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Application of osagp13 gene and its RNA interference fragments controlling rice flowering

An RNA interference and fragment technology, applied in the field of plant genetic engineering, can solve the problems of unclear molecular biological mechanism, and the gene for rice flowering has not been isolated or cloned, and achieves simple and mature implementation procedures, early heading and flowering period, and operability. strong effect

Inactive Publication Date: 2014-10-22
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Most of the genes that have been reported to control rice flowering time play a role by controlling the expression of the florigen gene Hd3a / RFT1, but no genes that affect rice flowering by regulating the expression of OsFTL1 have been isolated or cloned, and the molecular biology related to this pathway Mechanism is unclear

Method used

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  • Application of osagp13 gene and its RNA interference fragments controlling rice flowering
  • Application of osagp13 gene and its RNA interference fragments controlling rice flowering
  • Application of osagp13 gene and its RNA interference fragments controlling rice flowering

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1 RNA Extraction and Reverse Transcription

[0023] The mortar, pestle, stainless steel spoon and other utensils needed in the RNA extraction process can be used after baking in an oven at 180°C for 18 hours. Quick-freeze the pestle and mortar with liquid nitrogen, place the young rice flower spikes (<3cm) in the mortar, and use the pestle to grind the material into powder (repeatedly add liquid nitrogen 2-3 times); Quick-freeze the stainless steel medicine spoon with liquid nitrogen, transfer 50-100mg of powdered material to a 1.5mL RNase-free EP tube containing 1mL RNA-solv extract (OMEGA), mix it upside down, and let stand at room temperature for 5min; add 0.2mL chloroform, shake vigorously for 15s, let stand on ice for 10min; centrifuge at 12000g 4°C for 10min, transfer 80% supernatant to a new 1.5mL RNase-free EP tube; add 500μL isopropanol, mix by inverting and place at room temperature 10min; centrifuge at 12,000g at 4°C for 10min; discard the supernatan...

Embodiment 2

[0027] Embodiment 2 Construction of OsAGP13-RNAi vector

[0028] The primer sequences used in Example 2 are shown in Table 1, and the primers were all synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0029] Table 1 Primer list

[0030]

[0031] (1) Obtaining OsAGP13-RNAi fragment (RNA interference fragment of OsAGP13 gene):

[0032] Using <3cm rice panicle cDNA as template, PCR reaction was carried out using primers OsAGP13-RNAi FP and OsAGP13-RNAi RP synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0033] PCR reaction system: dd H 2 O34μL, 10×Taq buffer5μL, MgCl 2 (25mM) 4μL, dNTPs (10mM) 1μL, OsAGP13-RNAi FP (10μM) 2μL, OsAGP13-RNAi RP (10μM) 2μL, cDNA 1μL, Taq DNA polymerase (1U / μL) 1μL; total volume 50μL, PCR reaction system Components were from Fermentas.

[0034] PCR reaction program: 94°C 2min; 94°C 20sec, 55°C 30sec, 72°C 1min, 30 cycles; 72°C 5min; 4°C storage. After the reaction, the OsAGP13-RNAi fragment was purified with a PCR product r...

Embodiment 3

[0037] Example 3 Agrobacterium-mediated genetic transformation of rice callus

[0038] (1) Induction of callus and subculture of rice seeds: Peel off the glumes of "Nipponbare" seeds at the wax ripening stage of rice, and place them in a 37°C incubator to dry for 12 hours; After washing with sterile water for 4 times, drain the water; use 70% (v / v) alcohol to shake and wash at a speed of 100rpm for 1 min, then pour off the alcohol quickly; after washing with sterile water for 4 times, drain the water ; Add mercury chloride with a concentration of 0.1% (m / v) (the seeds can be soaked in mercury chloride), place on a shaker and wash at a speed of 90rpm for 20 minutes, and use sterile water to wash 4-5 times after rinsing Pour off the sterile water, spread the rice seeds evenly on the sterile filter paper to remove the residual moisture on the surface, dry them for 30 minutes, inoculate the dried seeds into the induction medium, and place them in a dark incubator at 30°C. Cultiva...

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Abstract

The invention relates to an OsAGP13 (Oryza sativa Annotation Gigantea Project 13) gene for controlling rice flowering and application of an RNA (Ribonucleic Acid) interference fragment of the OsAGP13 gene, belonging to the field of plant genetic engineering; the nucleotide sequence of the RNA interference fragment of the OsAGP13 gene is represented by SEQIDNO:1; an OsAGP13-RNAi carrier is constructed by utilizing a genetic engineering technology; an OsAGP13-RNAi transgenic plant having an advanced heading and flowering period is obtained through a transgenic technology; the invention also proves that expression of an OsFTL1 gene is inhibited by the OsAGP13 gene; in the OsAGP13-RNAi plant, due to inhibition of expression of the OsAGP13 gene, high expression of the OsFTL1 gene is caused, so that rice is promoted to flower in advance; and the result shows that rice flowering can be controlled by the OsAGP13 gene. The OsAGP13 gene and the interference fragment of the OsAGP13 gene disclosed by the invention can be used for researching molecular biological mechanisms influencing rice flowering and controlling rice flowering.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to an application of OsAGP13 gene and RNA interference fragment thereof for controlling flowering of rice. Background technique [0002] Photoperiod is a determinant factor affecting plant flowering. Rice is a typical short-day plant, and rice grown under short-day conditions will flower earlier. A series of genes affecting rice flowering have been cloned from rice: (1) The expression pattern of OsGI (Oryza sativa Gigantea) gene is controlled by circadian rhythm. Overexpression of the OsGI gene, whether in long-day or short-day conditions, rice flowering is delayed, indicating that OsGI can inhibit rice flowering (Hayama et al., Adaptation of photoperiodic control pathways produces short-day flowering in rice.Nature, 422:719 -722.). (2) The expression of the Hd1 (Heading date1) gene is regulated by the circadian rhythm, and rice synchronizes the light signal with the circadi...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63A01H5/00
Inventor 赵洁马浩力马腾飞王昕张嘉
Owner WUHAN UNIV
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