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Method for improving content of amylose in rice by using RNA interference

An amylose content, RNA interference technology, applied in the direction of recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve the problems of weak inhibition of endogenous gene expression, reduction of target gene expression, poor genetic stability of inhibition effect, etc. Achieve the effect of small RNA interference fragments, high genetic stability, and convenient agronomic traits

Inactive Publication Date: 2009-09-02
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology

However, the application of antisense technology has certain limitations. Its inhibition of endogenous gene expression is weak, and it often produces transitional phenotypes, which will hinder the accurate judgment of the function of the target gene.
The genetic stability of its inhibitory effect is poor, and it cannot stably and reliably reduce the expression of the target gene
[0007] 2. Gene knockout technology and gene conversion technology mediated by DNA / RNA chimeric molecules, but this method can only study one gene at a time, and cannot effectively knock out multi-gene families
Some genes that play a key role in the development of animals and plants, if they are heritably modified by gene knockout or mutation at the DNA level, will be prematurely silenced and produce lethal phenotypes, so they cannot be studied in depth

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  • Method for improving content of amylose in rice by using RNA interference

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Embodiment Construction

[0024] Embodiments of the present invention will be specifically described below.

[0025] 1. Obtaining RBE3 gene-specific RNAi fragments

[0026] (1), utilize CTAB method to carry out the extraction of rice genome DNA.

[0027] Take 0.5 g of young rice leaves, add liquid nitrogen to pulverize, add 2 mL of 2% 2×CTAB extract solution kept at 65°C, mix well, and keep at 65°C for 30-60 minutes. Add an equal volume of chloroform / isoamyl alcohol (24:1), mix gently by inversion, centrifuge at 10000r / min for 5min. Take the supernatant, add 1 / 10 volume (about 0.2 mL) of CTAB / NaCl solution at 65°C, and mix by inverting. Extract with an equal volume of chloroform / isoamyl alcohol (24:1), centrifuge at 10000r / min for 5min. Take the supernatant, add (exactly) equal volume of CTAB precipitation solution, invert and mix well, if precipitation is visible, proceed to the next step, otherwise, keep warm at 65°C for 30min. 4°C, 2700r / min, centrifuge for 5min. Remove the supernatant and resu...

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Abstract

The invention discloses a method for improving content of amylose in rice by using RNA interference in the technical field of biology. The method comprises: cloning a Q enzyme RBE3 genetic fragment from the rice as an RNA interference fragment; starting an expression by using an endosperm specific promoter; constructing a siRNA expression vector of RBE3 gene; and obtaining transgenic rice interfering the RBE3 gene by using a method of Agrobacterium-mediated transformation. RT-PCR and Southern are hybridized to check the integration and expression conditions of a target gene; an iodine development process for measuring the content of endosperm amylose of transgenic rice strains shows that the average improvement magnitude of the amylose of the transgenic plants is 140 percent compared with non-transgenic strains, and reaches 238 percent to the maximum; and after screening, the transgenic rice strains of which seed amylose is remarkably improved are obtained. The method for improving the content of the amylose in the rice lays a foundation for producing the amylose on a large scale by using the transgenic rice.

Description

technical field [0001] The invention relates to a method for increasing the amylose content in rice seeds in the field of biotechnology, in particular to a method for increasing the amylose content in rice seeds by using RNA interference. Background technique [0002] Starch is the main storage form of carbohydrates in higher plants and the most important component of food crop products. The storage starch of plants mainly consists of two components: amylose and amylopectin. The ratio of amylose and amylopectin determines the use of starch. Amylopectin is mainly used in the food industry, while amylose is widely used in industry, involving more than 30 industries such as food, medical, textile, papermaking, and environmental protection. field. The function of amylose, especially the amylose after physical and chemical modification, is further strengthened. If the amylose is dissolved and combined with hydrogen bonds, it can form a rigid opaque colloid. This feature is used...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
Inventor 程备久张建江海洋夏眠朱苏文汪洁明
Owner ANHUI AGRICULTURAL UNIVERSITY
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