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Long non-coding RNA gene ROVULE and application thereof in regulation of endosperm development of rice

A long-chain non-coding, gene technology, applied in the field of plant genetic engineering

Pending Publication Date: 2020-01-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although scholars at home and abroad have done a lot of research on rice breeding to increase yield in recent years, most of them are limited to protein-coding genes and a small number of small RNA molecules (micro-RNA) on the regulation of yield shape, while related LncRNAs in regulating endosperm development. Production is rarely reported

Method used

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  • Long non-coding RNA gene ROVULE and application thereof in regulation of endosperm development of rice
  • Long non-coding RNA gene ROVULE and application thereof in regulation of endosperm development of rice
  • Long non-coding RNA gene ROVULE and application thereof in regulation of endosperm development of rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Obtainment and functional identification of in situ activated plants by ROVULE

[0035] The present invention found that lncRNA ROVULE (expression Restricted to OVULEs), which is hardly expressed in other tissues and specifically expressed in ovules, is located on the positive-sense DNA strand of rice chromosome 8, and its gene covers a range from 22380590bp to 22383406bp. Its sequence is shown in SEQ ID NO: 1, and its length is 2817bp. The ROVULE gene can transcribe 4 variants, which are: variant 1 is 777nt in length, and its sequence is shown in SEQ ID NO: 2; variant 2 is 1383 nt in length, and its sequence is shown in SEQ ID NO: 3; The length of variant 3 is 840nt, and the sequence is shown in SEQ ID NO: 4; the length of variant 4 is 423nt, and the sequence is shown in SEQ ID NO: 5 ( figure 1 A).

[0036] The rice gene ACTIN2 was used as an internal reference to detect the expression levels of ROVULE variants 1-4, and the primers for ROVULE variants 1-4 w...

Embodiment 2

[0046] Example 2 Analysis of Phenotypes Related to Endosperm Development of ROVULE Activation Mutant Plants

[0047] In order to observe rice endosperm development and yield-related traits, mutant ROVULEox-1 and wild-type rice were grown outdoors. When the rice grows to the flowering stage, mark the florets that just bloom at noon every day. The corresponding materials were removed and fixed at regular intervals for subsequent experiments.

[0048] 1. In order to observe the fertility of pollen grains, get mature single-cell stage pollen grains, I 2 - After IK staining, take pictures under the microscope, the results are as follows figure 2 As shown, the ROVULE mutant has reduced sportiness, overall reduced panicle type, less number of primary branches, reduced total grain number, significantly decreased seed setting rate, and overall reduced yield phenotype. The specific performance is that the whole plant of the ROVULE mutant is smaller ( figure 2 A); spike type become...

Embodiment 3

[0056] Example 3 Construction, transformation and related phenotype analysis of ROVULE overexpression plants

[0057] The cDNA was reverse-transcribed using Zhonghua 11 wild-type rice panicle total RNA, and the first two exons shared by ROVULE variant 1 and variant 2 were amplified. The full-length DNA sequence is 640bp, and the sequence is as shown in SEQ ID NO: 6 Shown; the primers used for amplification are:

[0058] F: 5'-GAGAACACGGGGGACTCTAGCCGCGCACGCAGAAAA-3',

[0059] R: 5'-TCACCTGTAATTCACACGTGCTCGAGGAAGCCGACCTCC-3';

[0060] The 1390 35S promoter vector restriction site uses BamHI and EcoRI, and uses homologous recombination to recombine the first two exons of ROVULE into the vector. The ROVULE overexpression vector guided by the 1390 35S promoter was used; the callus genetic transformation method mediated by Agrobacterium was used for transgenic treatment, and the corresponding vector was transferred into rice; the transformant was screened with hygromycin (Hyg) to ...

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Abstract

The invention discloses a long non-coding RNA gene ROVULE and application thereof in regulation of rice endosperm development. The long non-coding RNA ROVULE gene is obtained from rice by cloning, anda transcript of the long non-coding RNA ROVULE gene participates in the endosperm development after rice pollination. Researches on biological functions of ROVULE prove that activation on expressionof partial fragments of the ROVULE is sufficient to remarkably reduce the setting rate of ROVULE. Further researches discover that the long non-coding RNA ROVULE does not influence fertility of stamens and pistils, but influences the developing process of endosperm after pollination, and indicate that the long non-coding RNA ROVULE can negatively regulate the endosperm development after rice pollination. The long non-coding RNA ROVULE can provide a new strategy and genetic resource to cultivation and production increase of rice and has important theoretical significance and application value.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, and more specifically, relates to long-chain non-coding RNA gene ROVULE and its application in regulating rice endosperm development. Background technique [0002] Endosperm is a unique tissue of angiosperms. It is a triploid formed by the combination of a diploid central cell and a sperm cell in the embryo sac during double fertilization. It is an important material for plant genetic research. At the same time, endosperm is the most important place for the storage of nutrients in seeds, providing food, nutrition and energy for human beings. The formation and development of endosperm directly affect the formation of crop yield and quality. Discovering genes related to endosperm development and clarifying their functions and regulatory mechanisms is of great significance for understanding the process of plant seed development, and it is also of great significance for guiding the...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/46
CPCC12N15/113C12N15/8261C12N15/8234
Inventor 周燕飞于洋张玉婵陈月琴
Owner SUN YAT SEN UNIV
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