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A gene ospk3 controlling rice thousand-grain weight and its application

A thousand-grain weight and gene technology, applied to the gene OsPK3 to control the thousand-grain weight of rice and its application field, to achieve the effect of smaller seed thickness and lower thousand-grain weight

Active Publication Date: 2020-05-26
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cloning and functional analysis of these grain weight genes provide more and more basic materials for molecular design and breeding based on improved grain shape, rice yield and quality, but there is no report that the gene OsPK3 has the function of controlling rice thousand-grain weight

Method used

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  • A gene ospk3 controlling rice thousand-grain weight and its application
  • A gene ospk3 controlling rice thousand-grain weight and its application
  • A gene ospk3 controlling rice thousand-grain weight and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Wild type and mutant seed physiological index determination

[0031] (1) Rice material

[0032] The wild-type material is japonica rice variety Dong jin, and the mutant is ospk3 (pyruvate kinase 3).

[0033] (2) Determination of physiological indicators of wild type and mutant seeds

[0034] ① Determination of total starch and soluble sugar content

[0035] Weigh 0.1g of the dried sample crushed through a 100-mesh sieve, put it in a 10ml centrifuge tube, add 5ml of distilled water, extract in boiling water for 30min, and centrifuge at 4500r / min for 5min after cooling, absorb the supernatant, and pour it into the sediment Add 5ml of water, extract in a boiling water bath for 10min, then centrifuge at 4500r / min for 4-5min, absorb the supernatant, combine the two supernatants into a 10ml centrifuge tube and dilute to 10ml with distilled water for the determination of soluble sugar content.

[0036] Add 2ml of water to the precipitate, mix well, gelatinize in a...

Embodiment 2

[0043] Map-position cloning of embodiment 2 OsPK3 gene

[0044] (1) Genetic analysis and positioning population

[0045] Use ospk3 and Nip (Nipponbare) to construct the F2 population, and backcross ospk3 and wild-type Dongjin, and then use the SSR microsatellite polymorphism marker to initially locate the gene Ospk3 through the method of map-based cloning, and then fine-tune the gene positioning, see the results Figure 4 .

[0046] Genetic analysis of F2 populations showed that the plant height of the two F2 populations showed a segregation ratio of 3:1, indicating that the mutants may be controlled by a pair of recessive single genes. Through the F2 population of ospk3 and Nip, the near-isogenic pool and SSR microsatellite polymorphism markers, the gene was mapped between RM1113-RM127 of rice chromosome 4, and the Indel marker was further developed to fine-map the gene to AI45 and In the 57KB interval between the AI51 markers, there are 10 ORFs in this interval, and the e...

Embodiment 3

[0060] RNA was extracted from rice roots, stems, leaves, leaf sheaths, young spikes, glumes, and 3d and 6d after flowering caryopses, and the expression levels were analyzed by QPCR after reverse transcription. The results are shown in Figure 5 ,Depend on Figure 5 It can be seen that the OsPK3 gene is constitutively expressed and expressed in all tissues of rice, but the expression level is the highest in leaves. Therefore, this gene may affect the material metabolism of rice source organs and sink organs at the same time, and eventually lead to changes in thousand-grain weight. The crispr / cas9 knockout vector construction and genetic transformation of embodiment 3OsPK3

[0061] (1) Design and selection of guide RNA (guide RNA, gRNA) target site sequence

[0062] According to the genome sequence of OsPK3 (as shown in SEQ ID NO: 2), design, select and synthesize two guide RNA target sequences of OsPK3.

[0063] (2) Construction of CRISPR / Cas9-gRNA vector with two target sit...

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Abstract

The invention discloses a gene OsPK3 for controlling 1000-grain weight of rice. The nucleotide sequence of the gene is the nucleotide sequence which is shown in SEQ ID NO:2, or the nucleotide sequencewhich is shown in the SEQ ID NO:2 and can encode the same functional protein by substituting, deleting and / or adding one or more nucleotides. According to the gene OsPK3 for controlling the 1000-grain weight of the rice, a new gene OsPK3 is positioned through a map-based cloning method, and a transgenic mutant strain is formed by knocking out the gene OsPK3 by utilizing a CRISPR / Cas9 system; whenthe mutant plant is compared with a wild type, the seed grain thickness is decreased, the 1000-grain weight is remarkably reduced, and the plant height is reduced, which show that the gene is the gene for controlling the 1000-grain weight of the rice and can be used for high-yield and stable-yield breeding of the rice.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and genetic breeding, and specifically relates to a gene OsPK3 for controlling thousand-grain weight of rice and its application. Background technique [0002] Rice is one of the three most important food crops in the world, and is the basic food for more than half of the world's population. However, with the continuous growth of population and the sharp reduction of arable land, food production is still facing enormous pressure. Therefore, further increasing the yield of rice per unit area is an important way to solve this problem, which is very important for ensuring food security in my country and promoting agricultural economic development. is of great significance. The number of effective panicles, the number of grains per panicle and the weight of thousand grains are the three basic elements of rice yield. The thousand-grain weight is determined by the grain length, grain width ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/82A01H5/10A01H6/46
CPCC12N9/1205C12N15/8261C12Y207/0104
Inventor 涂斌胡丽李佳莲李仕贵马炳田王玉平钦鹏陈薇兰郑灵
Owner SICHUAN AGRI UNIV