Application of rice histone methyltransferase in enhancing crop drought resistance and improving yield of single plant

A methyltransferase and histone technology, applied in the field of biological genetic engineering, can solve the problems affecting plant drought resistance and yield traits, achieve high agricultural application value, improve stress resistance and yield traits, and have broad application prospects. Effect

Active Publication Date: 2021-02-09
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Previous studies on rice SDG708 confirmed that it was involved in the process of plant flowering regulation, and transcriptome data analysis also showed that this gene may be involved in rice abiotic stress response (New Phytol, 2016, 210: 577–588), but SDG708 and its establishment How the methylation modification of H3K36 regulates the drought resistance and agronomic traits of rice has not yet been reported. At the same time, whether changing the expression level of SDG708 can affect the drought resistance and yield traits of plants is also worthy of attention

Method used

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  • Application of rice histone methyltransferase in enhancing crop drought resistance and improving yield of single plant
  • Application of rice histone methyltransferase in enhancing crop drought resistance and improving yield of single plant
  • Application of rice histone methyltransferase in enhancing crop drought resistance and improving yield of single plant

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1. Rice histone lysine methyltransferase gene SDG708 the acquisition

[0023] Obtained from the Rice Genome Database (http: / / rice.plantbiology.msu.edu) Os04g0429100 Gene sequence, design a pair of primers according to the 5' and 3' end sequences, the primer sequences are: 5'-ATGGAGGAAGAGCGCATGGA-3' (SEQ ID No: 3), and 5'- TCATGGACCGTTTTCAAGT-3' (SEQ ID No: 4) .

[0024] Extract the total RNA of rice seedlings (Promega, SV total RNA isolation system), use the total RNA of rice as a template, and use AMV reverse transcriptase (TaKaRa) to synthesize cDNA (according to the user manual of Plant RT-PCR Kit 2.01 (TaKaRa)) . Using cDNA as Template, PCR Amplification of Rice Histone Lysine Methyltransferase Gene SDG708full-length cDNA sequence. The 50ul PCR reaction system contains: template 2ul, high-fidelity enzyme KOD plus (TOYOBO) 1ul, 10× buffer 5ul, 2.5uM dNTP 8ul, 20uM 5' and 3' primers 1ul each, water 32ul. The reaction conditions were: pre-denaturation ...

Embodiment 2

[0025] Example 2. SDG708 Obtaining of Overexpression and RNA Interference Mutant Plants

[0026] one, SDG708 Construction of overexpression vector

[0027] In order to analyze the function of SDG708 gene, to SDG708 gene as the target gene, construct SDG708 Gene overexpression vector. Obtained with embodiment 1 pUC19-SDG708 As a template, two pairs of different primers were used for PCR amplification SDG708 Nucleotide sequence from position 1 to position 1557 at the 5' end. The primer pairs used were:

[0028] 5'-GTCGACATGGAGGAAGAGCGCATGGA-3' (SEQ ID No: 5),

[0029] 5'-GGATCCTCATGGACCGTTTTTCCAAGT-3' (SEQ ID No: 6),

[0030] The resulting PCR product is SDG708-CDS . use Sal I and Bam H I digest the product and recover. use Sal I and Bam H I carry Ubiquitin Genetic transformation vector with gene promoter and EYFP tag pU1301 ( pU1301 It is rebuilt on the basis of the commonly used plant genetic transformation vector pCAMBIA1301 in the world, ...

Embodiment 3

[0046] Example 3. SDG708 Identification of osmotic stress phenotypes in transgenic plants

[0047] In order to further verify the function of SDG708 in the response to drought stress, 20% PEG8000 solution was used to cause short-term osmotic stress to simulate the real drought stress environment. First, the rice seedlings that had grown normally for 21 days under short-day sunlight were transferred to a nutrient solution containing 20% ​​PEG8000 to simulate a drought environment, and the stress treatment was continued for 7 days until the wilting phenotype appeared, and then all the rice seedlings were transferred to the normal culture solution The seedlings were restored to growth for 7 days, and the damage of seedlings under PEG stress was compared. like figure 1 As shown, relative to the wild type, the mutant 708Ri-1 and 708Ri-2 All showed more obvious damage after PEG stress, on the contrary 708OE-1 and 708OE-2 The two overexpression materials exhibited stronge...

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Abstract

The invention belongs to the field of biological gene engineering, and particularly relates to application of rice histone H3K36 methyltransferase in the aspects of enhancing drought resistance of rice and improving yield of a single plant. According to the application, a nucleotide sequence of rice histone H3K36 methyltransferase SDG708 is cloned, a recombinant expression vector is constructed, and transformation is carried out through an agrobacterium transformation method; by connecting a corn ubiquitin gene promoter, the SDG708 gene is continuously overexpressed in transgenic rice; and theSDG708 expression quantity down-regulation mutant is obtained by constructing an antisense RNA vector of a gene. A rice SDG708 overexpression transgenic plant shows obvious resistance under simulatedosmotic pressure stress, the plant resistance is also obviously improved under a soil drought condition, and the yield of a single rice plant can be obviously increased under normal and drought stress conditions. The application provides a high-quality gene and a breeding material for research on drought resistance and yield trait improvement of the rice, and has high practical application value.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and specifically relates to the application of a histone H3K36 methyltransferase (SDG708) related to the regulation of rice epigenetics in osmotic stress resistance and drought stress resistance, and also relates to the Application of overexpression plants in improving yield traits under normal and drought stress conditions. Background technique [0002] Epigenetics mainly studies the mechanism of gene expression differences under the condition of unchanged genotype. Chromatin structure and function are regulated by multiple epigenetic mechanisms, including histone modification, DNA methylation, ATP-dependent chromatin remodeling, replacement of histone variants, and regulation of non-coding RNAs. Histone lysine methylation modification is a histone modification that has been widely studied in recent years. It plays an important role in regulating chromatin structure and g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00A01H6/46
CPCC12N15/8273C12N15/8261
Inventor 董爱武俞瑜陈凯刘兵
Owner FUDAN UNIV
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