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Plant abiotic stress inducible expression promoter pTaPI1A and application thereof

A technology of abiotic stress and inducible expression is applied in the field of abiotic stress inducible expression promoter pTaPIP1A in plants, which can solve the problems of reduced yield, slow plant growth, and impact on plant development.

Active Publication Date: 2016-05-25
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Constitutive promoters can efficiently express the target gene throughout the growth and development of the plant, but it is accompanied by a large amount of nutrient consumption, which affects the development of the plant, manifested as slow plant growth, short stature, and reduced yield.

Method used

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  • Plant abiotic stress inducible expression promoter pTaPI1A and application thereof
  • Plant abiotic stress inducible expression promoter pTaPI1A and application thereof
  • Plant abiotic stress inducible expression promoter pTaPI1A and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Promoter pTaPIP1A clone

[0022] (1) Extract the gDNA of Wheat Konon 9204

[0023] Use the Plant Genomic DNA Extraction Kit (Tiangen, DP305) to extract DNA, and the specific steps are as follows: ①Take about 100 mg of leaves of Wheat Kenon 9204, add liquid nitrogen and grind them thoroughly; ②Quickly transfer the ground powder to a pre-installed 700μl, 65°C preheated buffer GP1 centrifuge tube (before the experiment, add β-mercaptoethanol to the preheated GP1 to make the final volume percentage concentration 0.1%), quickly invert and mix well, then put the centrifuge tube in Water bath at 65°C for 20 minutes. During the water bath, invert the centrifuge tube to mix the sample several times; ③Add 700μl chloroform, mix thoroughly, and centrifuge at 12000rpm for 5 minutes; ④Carefully transfer the upper aqueous phase obtained in the previous step into a new centrifuge tube, add 700μl buffer GP2, mix thoroughly; ⑤ transfer the mixed liquid into the adsorption col...

Embodiment 2

[0034] Example 2 Promoter pTaPIP1A Induced downstream genes TaPIP1A Specific analysis of expression in wheat tissues

[0035] (1) Material cultivation: Wheat was planted in the field, and roots (root), stem (stem), flag leaf (flagleaf), leaf rachis (rachis), ear (heading spike), cob (cob), and lemma ( Tissues of lemma), lemma (glumelle), stamen, pistil (pistil) and awn (awn) were frozen in liquid nitrogen and stored at -80°C.

[0036] (2) Use Trizol (Invitrogen) to extract total RNA, the specific steps are as follows: ① Grind 50-100 mg wheat tissue with liquid nitrogen, transfer it to an EP tube containing 1 ml Trizol reagent after grinding, mix well, and let stand at room temperature for 5 minutes; ② Add 0.2ml chloroform, mix well, let stand at room temperature for 2-3min; centrifuge at 12000g for 15min at 4°C; ③Transfer the upper colorless aqueous phase to a clean 1.5ml EP tube, add 0.5ml isopropanol, mix After uniformity, place at room temperature for 10 minutes; at 4°C...

Embodiment 3

[0044] Example 3 Promoter pTaPIP1A Induction of downstream target gene expression under drought stress

[0045] (1) Material cultivation and PEG-6000 artificial simulated drought treatment:

[0046] Kenon 9204 wheat seeds were sown in Petri dishes covered with 2 layers of filter paper, ddH 2 O soaking, germination for 4 days, endosperm removal, Hogland culture until the 10th day, the seedlings with consistent growth were selected and treated with PEG-6000 with a mass percentage concentration of 20%, 0h, 1h, 3h, 6h, 12h, 24h, 48h, Samples were taken at 72 hours, and stored at -80°C after freezing in liquid nitrogen;

[0047] (2) Use Trizol to extract total RNA, see step (2) of Example 2 for specific steps;

[0048] (3) Using PrimeScript TM RTreagentKitwithgDNAEraser (TaKaRa, RR047A) was used to synthesize the first-strand cDNA. For specific steps, see step (3) of Example 2;

[0049] (4) Use SYBR ? PremixExTaqII (TaKaRa, RR820A) kit was used for real-time fluorescent quant...

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Abstract

The invention discloses a plant abiotic stress inducible expression promoter pTaPI1A. The nucleotide sequence of the plant abiotic stress inducible expression promoter pTaPI1A is as indicated in SEQ ID NO: 1. The invention further discloses a recombinant expression vector containing the promoter pTaPI1A and application of the promoter pTaPI1A to plant breeding. According to the application, the promoter with the nucleotide sequence as indicated in SEQ ID NO: 1 is integrated with a target gene, a recombination inducible expression vector is constructed, the recombination inducible expression vector is transformed into plant cells or tissues or organs for breeding, a plant overground part expression target gene is induced under the abiotic stress condition, thereby new plant varieties which express the target gene under the abiotic stress condition especially under drought and high salt stress, and great significance is achieved for plant genetic engineering breeding especially for wheat genetic engineering breeding.

Description

technical field [0001] The invention relates to the technical field of biological genetic engineering, in particular to a plant abiotic stress-induced expression promoter pTaPIP1A and its application. Background technique [0002] Abiotic stress has a great impact on the yield of crops. Among various abiotic stresses, salt stress and drought stress often occur at the same time, causing dehydration of plant cells and breaking the ion balance, which in turn leads to a decrease in crop yield. Therefore, cultivating new varieties of drought-resistant and salt-tolerant crops through genetic engineering has become one of the current urgent tasks. [0003] In the process of genetic engineering breeding, the commonly used promoters include constitutive promoters and inducible promoters. The constitutive promoters include CaMV35S promoter, Ubiqitin promoter and so on. Constitutive promoters can efficiently express the target gene throughout the growth and development of the plant,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
CPCC07K14/415C12N15/8273
Inventor 张玮韩洁李俊明纪军崔法苏倩男刘磊
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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