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Application of rice SPL6 gene in inhibiting degeneration of spike top
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A rice, gene technology, applied in the field of genetic engineering, can solve the problems of yield and quality degradation
Active Publication Date: 2019-10-22
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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However, in rice production, with the passage of time and the number of sowing generations, many high-quality rice species will encounter a tendency to deteriorate in yield and quality
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Embodiment 1
[0049] Example 1: Identification of Mutants
[0050] We ordered spl6 allelic mutants, the supplier is the Rice T-DNAInsertionSequence Database (RISD DB), purchased T-DNAinsertion mutants spl6-1 and spl6-2, the genetic background is rice variety Dongjin, and it was They were named spl6-1 and spl6-2, respectively. The study found that the phenotype of spl6-2 and spl6-1 on the ear is consistent, that is, the same death phenotype at the top of the ear. Sequencing results showed that the T-DNA in spl6-2 and spl6-1 were respectively inserted on the 10th and 11th exons of the gene SPL6 (such as figure 2 shown in a). Transcript level identification was carried out by RT-PCR, and the identified primer sequence was:
[0051] Forward 5'-TTGAAGATGGTGGATTTAGCAACGG-3' (SEQ ID NO: 3);
[0052] Reverse 5'-AACCTTTGGACAGGTGTGGAGTAGC-3' (SEQ ID NO: 4).
[0053] PCR conditions: pre-denaturation at 94°C for 5min; 32 cycles (94°C, 30s; 57°C, 30s; 72°C, 1min); 72°C, 5min; 16°C, hold.
[0056] 2.1 The sequence fragment SEQ ID NO: 1 containing the SPL6 coding frame (CDS) and the SPL6 promoter sequence fragment (SEQ ID NO: 2) of the first 2130 bp of the ATG were cloned by RT-PCR respectively, using the kit PrimeScript RT reagent Kit with gDNA Eraser and KOD-Plus sequentially connected the two fragments into the vector pCAMBIA2300 (supplier is CAMBIA) to obtain the genetic complementationplasmid spl6-Comp.
[0057] Using PrimeScript RT reagent Kit with gDNA Eraser Kit and KOD-Plus, clone the SPL6 coding frame sequence by RT-PCR, the conditions are:
[0058] Forward primer: 5'-TCTAGAGTAGATCCTTTTACGCGGTGTGC-3' (SEQ ID NO:5),
[0059] Reverse primer: 5'-GTCGACCTCCTCACATTGGTCCACGTTCT-3' (SEQ ID NO: 6).
[0060] PCR conditions: pre-denaturation at 94°C for 2min; 32 cycles (94°C, 30s; 57°C, 30s; 68°C, 3min); 68°C, 5min; 16°C, hold.
[0061] The SPL6 promoter sequence was cloned by RT-PCR using the KOD-Plus ...
Embodiment 3
[0067] Example 3: Investigation of normal WT and mutant agronomic traits
[0068] In order to study the specific effect of the SPL6 mutation on the panicle, we investigated the panicle length, primary branch, secondary branch, and grain per panicle number, seed setting rate, and thousand-grain weight. see figure 2 and image 3 , the investigation results of these agronomic characters showed that under normal field growth conditions, the panicle development of the mutants spl6-1 and spl6-2 was severely inhibited, and the top of the panicle died before heading, and the greatly reduced seed setting rate was very important for rice Yield has a major impact.
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Abstract
The invention discloses an application of a rice SPL6 gene in inhibiting degeneration of a rice spike top, wherein the application includes the following steps: 1) extracting a rice genome, and carrying out gene sequencing; 2) carrying out sequence alignment with a wild-type rice genome with normal SPL6 gene, and screening a mutant spl6-Mut including spl6-1 and spl6-2 and having the SPL6 gene mutated; 3) cloning a sequence fragment containing an SPL6 coding frame and an SPL6 promoter sequence fragment by RT-PCR, and then successively connecting the two fragments into a plant binary expressionvector, to obtain a genetic complementary plasmid spl6-Comp equivalent to the wild type SPL6 gene; 4) transferring the obtained spl6-Comp plasmid into the mutant spl6-1 plant through an agrobacteriummediated transgenic method, and identifying the obtained transgenic plant as a positive plant in genome level and transcription level; and 5) obtaining transgenic rice having normal spike in mature period and having the SPL6 gene restoring into normal SPL6 gene through plant cultivation. The experiments show that the SPL6 can restore a spike death phenotype of the spl6-1 mutant.
Description
technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the application of rice SPL6 gene in suppressing ear top degeneration, in particular to the application of rice SPL6 gene in inhibiting ear top cell death and ear baldness. Background technique [0002] Rice (Oryza sativa L.) is one of the most important food crops in the world, and more than half of the world's population uses rice as a staple food. In my country, rice accounts for more than half of the total grain output, and rice production is directly related to the country's food security and national economy and people's livelihood. Due to the large population in my country and the decrease of cultivated land year by year, the importance of maintaining high rice yield and stable genetic traits is self-evident, and it is also a subject that my country's rice breeding has been working on for a long time. However, in rice production, many high-quality rice speci...
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IPC IPC(8): C12N15/82A01H5/00A01H6/46C12Q1/6841
CPCC12N15/8205C12N15/8261C12Q1/6841C12Q2531/113
Inventor 郭房庆王庆龙孙爱珍陈丽莎
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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