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Rice alpha-amylase and coding genes and application thereof

A technology of amylase and rice, which is applied in the fields of application, hydrolase, genetic engineering, etc., and can solve problems such as few reports

Inactive Publication Date: 2019-03-26
SHENZHEN INST OF MOLECULAR CROP DESIGN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to utilize the above technology, α-amylase genes from different sources, such as rice, an important crop, are needed, but related studies are rarely reported

Method used

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  • Rice alpha-amylase and coding genes and application thereof
  • Rice alpha-amylase and coding genes and application thereof
  • Rice alpha-amylase and coding genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1. Acquisition of rice α-amylase gene OsAA and analysis of its tissue expression pattern

[0065] 1. Extraction of rice Nipponbare RNA

[0066] Use the Trizol Reagent method to extract Nipponbare RNA: weigh each tissue of Nipponbare (root, stem, leaf, pistil, lemma, pada, anther and endosperm) into liquid nitrogen, take it out, grind it into powder with a mortar, and then add 1ml TrizolReagent (TransGen Biotech), shake vigorously, then add 0.2ml chloroform, shake vigorously for 15s, let stand at room temperature for 3min; centrifuge at 12000rmp 4°C for 15min; carefully take out the centrifuge tube from the centrifuge, transfer 0.6ml supernatant to another Put in a new centrifuge tube; add an equal volume of isopropanol to the supernatant, invert the centrifuge tube up and down to mix thoroughly, and let it stand at room temperature for 10 minutes; centrifuge at 12000rmp 4°C for 10 minutes; discard the supernatant, and a white gelatinous precipitate appears at th...

Embodiment 2

[0085] Cloning of embodiment 2, PG47 promoter and BT1 transit peptide gene

[0086] The PG47 promoter (as shown in SEQ ID NO: 2 in the sequence listing) and the BT1 transit peptide gene (as shown in SEQ ID NO: 3 in the sequence listing) were obtained from the pZhen18B vector plasmid (Chang, Z., Chen, Z., Wang, N., Xie, G., Lu, J., Yan, W., Zhou, J., Tang, X., and Deng, X.W.. Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear Amplified from male sterilitygene.Proc Natl Acad Sci USA.2016.113,14145-14150.). The primers required for PG47 promoter amplification are shown in SEQ ID NO: 9-10 in the sequence table, wherein the forward primer has a HindIII restriction site, and the reverse primer has a SacI restriction site; BT1 transit peptide gene The primers required for amplification are shown in SEQ ID NO: 11-12 in the sequence listing, wherein the forward primer has a SacI restriction site. The primers were designed using Primer...

Embodiment 3

[0089] Example 3, Construction of recombinant expression vector pSZYJY-02 containing rice α-amylase gene OsAA

[0090] 1. Construction of recombinant expression vector pSZYJY-01 containing rice PG47 promoter

[0091] Insert the PG47 promoter of the amplified product in Example 2 into the EcoRI and SacI restriction sites of the pCAMBIA1300 vector, and the construction process is as follows figure 2 shown. That is, the amplified product of the PG47 promoter and the pCAMBIA1300 vector were digested with HindIII and SacI respectively and recovered, and then the recovered product was ligated, and the ligation system was as follows:

[0092] PG47 promoter PCR product (50ng) 2ul

[0093] Digested pCAMBIA1300 vector product (50ng) 2ul

[0094] 5X In-Fusion HD Enzyme Premix (TaKaRa) 1ul

[0095] Connect at 50°C for 20 minutes.

[0096] All the ligation products above were heat-shock transformed into Escherichia coli competent cells, positive clones were selected for sequencing, and...

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Abstract

The invention provides rice alpha-amylase and application of coding genes of the rice alpha-amylase in pollen fertility control and belongs to the field of biotechnology. The method comprises the following steps: by cloning a nucleotide sequence of Nipponbare rice alpha-amylase, constructing a recombinant expression vector and performing genetic transformation. Under drive of a PG47 promoter, alpha-amylase is specifically expressed at the later pollen development stage; and meanwhile, the alpha-amylase is capable of degrading starch in pollen grains to cause transgenic pollen sterility of therice under guidance of a BT1 transfer signal peptide, the accuracy is high, and the condition that transgenic elements of transgenic crops are spread to other crop varieties by virtue of pollens is effectively avoided. The alpha-amylase disclosed by the invention can be used for maintaining a homozygous recessive state of male sterility plants, so that the artificial emasculation step is saved inthe hybrid seed production process, extensive propagation of sterile lines can be realized, and the labor input and the influence on yield can be reduced, so that the rice alpha-amylase has wide application prospects in the aspects of crop planting resource improvement, genetic breeding and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the application of a rice α-amylase and its coding gene in the control of pollen fertility, and also relates to the method of combining the hydrolysis function of the α-amylase on starch to affect the male fertility of rice application. By using modern biotechnology and applying it to the hybrid seed production technology system, it can not only ensure the quality of seed production, but also improve the efficiency of seed production; it can also effectively prevent the spread of transgenes. Background technique [0002] Hybrid rice breeding is the main way to effectively improve rice yield and quality. Due to the application of hybridization technology, the aggregation of various traits such as yield increase, disease resistance, insect resistance, drought resistance, herbicide resistance, waterlogging resistance, cold resistance, salt resistance and lodging resistance h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/26C12N15/56A01H5/00A01H6/46
CPCC12N9/2414C12N15/8245C12N15/8289C12Y302/01001
Inventor 唐晓艳王梦龙彭小群严维尹峰陈竹锋
Owner SHENZHEN INST OF MOLECULAR CROP DESIGN
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