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Use of UDPG pyrophosphorylase in rice

A technology of uridine diphosphate and glucose coke, applied in the directions of application, biochemical equipment and methods, and introduction of foreign genetic material using a carrier, can solve the problems of changing rice pollen fertility, rice yield traits and other problems, and achieve complete abortion and plant growth. Tall, vigorous growth effect

Inactive Publication Date: 2005-05-11
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, people have not found that the pollen fertility of rice can be changed and the yield traits of rice can be improved by regulating the expression of uridine diphosphate glucose pyrophosphorylase gene

Method used

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  • Use of UDPG pyrophosphorylase in rice
  • Use of UDPG pyrophosphorylase in rice
  • Use of UDPG pyrophosphorylase in rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Enhanced expression vector construction of uridine diphosphate glucose pyrophosphorylase gene

[0050] like figure 1 As shown in A, two primers were used in this experiment, the nucleic acid sequences of which were UGPF (5'-ATTGGATCCATGGCGGTCGCCGCCGACGTG-3') and UGPR (5'-CCGGGATCCTCAAAGATCCTCCGGACCGTTG-3'). Using UGPF / UGPR as a primer combination, using the plasmid cloned with the full-length gene of rice uridine diphosphate glucose pyrophosphorylase as a template DNA, by PCR (94°C 3min; 94°C 1min, 68°C 1min30s, 26cycles; 68°C 10min ) to synthesize and obtain the full-length gene of rice uridine diphosphate glucose pyrophosphorylase. Cut the uridine diphosphate glucose pyrophosphorylase gene with restriction endonuclease BamHI at 37°C, and connect it into the intermediate vector pU1301 digested with restriction endonuclease BamH1 and dephosphorylated by alkaline phosphatase , so that the uridine diphosphate glucose pyrophosphorylase gene is placed between t...

Embodiment 2

[0051] Example 2: RNAi vector construction of uridine diphosphate glucose pyrophosphorylase gene

[0052] like figure 1 As shown in B, four kinds of primers were used in this experiment, and their nucleic acid sequences are respectively:

[0053] F1 (5'-TCTCTCGAGTCTAGACCTTATTGTGATTC 3')

[0054]R1 (5'-ATTAAGCTTATTCACATGCTCATCAGGGAC 3')

[0055] F2 (5'-CCTCTCGAGCCTTATTGTGATTCAAATTGAG 3')

[0056] R2 (5'-GAAAAGCTTGAATTCATTCACATGCTCATC 3')

[0057] A. The partial coding region of the uridine diphosphate glucose pyrophosphorylase gene is forward inserted into the first polylinker site of the pHANNIBAL vector:

[0058] 1. Amplification of the target fragment Using the primer pair F1 / R1 to amplify the coding region of the uridine diphosphate glucose pyrophosphorylase gene;

[0059] 2 Digestion and ligation The purified PCR product was double digested with Xho I and EcoR I and then ligated to the same double digested pHANNIBAL vector;

[0060] 3 Identification of recombinant pl...

Embodiment 3

[0067] Example 3: Agrobacterium-mediated transformation and screening of transgenic rice plants

[0068] A. Callus induction

[0069] 1. Peel off the glumes and glumes of the seeds, select mature and plump rice seeds, and put them into a 50ml centrifuge tube.

[0070] 2. Wash 3 times with 30ml distilled water, soak in 70% ethanol for 2min, wash 3 times with distilled water, and then use 0.15% HgCl 2 Soak for 15-20min while shaking at 100rpm.

[0071] 3. Open the centrifuge tube on a sterile table and pour off the HgCl 2 , After washing the seeds with sterile water for 4 or 5 times, pour the seeds on sterilized filter paper and let them stand for about 1 hour.

[0072] 4. Put the seeds into N6 solid medium (10 grains / 25ml / bottle), with the embryos facing up or in contact with the medium, culture in the dark at 28° C. for 4 weeks, and induce rice callus.

[0073] 5. Observe the induced callus, separate the light yellow, dense embryogenic callus from the small shield, transfe...

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Abstract

The invention was involved in the application of uridine 5'diphosphate glucose pyrophosphorylase gene in the rice field. Uridine 5'diphosphate glucose pyrophosphorylase gene was connected with intensified or inhibited expression vector, which was intensified or, inhibited and transferred expression vector into rice, then, uridine 5'diphosphate glucose pyrophosphorylase gene in rice was intensified or inhibited. The rice that was intensified by uridine 5'diphosphate glucose pyrophosphorylase gene had rapid growth, long ears, many seeds and high yields per plant, while the rice that was inhibited by uridine 5'diphosphate glucose pyrophosphorylase gene showed that male sterility was barren and its pollen was with 100% sterility. It was with self-sterility while cross was normal. The invention was found to provide a new path for improving rice species applying uridine 5'diphosphate glucose pyrophosphorylase gene.

Description

technical field [0001] The invention belongs to the field of transgenic plants and relates to the application of a uridine diphosphate glucose pyrophosphorylase gene. Specifically, the present invention relates to the use of the uridine diphosphate glucose pyrophosphorylase gene to change the pollen fertility and yield traits of rice, and its application in breeding new sterile lines and high-yielding new varieties. Background technique [0002] UDP-Glucose Pyrophosphorylase (UGPase) was first discovered in yeast cells by Munch Petersen in 1953 (Munch Petersen A, Kalckar HM, Cutolo E et al 1953 Uridyl transferase and the formation of uridine triphosphate . Nature 172:1036-1037). The protein is widely distributed in nature, and has been found in many animals and plants and has been isolated and purified. Uridine diphosphate glucose pyrophosphorylase catalyzes a magnesium-dependent reversible reaction that transfers uridine from uridine diphosphate gl...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N15/54C12N15/82
Inventor 何光存陈荣智祝莉莉何瑞锋
Owner WUHAN UNIV
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