Millet α-amylase and its coding gene and application

A technology of α-amylase and coding gene, which is applied in the fields 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 manual detasseling steps and maintain Precise effects on maintenance and reproduction, gene expression levels

Active Publication Date: 2017-12-12
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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  • Millet α-amylase and its coding gene and application
  • Millet α-amylase and its coding gene and application
  • Millet α-amylase and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Obtaining millet α-amylase gene

[0051] 1. Extraction of Xiaomi RNA

[0052] Use Biozol Reagent method to extract millet RNA: Weigh 0.1g of fresh millet ear tissue and 1ml BiozolReagent, mix well, and let stand for 5min at room temperature; add 0.2ml chloroform per 1ml Biozol Reagent, shake vigorously for 15s, wait until the solution is fully emulsified, and then Let stand at room temperature for 5 min, 12000 rpm, and centrifuge at 4°C for 15 min; carefully remove the centrifuge tube from the centrifuge, and transfer the supernatant to another new centrifuge tube; add an equal volume of isopropanol to the supernatant and invert the centrifuge tube upside down After mixing well, let stand at room temperature for 10min, 12000rpm, and centrifuge for 10min at 4℃; discard the supernatant, the tube wall can be white precipitate, add 0.5ml 75% ethanol (prepared with DEPC water after sterilization) to wash, invert and mix well Centrifuge at 10000rmp, 4℃ for 5min; repeat ...

Embodiment 2

[0071] Example 2 Construction of the recombinant expression vector DX2182-XMAA of millet α-amylase

[0072] See the build process figure 1 The vector DX2182 is pre-constructed with a male gamete preferential promoter PG47 with a sequence length of 2732 bp shown in SEQ ID No. 3 and a transduction peptide with a sequence length of 216 bp shown in SEQ ID No. 2. Insert the amplified product of Example 1 into the DX2182 vector Mlu1 and Sac1 restriction sites, which are the male gamete preferential promoter and the downstream of the transduction peptide.

[0073] The primers SEQ ID NO: 5-6 amplify the PCR product by 1% agarose gel electrophoresis to recover the product of about 1300 bp. The vector DX2182 was digested with Mlu1 and Sac1, and the linear digested vector was recovered.

[0074] The 2X ligation kit connects the abortion gene to DX2182, the 10ul system is as follows:

[0075] 2.5μ1α-amylase PCR product (50ng), 2.5μ1 digestion vector (100ng), 5μ1 Ligation Mix, ligation procedure:...

Embodiment 3

[0077] Example 3 Obtaining transgenic rice plants

[0078] Agrobacterium EHA105 stored at -70°C was streaked on a plate containing Kan (50μg / ml) + Rif (25μg / ml) + streptomycin (50μg / ml) and cultured at 28°C. Pick a single colony and inoculate it in 50ml YEB liquid medium, shake culture at 220rpm 28℃ for 12-16hr. Take 2ml of bacterial solution and transfer it to 100ml (containing antibiotics) YEP liquid medium, and shake culture to OD at 28℃ and 220rpm 600 = 0.5. Pre-cooling on ice for 10 minutes, 5000 rpm for 10 minutes (refrigerated centrifuge is pre-cooled to 4°C). Wash twice with sterile deionized water (10ml each time), wash once with 10% glycerol and dissolve it in 3ml 10% glycerol. Take 100 μl of competent cells plus 1 μl of the DX2182-XMAA plasmid obtained in Example 2, and transform with 2.5KV electric shock. Cultured on YEP culture plate containing kanamycin and rifampicin at 28°C, selected positive clones, and verified by PCR with DX2182-XMAA vector specific primer S...

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Abstract

The invention provides millet alpha-amylase as well as an encoding gene and application thereof, belonging to the technical field of gene engineering. An amino acid sequence of the millet alpha-amylase is shown by SEQ ID No.4 or is an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acids in the sequence and has the same function, and a gene encoded nucleotide sequence of the millet alpha-amylase is shown by SEQ ID No.1 or a sequence with 80% of homology with this sequence. Under the driving of a pollen specific promoter, the millet alpha-amylase provided by the invention degrades starch in advance in a pollen developmental phase, germination energy for pollen cannot be provided, and the germination of the pollen is restrained, resulting in male abortion of crops. The millet alpha-amylase provided by the invention can be used for keeping the homozygosis recessive state of a male sterile plant, and meanwhile, a step of artificial stamen removal during hybrid seed production is saved, so that the millet alpha-amylase has broad application prospect in the aspects of crop germplasm resource improvement and genetic breeding.

Description

Technical field [0001] The invention belongs to the technical field of plant molecular biology and genetic engineering, and specifically relates to an application of millet alpha amylase in causing pollen abortion, and also relates to combining the application, using modern biotechnology, and applying it to a hybrid seed production technology system to ensure Seed production quality, and improve seed production efficiency; it can also be used 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 yield, but there are still some limiting factors, such as the difficulty of combining good quality and the small proportion of hybrid crops. Although almost all crops have the greatest possible use of heterosis, they still have great potential. Therefore, many scholars have conducted research, especially in rice. The main way of heterosis is from "three-line method" to "two-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/32C12N15/82A01H4/00A01H5/00
CPCA01H4/00C12N9/2422C12N15/8289C12Y302/01001
Inventor 黄培劲张维金雄霞陈思兰吴永忠
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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