027] 2 μg of depleted double-stranded cDNA of peanut early embryos under calcium-deficient and sufficient conditions were taken respectively, and probe-labeled with DIG-dUTP (Roche) according to DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). Take 1 μl of the amplified library stored at -70°C and dilute to 10 with LB liquid medium 3 , 10 4 , 10 5 , take 1 μl respectively, add 100 μl liquid LB, smear on the LB solid medium plate containing 100 μg / ml ampicillin, after culturing overnight, calculate the number of grown clones. Spread the Hybond-N+ nylon membrane (Amersham) on the LB plate containing 100 μg / ml ampicillin, and try to avoid generating air bubbles. According to the titer detection results, take an appropriate amount of library bacteria solution according to 2000 clones per plate and evenly smear the plate, dry it on the ul...

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Abstract

The invention relates to a method for isolating and regulating peanut embryo development genes, which belongs to the field of functional genomics. AhHsfa4A The gene sequence is shown as SEQ ID NO: 1, which will be screened to obtain the closely related important genes that are induced by calcium to cause programmed death of peanut early germ cells AhHsfa4A , and obtained from the constructed whole-tissue full-length library by improved RACE technology AhHsfa4A The full sequence of the gene. The present invention establishes a method (SSHaLL) for efficiently screening important genes of peanut early embryos induced by calcium deficiency, and uses this method to screen an important gene that causes programmed death of early peanut embryos AhHsfa4A And its expression was identified. The invention has laid a good foundation for efficient screening of important differentially expressed genes in peanut embryo development, thereby further clarifying the molecular mechanism of peanut embryo development.

Description

technical field [0001] The invention relates to a method for isolating and regulating peanut embryo development genes, belonging to the field of functional genomics. Background technique [0002] Plant growth, development, flowering, fruiting, senescence and death are accompanied by complex physiological and biochemical changes, which are the result of orderly expression and regulation of genes in plants in time and space. The differential expression of genes is closely related to the biological traits and functions of tissues and cells, and has become an important research topic in life sciences. Comparing the differences in gene expression between different cells or different genotypes is not only the basis for studying the molecular mechanism of life processes, but also the prerequisite for isolating and cloning target genes. Separation of differentially expressed genes is of great significance for understanding and revealing the growth and development rules of plants, a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 庄伟建陈华张冲蔡铁城周双彪邓烨
Owner FUJIAN AGRI & FORESTRY UNIV
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