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Mutant gene of rice nuclease gene OsGEN-L and application thereof

A technology of mutated genes and nucleases, applied in the fields of application, hydrolase, genetic engineering, etc., can solve problems such as difficulty in finding restorer lines, inconsistent fertility of interfering offspring, restrictions on heterosis utilization, etc., and achieves a highly practical effect

Inactive Publication Date: 2017-08-18
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the inconsistent fertility of the interfering offspring, and they are all dominant sterility, it is difficult to find restorer lines in breeding materials, which greatly restricts the utilization of heterosis

Method used

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  • Mutant gene of rice nuclease gene OsGEN-L and application thereof
  • Mutant gene of rice nuclease gene OsGEN-L and application thereof
  • Mutant gene of rice nuclease gene OsGEN-L and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Obtaining and morphological observation of embodiment 1, OsGEN-L mutant plant

[0023] From the winter of 2011 to the spring of 2012, calluses were induced and differentiated into seedlings with the embryos of Japan's sunny dry seeds. After rooting and training, the first-generation seedlings were transplanted to paddy fields for planting. When they matured, they were harvested in a single ear. The second generation was planted in 2013. After flowering The pollen fertility of the second-generation lines was observed in the second generation, and it was found that there were 5 sterile mutants in one line. The anthers of all the sterile plants had no pollen, and they did not self-fertilize when they matured, but after pollination with normal pollen Strong normal ( figure 1 ). Nipponbare and Zhongjiu B (indica rice) were used to cross the mutants with normal fertility and planted F in 2014 1 , found F 1 All fertile, planted F in 2015 2 The segregation ratios of fertile...

Embodiment 2

[0024] Location and cloning of embodiment 2, OsGEN-L gene

[0025] 1. Positioning groups

[0026] With the above sterile mutants, F 2 In order to locate the population, carry out the phenotype identification of individual plants in the field according to the fertility, and take the young leaves of 15 fertile plants for each plant, and take the F 2 The young leaves of all the male sterile plants were used for later use.

[0027] 2. Extraction of rice DNA

[0028] Parents were extracted using the improved CTAB method, including the following steps: Take 0.1-0.2 grams (about half a piece) of leaves and put them in a small mortar, add an appropriate amount of liquid nitrogen, grind them to powder immediately, put them into a 2m1 centrifuge tube, and add 700μL Put the 1.5×CTAB solution preheated at 100°C in a centrifuge tube, mix it carefully, put it in a 65°C water bath, take out the centrifuge tube after 20 minutes, add an equal volume of chloroform / isoamyl alcohol, mix vigoro...

Embodiment 3

[0035] Example 3, Functional Verification of OsGEN-L Gene

[0036] In order to further confirm that the OsGEN-L gene is the gene that causes the phenotype of the male sterile mutant, the OsGEN-L gene and its expression regulatory region (including 3626 bp exons and Contains, 2125bp upstream promoter and 1559bp downstream terminator), the nucleotide sequence is shown in SEQ ID NO.29. The specific operation is segmented amplification and connection into the pCAMBIA1301 vector: using the restriction site BstEII existing in the middle of the coding region, the restriction site BamHI is added to the end of the first half of the upstream primer C360F1, and the first half of the downstream primer C360R1 is located at the BstEII site Finally, the second half of the upstream primer C360F2 is located in front of the BstEII site, and the end of the second half of the downstream primer C360R2 adds a restriction site XbaI. The sequence of the cloning primers is shown in Table 1; first use ...

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Abstract

The invention relates to a mutant of a rice nuclease gene OsGEN-L and application thereof, which belong to the technical field of genetic engineering. In the mutant provided by the invention, the nuclease gene OsGEN-L is mutated into osgen-l, a nucleotide sequence of the normal OsGEN-L gene is as shown in SEQ ID NO. 1, and a nucleotide sequence of the mutant gene osgen-l of the OsGEN-L is as shown in SEQ ID NO. 4. The mutation is concretely characterized in that the first A base deletion after the 1053th bases of the OsGEN-L gene causes premature translation termination, and the mutation finally causes pollen sterility of the mutant, can be used for preparing recessive male sterile line, and plays an important role on genetic improvement, variety breeding and seed production of rice.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a rice nuclease gene OsGEN-L and a mutant gene osgen-1, and also relates to a mutant osgen-1 of the OsGEN-L gene, as well as a nuclease gene OsGEN-L gene and a mutant gene osgen-1 And the application of mutant osgen-l. Background technique [0002] Rice (Oryza sativa) is one of the main food crops, and about half of the world's population uses rice as a staple food. In my country, the sown area of ​​rice accounts for 30% of the sown area of ​​grain crops in the country, but the total output accounts for more than 42% of the grain crop output. The important reason is the successful application of rice heterosis. [0003] Nuclear male sterility is one of the effective ways to utilize crop heterosis. However, it is difficult to obtain a fully sterile population due to nuclear sterility, which limits its use in heterosis. Effectively solving the problem of 50% fertile...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/82A01H5/00
CPCC12N9/22C12N15/8289
Inventor 张毅杨昆吕俊石敏陈云鲜小华梁艳玲杜双林
Owner SOUTHWEST UNIVERSITY
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