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Application of plant α-amylase in causing pollen abortion

A technology of α-amylase and pollen abortion, which is applied in the field of plant molecular biology and genetic engineering, can solve the problems of lower pollen energy metabolism, insufficient starch accumulation, and few reports, so as to save the artificial detasseling step, Expression levels are precise, effects of maintenance and reproduction are maintained

Active Publication Date: 2018-03-13
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the contrary, if amylase is overexpressed or silenced during pollen formation, it will reduce the level of pollen energy metabolism, resulting in insufficient starch accumulation, resulting in abortive pollen
However, there are few reports on rice pollen abortion genes.

Method used

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  • Application of plant α-amylase in causing pollen abortion
  • Application of plant α-amylase in causing pollen abortion
  • Application of plant α-amylase in causing pollen abortion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1 Rice α-amylase gene acquisition

[0057] 1. Extraction of rice Nipponbare RNA

[0058] Use the Biozol Reagent method to extract rice Nipponbare RNA: Weigh 0.1g Nipponbare fresh young ear tissue and mix it with 1ml Biozol Reagent, and let it stand at room temperature for 5 minutes; add 0.2ml chloroform for every 1ml Biozol Reagent, shake vigorously for 15s, and wait until the solution is fully emulsified, Let stand at room temperature for 5 minutes, centrifuge at 12000rmp, 4°C for 15 minutes; carefully take out the centrifuge tube from the centrifuge, absorb the supernatant and transfer it to another new centrifuge tube; add an equal volume of isopropanol to the supernatant, and centrifuge upside down After the tube is fully mixed, let it stand at room temperature for 10 minutes, centrifuge at 12000 rpm for 10 minutes at 4°C; discard the supernatant, a white precipitate may appear on the tube wall, add 0.5ml of 75% ethanol (prepared with sterilized DEPC wate...

Embodiment 2

[0073] Example 2 Constructing the recombinant expression vector DX2182-α-Amylase of rice α-amylase

[0074] See the build process figure 1 , Insert the amplified product of Example 1 into the DX2182 vector Mlu1 and Sac1 restriction sites.

[0075] Primers SEQ ID NO: 5 and SEQ ID NO: 6 amplify the PCR product and recover a product of about 1300 bp by electrophoresis on 1% agarose gel. The vector DX2182 was digested with Mlu1 and Sac1, and the linearized vector was recovered.

[0076] 2X ligation kit connects the abortion gene to DX2182, the 10ul system is as follows:

[0077] 2.5 μl of α-amylase PCR product (50 ng), 2.5 μl of enzyme-cut vector (100 ng), 5 μl of Ligation Mix, ligation procedure: 50°C for 60 minutes.

[0078] Transformation: Take 2 μl of the above ligation product and transform Escherichia coli competent cells by electroporation. Select positive clones for sequencing. Named DX2182-α-Amylase. The DX2182-α-Amylase vector contains a hygromycin resistance gene, ...

Embodiment 3

[0079] The acquisition of embodiment 3 transgenic rice plants

[0080] Agrobacterium EHA105 stored at -70°C was streaked on a plate containing Kan (50 μg / ml) + Rif (25 μg / ml) + streptomycin (50 μg / ml) and cultured at 28°C. Pick a single colony and inoculate it in 50ml of YEB liquid medium, shake it at 220rpm at 28°C for 12-16hr. Take 2ml of the bacterial liquid and transfer it to 100ml (containing antibiotics) YEB liquid medium, shake and cultivate at 28°C and 220rpm until OD600=0.5. Pre-cool on ice for 10 minutes, 5000rpm 10min (refrigerated centrifuge pre-cooled to 4°C). Wash twice with sterile deionized water (10ml each time), wash once with 10% glycerol and dissolve in 3ml 10% glycerol. Take 100 μl of competent cells and add 1 μl of the DX2182-α-Amylase plasmid obtained in Example 1, and transform by electroporation at 2.5KV. Culture at 28°C on a YEB culture plate containing kanamycin and rifampicin, select positive clones, and verify by PCR with DX2182-α-Amylase vector...

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Abstract

The invention provides an application of application of plant alpha-diastase in pollen abortion, and belongs to the technical field of genetic engineering. Through introducing polynucleotide of the plant alpha-diastase, recombinant vector and recombinant vector are structured; a pollen specific promoter drives the plant alpha-diastase to degrade starch in advance during the pollen developmental phase; the pollen sprouting energy cannot be provided, and the pollen sprouting is contained, thus abortion is generated; the transgenetic seedling of transgene pollen abortion is obtained. The plant alpha-diastase can be used as the pollen abortion gene and can be originated from genes of rice endogenesis, millet, broomcorn, barley, wheat and other gramineous plants; the plant alpha-diastase is very good for transgenic plant of pollen abortion. The plant alpha-diastase gene has accurate expression level and can control the spreading of transgenosis; the plant alpha-diastase gene also can keep and breed a male sterility line while leave out the artificial castration step during the hybrid seed production process; the application prospect is wide.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biology and genetic engineering, and specifically relates to the application of a plant α-amylase in causing pollen abortion, and also relates to combining the nutrition and utilizing modern biotechnology to apply it to a hybrid seed production technology system to ensure Seed production quality, and improve seed production efficiency; can also be applied to prevent the spread of transgenes. Background technique [0002] Utilization of plant heterosis is one of the most economical and effective ways to increase grain yield, but there are still some limiting factors, such as the difficulty of combining good and bad crops, and the small proportion of hybrid crops. Although almost all crops have exploited heterosis to the greatest possible extent, there is still enormous potential. Therefore, many scholars have conducted research, especially in rice, the main way of heterosis has been change...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/32A01H5/00A01H4/00
CPCA01H4/00C12N9/2422C12N15/827
Inventor 黄培劲吴永忠张维金雄霞陈思兰安保光李新鹏龙湍曾翔
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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