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Apricot cold-resist gene transformation method

A gene transformation and gene technology, applied in the field of biotechnology and modern agriculture, can solve the problems that the transgene is limited by the genetic transformation system, and it is difficult to genetically transform and screen multi-genotype receptors, so as to enrich new germplasm resources and improve The effect of cold resistance properties

Inactive Publication Date: 2014-01-22
TAISHAN RES INST OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the Agrobacterium-mediated, gene gun and other methods used in transgenics mainly realize gene introduction through the establishment of tissue culture systems. Difficult to achieve genetic transformation and screening of multi-genotype receptors

Method used

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  • Apricot cold-resist gene transformation method
  • Apricot cold-resist gene transformation method
  • Apricot cold-resist gene transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Molecular detection of transgenic apricot tissue culture seedlings:

[0038] Take about 0.3 g of young apricot leaf tissue, extract plant DNA by CTAB method, dissolve in TE solution, and store at -20°C. The extracted transgenic apricot leaf DNA was used as a template, the plasmid DNA was used as a positive control, and the untransformed apricot leaf DNA was used as a negative control for PCR amplification detection.

[0039] The target gene primer sequence is:

[0040] Primer 1: 5′-ATGTCGGATGATGAAAGGGAGGAGAAGGA-3′,

[0041] Primer 2: 5'-TAGGGAAGGGAGGTCAATCTTGAGGTGT-3'.

[0042] The reaction system is: 10×Taq Buffer (Mg2+) 2.0ul, dNTP1.0ul, primer 0.8ul, template 1.0ul, TaqDNA polymerase 0.3ul, ddH 2 O15.1ul amplification program is: pre-denaturation at 94°C, 4min; denaturation at 94°C, 45s; annealing at 55°C, 45s; extension at 72°C, 1min, final extension at 72°C, 5min; 1.0% agar for 35 cycles of amplification products Glucose gel electrophoresis, EB staining observa...

Embodiment 2

[0045] Effect of different exogenous DNA concentrations on gene transformation rate:

[0046] PCR molecular detection was carried out on the 7th, 30th, and 60th days after the transformation, respectively, and 10 fruits were randomly selected each time. The DNA of the whole young fruit was detected and extracted on the 7th day, and the DNA of the seeds was extracted after the 30th day. After the detection, the positive rate was counted, and the results are shown in Table 2. The introduction effect of the pollen mixed with the exogenous gene was significantly higher than that of the bombarded pollen by the gene gun.

[0047] Table 2 The impact of different processing methods on the gene conversion rate

[0048]

[0049] Depend on Figure 4 It can be seen that when the concentration of imported DNA is 500 μg / mL, the positive rate of fruit setting is the highest, reaching 75% at the 7th day, and 22.5% after 60 days. The DNA concentration had the least effect on the growth of...

Embodiment 3

[0051] The influence of different ways to deal with the stigma on the gene conversion rate:

[0052] After the pollen was pollinated in different ways, the stigma was subjected to three treatments of excising 0, 1 / 2, and completely excision, and the AmEBP1 plasmid DNA was introduced at a concentration of 500 μg / mL. For the detection, 10 fruits were randomly selected each time, the DNA of the whole young fruit was detected and extracted on the 7th day, and the DNA of the seeds was extracted after the 30th day, and the positive rate was counted after PCR detection. The results are shown in Table 3.

[0053] It can be seen from Table 4 that when the concentration of introduced DNA is 500 μg / mL, the positive rate of fruit setting after cutting off 1 / 2 style is the highest, reaching 75% at 7 days, and 30% after 60 days, indicating that cutting 1 / 2 style, transgenic The highest success rate. After cutting off 1 / 2 style, the distance of pollen entering the embryo sac is shortened, a...

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Abstract

The invention relates to an apricot cold-resist gene transformation method, and belongs to the technical field of biotechnology and modern agriculture. According to the apricot cold-resist gene transformation method, a pollen tube is adopted to introduce and transform apricot cold-resist genes and the method comprises the steps of (1) extracting plasmid DNA, (2) collecting and processing pollen to be pollinated, (3) conducting cross pollination on receptors by the utilization of the pollens containing the target genes, (4) obtaining and conducting propagation expanding on transgenic plants and (5) detecting transgenic apricot tissue cultured seedlings. According to the apricot cold-resist gene transformation method, the apricot cold-resist genes are successfully transformed through the pollen tube pathway method, the transgenic plants are obtained, the cold-resist characteristic of apricot trees is improved, and meanwhile, novel genetic resources of cold-resist apricots are enriched.

Description

technical field [0001] The invention relates to a method for transforming an apricot cold-resistant gene, in particular to a method for introducing a gene into an apricot pollen tube, and belongs to the fields of biotechnology and modern agricultural technology. Background technique [0002] Low temperature is an important factor limiting the growth, development and geographical distribution of plants. Plant frost damage will cause great economic losses to agriculture and planting. According to statistics, in our country, there is a periodical freeze damage about every ten years or so. The types of freeze damage that often occur include freeze damage to overwintering crops, freeze damage to fruit trees, and freeze damage to economic trees. In contrast, the freezing damage to crops, vegetables, and flowers only affects the current year, while the freezing damage to fruit trees and economic trees not only causes huge losses in the current year, but also will continue to be af...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12Q1/68A01H1/02A01H5/00
Inventor 冯殿齐牛庆霖刘静赵进红罗磊王玉山黄艳艳张虹
Owner TAISHAN RES INST OF FORESTRY
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