Method for genetic transformation of pineapple somatic embryogenic receptor kinase gene AcSERK1 and application thereof

A genetic transformation method and receptor-like kinase technology, applied in biochemical equipment and methods, applications, horticultural methods, etc., can solve the problem of low genetic transformation efficiency, few and long gene function research reports, and it takes at least 7 months To achieve the effects of improving the efficiency of genetic transformation, promoting the occurrence of somatic embryos, and promoting the formation of pineapple embryogenic cells

Inactive Publication Date: 2018-01-09
SOUTH CHINA AGRI UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Some researchers used callus and leaves as recipients to introduce exogenous genes into pineapple through Agrobacterium-mediated or gene gun (Firoozabady et al., 2006; Koet al., 2006; Sripaoraya et al., 2001; Zhou et al., 2001; Espinosa et al., 2002), but in general, the genetic transformation efficiency is not high (average 3%), and the time period for obtaining transgenic plants is longer, at least 7 months (Firoozabady et al. , 2006); some researchers have cloned bromelain gene and nifH gene (Sawano et al., 2002; Ando et al., 2005; Leon et al., 2002), but the current research on gene function from pineapple There are few reports, especially no genes related to pineapple somatic embryogenesis AcSERK1 Functional Verification Report

Method used

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  • Method for genetic transformation of pineapple somatic embryogenic receptor kinase gene AcSERK1 and application thereof
  • Method for genetic transformation of pineapple somatic embryogenic receptor kinase gene AcSERK1 and application thereof
  • Method for genetic transformation of pineapple somatic embryogenic receptor kinase gene AcSERK1 and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment

[0026] Embodiment adopts the following steps to realize pineapple somatic embryogenesis receptor kinase gene AcSERK1 genetic transformation of

[0027] 1. Construction basis of plant sense expression vector pFGC5941-35s-AcSERK1 AcSERK1 cDNA (GeneBank accession number: HM236375) sequence, designed with Nco I and Xba Gene-specific primers for I restriction sites (see Table 2) to contain AcSERK1 The full-length pMD19-T of the cDNA was used as a template, and the amplification contained AcSERK1 The target fragment of the ORF, and the PCR product was recovered with the Takara recovery kit. use Nco I and Xba I Simultaneously carry out double enzyme digestion on the recovered PCR product and pFGC-5941, recover them separately, perform ligation, transformation, and bacterial liquid PCR detection (see Table 2 for carrier-specific primers), positive clones are sent to Shanghai Sangong for sequencing and identification, and no migration Code mutation to obtain a recombin...

Embodiment 2

[0040] Example 2 Application of AcSERK1 gene: The following steps were used to verify the effect of the transfer of AcSERK1 gene on pineapple somatic embryogenesis.

[0041] 1. Molecular detection of transgenic plants PCR detection method: extract genomic DNA from leaves of transformed plants, DNA from leaves of non-transformed plants (wild type) is a negative control, pFGC5941- AcSERK1 Plasmid DNA was used as a positive control, and the primers were bar gene primers (see Table 2) and vector-specific primers (see Table 2). Prepare PCR amplification reaction solution (25 μL system): 10×PCR Buffer (Mg 2+ ) 2.5 μL, dNTP (2.5mM) 2 μL, P1 (2.5mM) 1 μL, P2 (2.5mM) 1 μL, Taq Enzyme 0.2 μL, Template DNA 1 μL, ddH 2 O was added to 25 μL. The reaction conditions were: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, and extension at 72°C for 10 min. PCR amplification products were detected by ...

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Abstract

The invention discloses a method for genetic transformation of pineapple somatic embryogenic receptor kinase gene AcSERK1 and application thereof. The genetic transformation method of the invention comprises the following steps: 1) construction of a sense expression vector of pFGC5941-35s-AcSERK1; 2) preparation of callus and callus tolerance test to PPT; 3) preparation of agrobacterium infectionsolution suspended by MS; 4) pre-culture, infection, co-culture and PPT screening culture; 5) PPT resistant seedling rooting and transplanting. Transgenic plants obtained by the genetic transformationmethod of the invention are subjected to PCR and Southern blot and tested by RT-PCR, confirming that the AcSERK1 had been inserted into pineapple genomic DNA and overexpression is occurred in pineapple somatic embryos. Functional validation shows that the number of embryogenic callus particle and somatic embryos obtained by callus which overexpresses the AcSERK1 after somatic embryo induction issignificantly higher than that of wild type, indicating that overexpression of the AcSERK1 has significant promotion effect on formation of pineapple embryogenic cells and somatic embryogenesis.

Description

technical field [0001] The invention relates to a genetic transformation method of pineapple somatic embryogenesis receptor kinase gene AcSERK1 and its application in pineapple somatic embryogenesis, belonging to the field of biotechnology. Background technique [0002] Pineapple( Ananascomosus ), also known as pineapple, is one of the four major tropical fruit trees in the world and is also the main tropical fruit tree in my country. Except that the fruit is edible, the plant can be used for viewing and the leaves can separate the fiber. There are more than 100 edible pineapple varieties around the world, which are divided into four categories: Caine, Queen, Spain and Puerto Rico. Pineapple is a self-incompatible plant. Not only is the same species incompatible, but even pollination among different species in the same category cannot produce seeds. After the fruit is harvested, it needs to suck buds for reproduction and renewal. Because each plant of most fine cultivars c...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/54A01H6/22A01H4/00
Inventor 许文天何业华高玉尧马均郭翠红陈程杰林文秋
Owner SOUTH CHINA AGRI UNIV
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