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Barley alpha-amylase as well as coding gene and application thereof

A technology of amylase and barley, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of lower pollen energy metabolism, abortion, insufficient starch accumulation, etc., and achieve the elimination of manual detasseling steps and expression regulation Precise, Promising Effects

Active Publication Date: 2020-03-10
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 aborted pollen.

Method used

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  • Barley alpha-amylase as well as coding gene and application thereof
  • Barley alpha-amylase as well as coding gene and application thereof
  • Barley alpha-amylase as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Barley α-amylase acquisition

[0051] 1. Extraction of barley RNA

[0052] Use the Biozol Reagent method to extract barley RNA: weigh 0.1g of fresh barley ear tissue, add 1ml of Biozol Reagent to mix, and let stand at room temperature for 5 minutes; add 0.2ml of chloroform to each 1ml of Biozol Reagent, shake vigorously for 15s, and wait until the solution is fully emulsified, then Let stand at room temperature for 5 minutes, centrifuge at 12,000 rpm, 4°C for 15 minutes; carefully remove 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 turn the centrifuge tube upside down After fully mixing, let 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 (made with RNase-free water) to wash, invert and mix well, C...

Embodiment 2

[0070] Example 2 Construction of pollen abortion gene plant binary expression vector DX2182-HVAA1

[0071] See the build process figure 1 , the amplified product of Example 1, i.e. primer SEQ ID NO: 7-8 amplified PCR product 1% agarose gel electrophoresis reclaims about 1300bp product, inserts DX2182 (disclosed in Chinese patent CN106434673A, invention name: a kind of plant Anther-specific promoter PCHF15 and its application) in the linear restriction vector of MluI and SacI. The DX2182 vector already contains the pG47-optimized promoter and terminator, and is located on both sides of the MluI and SacI restriction sites, so MluI and SacI digest the vector DX2182, recover the linear restriction vector, and amplify as in Example 1 The PCR products were connected in a certain ratio, and finally a binary vector containing the pollen-specific expression cassette of HVAA1 was constructed.

[0072] 2X ligation kit ligates abortion gene to DX2182, 10μl system is as follows:

[0073...

Embodiment 3

[0078] Example 3 Creation of HVAA1 transgenic rice

[0079] Agrobacterium EHA105 stored at -70°C was streaked on a YEP plate containing Rif (25 μg / ml) + streptomycin (50 μg / ml), and cultured at 28°C. Pick a single colony and inoculate it in 50ml of YEP liquid medium containing the above-mentioned antibiotics, shake and culture at 220rpm at 28°C for 12-16h. Take 2ml of the bacterial liquid and transfer it to 100ml (containing the above antibiotics) YEP liquid medium, shake and culture at 28°C 220rpm to OD 600 = 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% glycerin. Take 100 μl of competent cells and add 1 μl of the DX2182-HVAA1 plasmid obtained in Example 2, and transform by electric shock at 2.5KV. Cultivate on a YEP culture plate containing kanamycin, rifampicin and streptomycin, select positive clones, and use DX...

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Abstract

The invention relates to a barley alpha-amylase as well as a coding gene and application thereof. The amino acid sequence of the barley alpha-amylase provided by the invention is shown in SEQ ID No.6or is an amino acid sequence having the same function obtained by substituting, deleting or adding one or several amino acids in the sequence, and the gene-coded nucleotide sequence is shown in SEQ IDNo. 1 or a sequence with 80% homology to the sequence. The barley alpha-amylase provided by the invention is expressed under driving of a pollen-specific promoter in the development period of pollen,can degrade starch in advance, and cannot provide energy for pollen germination, thereby suppressing the pollen germination and causing pollen abortion; and the barley alpha-amylase can effectively prevent transgenic pollen from escaping, can be used to maintain the homozygous recessive state of male sterile plants and omit an artificial emasculation step in the hybrid seed production process, and has broad application prospects in the fields of crop germplasm resource improvement and genetic breeding.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and specifically relates to pollen-specific expression of α-amylase, which can cause pollen abortion, and is applied to a hybrid seed production technology system using modern biotechnology to ensure the quality of seed production and improve the efficiency of seed production , can also be applied to prevent the spread of transgenes. Background technique [0002] The utilization of plant heterosis is one of the most economical and effective ways to increase grain production. Although almost all crops use heterosis as much as possible, there are still some limiting factors, such as the difficulty of breeding good combinations and the high cost of hybrid seed production. The proportion of hybrid crops is still small due to the characteristics of high risk and high risk. Among the main crops, rice is the most important. The commercial application of heterosis is mainly through the "three-line ...

Claims

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Application Information

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IPC IPC(8): C12N9/32C12N15/56C12N15/82A01H5/02A01H6/46A01H1/02
CPCA01H1/02C12N9/2422C12N15/8289C12Y302/01001
Inventor 黄培劲吴永忠金雄霞安保光张维陈思兰曾翔
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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