Sorghum alpha-amylase and coding gene and application thereof

An amylase and sorghum technology, applied in the application, genetic engineering, enzymes and other directions, can solve the problems of abortion, insufficient starch accumulation, lowering the level of pollen energy metabolism, etc. Precise effects of expression regulation

Active Publication Date: 2020-03-27
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

Method used

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  • Sorghum alpha-amylase and coding gene and application thereof
  • Sorghum alpha-amylase and coding gene and application thereof
  • Sorghum alpha-amylase and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Sorghum α-amylase acquisition

[0046] 1. Extraction of sorghum RNA Use the Biozol Reagent method to extract sorghum RNA: weigh 0.1g fresh sorghum young ear tissue, add 1ml Biozol Reagent to mix, let stand at room temperature for 5min; add 0.2ml chloroform for every 1ml Biozol Reagent, shake vigorously for 15s, After the solution is fully emulsified, let it stand at room temperature for 5 minutes, centrifuge at 12,000 rpm, and 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 isocyanate to the supernatant. For propanol, invert the centrifuge tube upside down and mix thoroughly, let stand at room temperature for 10min, centrifuge at 12000rpm, 4°C for 10min; discard the supernatant, a white precipitate may appear on the tube wall, add 0.5ml of 75% ethanol (prepared with RNase-free water) ) wash, invert and mix, centrifuge at 10,000rpm, 4°C ...

Embodiment 2

[0049] Example 2 Construction of pollen abortion gene plant binary expression vector DX2182-SBAA2

[0050] 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 with Example 1 The PCR products were connected in a certain ratio, and finally a binary vector containing the pollen-specific expression cassette of SBAA2 was constructed.

[0051]The 2X ligation kit connects the abortion gene to DX2182, and the 10μl system is as fo...

Embodiment 3

[0052] Example 3 Creation of SBAA2 transgenic rice

[0053] 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 antibiotics, and shake 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 and 220rpm until 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% glycerol. Take 100 μl of competent cells and add 1 μl of the DX2182-SBAA2 plasmid obtained in Example 2, and transform by electroporation at 2.5KV. Culture on a YEP culture plate containing kanamycin, rifampicin and streptomycin, select positive clones, and use DX2182-SBAA2 c...

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Abstract

The invention relates to sorghum alpha-amylase and a coding gene and application thereof. The amino acid sequence of the sorghum alpha-amylase is as shown in SEQID No.6, or is an amino acid sequence which is obtained through replacement, deletion or addition of one or more amino acids in the sequence as shown in the SEQID No.6 and has the same functions as that as shown in the SEQID No.6, and thenucleotide sequence coded by the gene is as shown in SEQID No.1 or is as shown in a sequence having 80% homology with the sequence. When the sorghum alpha-amylase disclosed by the invention is expressed at the pollen development period under driving of a pollen specificity promoter, starch can be degraded in advance, energy cannot be provided for pollen germination, the pollen germination is restrained, and pollen abortion is caused. The sorghum alpha-amylase disclosed by the invention can effectively prevent transgenic pollen from escaping, and can also be used for maintaining the homozygosisrecessive state of male sterile plants; and besides, in a hybridization seed production process, an artificial emasculation step is omitted, so that the sorghum alpha-amylase has great application prospects in the respects of crop germplasm resource improvement and heredity breeding.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and specifically relates to pollen-specific expression of sorghum 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 seed production Efficiency, 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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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/26A01H5/00A01H6/46
CPCC12N9/2414C12N15/8245C12N15/8289C12Y302/01001
Inventor 黄培劲金雄霞安保光陈思兰张维李新鹏吴永忠
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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