Application of aquaporin gene in construction of interaction carrier capable of interacting with Hpa1Xoo

An aquaporin and carrier technology, applied in the biological field, can solve the problem that the reporter gene cannot be activated

Inactive Publication Date: 2012-01-04
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conversely, if there is no interaction between the tested proteins, BD and AD cannot be combined, and the transcription of the reporter gene cannot be activated.

Method used

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  • Application of aquaporin gene in construction of interaction carrier capable of interacting with Hpa1Xoo
  • Application of aquaporin gene in construction of interaction carrier capable of interacting with Hpa1Xoo
  • Application of aquaporin gene in construction of interaction carrier capable of interacting with Hpa1Xoo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Yeast two-hybrid system vector construction

[0044]Using ExTaq enzyme (purchased from takara) to amplify Hpa1 by PCR method from genomic DNA of rice bacterial leaf spot Xoo and its Hpa1Xoo protein N-terminal 53 amino acid deletion sequence - ΔNT, from Arabidopsis ecotype Col-0 (seed in http: / / www.arabidopsis.org - purchase, grow to two leaves) amplify PIP1 in genomic DNA; 4 (SEQ ID No.1), amplification primer is shown in Table 1, and amplification condition is as follows: Hpa1 Xoo : 95°C, 5min; 95°C, 30sec, 55°C, 45sec, 72°C, 1.5min, 30cycles; 72°C, 10min; ΔNT: 95°C, 5min; 95°C, 30sec, 55°C, 45sec, 72°C, 1min , 30cycles; 72°C, 10min; PIP1; 4: 95°C, 5min; 95°C, 1min, 57°C, 45sec, 72°C, 2min, 30cycles; 72°C, 10min.

[0045] Using the method of TA cloning, the Hpa1 Xoo , ΔNT and PIP1; 4 were cloned into pMD19-T simple vector (purchased by takara company), selected positive clones, sent samples for sequencing and saved positive clones, after the sequencing wa...

Embodiment 2

[0049] Example 2 Verification of yeast two-hybrid system

[0050] Extract pCL1, pGADT7-T, pGBKT7-53, pGBKT7-Lam (purchased by Clontech Laboratories, Inc.), pGADT7::PIP1; 4, pGBKT7::Hpa1 Xoo , pGBKT7::ΔNT, pGADT7::Hpa1 Xoo , pGADT7::ΔNT and pGBKT7::PIP1;4 plasmids.

[0051] The specific method is verified according to the method on Clontech Laboratories, Inc. Yeast Protocols Handbook FOR RESEARCH USE ONLY PT3024-1. The strain used is yeast Y190 (Clontech Laboratories, Inc. Yeast Protocols Handbook FOR RESEARCH USE ONLY PT3 024-1).

[0052] Spread a common sterilized filter paper in a Petri dish, add 2.5-5.0ml Z buffer / X-gal solution (see Yeast Protocols Handbook) to wet the filter paper; then put chromogenic filter paper (Whatman 5# filter paper or Grade 410 filter paper) cut into a rectangle, put it in a petri dish, add a small amount of sterilized water to moisten it, pick up the colony with a toothpick and gently draw a line on the filter paper, freeze it directly in liqu...

Embodiment 3

[0053] Example 3 Membrane yeast two-hybrid system PIP1; 4 and Hpa1 Xoo Vector construction

[0054] Use pGADT7::PIP1 obtained in Example 1; 4, pGBKT7::Hpa1 Xoo , pGBKT7::ΔNT as a template, using the primers in Table 2, wherein PIP1; 4 genes were amplified using 1PIP1; 4 and 2PIP1; 4 primer pairs respectively, and the amplification conditions were: 95°C, 5min; 95°C, 50sec, 61 ℃, 45sec, 72℃, 1min, 30cycles; 72℃, 10min, the amplified product was cloned into pMD19-T simple vector (takara company) by TA cloning, positive clones were selected, sent for sequencing and saved positive clones for sequencing After being correct, extract the plasmid of the positive clone, digest it with Sfi I (takara company), and cut the gel to recover the target fragment; connect the above-mentioned cloning products obtained by using 1PIP1; 4, 2PIP1; 4 amplification to pBT3-N, pBT3-STE respectively Plasmids (Dualsystems Biotech) obtained pBT3-N::PIP1; 4, pBT3-STE::PIP1; 4 (eg image 3 ); Hpa1 Xoo An...

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Abstract

The invention belongs to the field of biotechnology and discloses application of an aquaporin gene in the construction of an interaction carrier capable of interacting with Hpa1Xoo. The interaction relationship between a plant aquaporin PIP1;4 and the Hpa1Xoo is obtained through a yeast two-hybrid system, membrane yeast two hybrid, pull-down assay, bimolecular fluorescence complementation and other protein interaction technologies. Therefore, the aquaporin PIP1;4 as shown in SEQ ID No. 1 (sequence identity number 1) can be applied to the construction of the interaction carrier enabling an expression product of the aquaporin PIP1;4 to interact with the Hpa1Xoo.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a kind of aquaporin gene in construction and Hpa1 Xoo The application of interactive interaction vectors. Background of the invention [0002] Aquaporin (AQP) refers to the membrane intrinsic protein that can selectively and efficiently transport water molecules and some small molecular solutes on the cell membrane. It belongs to the MIP (major intrinsic protein) superfamily, with a molecular weight of 23-31ku. AQP1 protein is a typical aquaporin in mammals that only transports water molecules. According to the sequence conservation of N and C terminals, Arabidopsis thaliana aquaporins PIP1 and PIP2 were successively identified. So far, water has been found in various plants such as Arabidopsis thaliana, tobacco, corn, pea, rice, barley leaf epidermis, astringer, tomato root, sugar beet storage tissue, zucchini seed and sunflower hypocotyl parenchyma cells. Channel proteins, and AQP...

Claims

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Application Information

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IPC IPC(8): C12N15/81
Inventor 董汉松桑素玲刘昌来尤真真徐衡
Owner NANJING AGRICULTURAL UNIVERSITY
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