Method for creating hybrid compatible line between Asian cultivated rice and African cultivated rice and application of method

A technology for cultivating rice and hybrids, which is applied in the field of plant genetic engineering technology and crop genetics and breeding, can solve the problems of no verification, no provision, etc., and achieve the effect of improving rice yield and resistance

Active Publication Date: 2017-07-21
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the patent publication does not verify ORF12 exist S1 The function of hybrid sterility and whether it is necessary for rice pollen and

Method used

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  • Method for creating hybrid compatible line between Asian cultivated rice and African cultivated rice and application of method
  • Method for creating hybrid compatible line between Asian cultivated rice and African cultivated rice and application of method
  • Method for creating hybrid compatible line between Asian cultivated rice and African cultivated rice and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 OgTPR1 Construction of functional knockout vector

[0031] For Asian-African rice hybrid sterility gene OgTPR1 (SEQ ID NO: 1) coding sequence, design target sequence (SEQ ID NO: 4). Two complementary primers (U3- OgTPR1- F, OgTPR1- R) Formation of the target double-stranded linker (Table 1), ligated into the expression cassette of the sgRNA driven by the rice OsU3 promoter. The PCR-amplified sgRNA expression cassette fragment was cloned into the gene editing binary vector pYLCRISPR / Cas9Pubi-H (contained in the T-DNA region) to generate KO- OgTPR1 ( figure 1 B).

[0032]

Embodiment 2

[0033] Example 2 OgTPR1 Knockout line ogtpr1 Identification

[0034] The binary transformation vector KO- OgTPR1 Agrobacterium-mediated transformation of NIL-g to obtain T 0 transformant. The positive knockout mutant of the transgene was identified by PCR with primers HptF / HptR (Table 1) ( figure 2 A). Further, a fragment containing the target site from two independently transformed plants was amplified by PCR, and sequenced and analyzed OgTPR1 Mutation effect of gene, get mutant ogtpr1-1 and ogtpr1-2 ( figure 2 B). They produce frameshifts and premature stop codons due to single base insertions, i.e. loss-of-function truncated genes ogtpr1, As shown in SEQ ID NO: 2-3.

Embodiment 3

[0035] Example 3 ogtpr1 Knockout mutant T 1 Identification and phenotype observation of plants without transgenic individuals

[0036] to the above ogtpr1 Transgenic T 1 Plants were transgenic ( HPT Gene) PCR detection, isolated individuals without transgene ( image 3 A). Then observe 2 ogtpr1 The fertility of the mutants was found to be fully fertile in the pollen and spikelets of these mutants ( image 3 B).

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Abstract

The invention discloses a method for creating a hybrid compatible line between Asian cultivated rice and African cultivated rice and application of the method in rice distant hybrid vigor. The method is characterized in that an appropriate gene editing site is selected for the African rice candidate gene OgTPR1 of rice interspecific hybrid sterility locus S1, and fixed point editing technologies such as CRISPR/Cas9 are used to perform fixed point knockout on the OgTPR1; the fact that the knockout of the mutant gene ogtpr1 does not affect parent pollen and female gamete fertility is proved while the pollen and female gamete fertility of hybrid F1 hybridized by an ogtpr1 mutation line and the Asian cultivated rice is normal, that is to say, the genetic effect of interspecific hybrid sterility is eliminated. The method has the advantages that the locus S1-mediated interspecific hybrid reproductive disorder can be broken through effectively, and the method has high application value is the utilization of rice distant hybrid vigor and the increasing of grain yield.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering technology and crop genetic breeding technology, and specifically relates to a method for creating an interspecific hybrid affinity line between Asian cultivated rice and African cultivated rice and its application in the utilization of rice distant heterosis. Background technique [0002] The world is facing the contradiction of increasing population and decreasing arable land. Using crop heterosis to increase crop yield is of great significance to resolve the food crisis. The genetic relationship between the two parents is more distant, that is, the genetic difference between the two parents is greater, and the performance of hybrid offspring is more obvious, which is called heterosis. However, distant hybrids are usually accompanied by severe hybrid sterility, which greatly hinders the application of crop heterosis. Hybrid sterility is a common phenomenon of interspecies or subspecies ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N9/22C12N15/8213C12N15/8287
Inventor 陈乐天刘耀光谢勇尧黄健乐
Owner SOUTH CHINA AGRI UNIV
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