Potato KNOX transcription factor StKNOX1 gene, encoded protein and application thereof

A technology for transcription factor and protein encoding, applied in the field of potato KNOX transcription factor StKNOX1 gene, encoding protein

Active Publication Date: 2020-09-01
宁夏农林科学院农业生物技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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Method used

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  • Potato KNOX transcription factor StKNOX1 gene, encoded protein and application thereof
  • Potato KNOX transcription factor StKNOX1 gene, encoded protein and application thereof
  • Potato KNOX transcription factor StKNOX1 gene, encoded protein and application thereof

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Experimental program
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Embodiment 1

[0037] Cloning of Potato KNOX Transcription Factor StKNOX1 Gene

[0038] The present invention uses RT-PCR technology to isolate and clone the potato KNOX transcription factor StKNOX1 from the potato cultivar'Longshu 3'. The sequences of all primers are as follows:

[0039] TF: AGAGGTAATAAAAGATGCTT (SEQ ID NO. 2);

[0040] TR: TTAAAATGAATCTATAAATTA (SEQ ID NO. 3).

[0041] The specific cloning method is as follows: first extract the total RNA of potato leaves with the kit, see the instructions for the specific process, and then use the reverse transcription kit (purchased from Tiangen) to reverse transcription of the mRNA into total cDNA. This template uses TF and TR as primers for PCR amplification. The PCR amplification reaction program is: 95°C pre-denaturation for 5 minutes; 95°C denaturation for 30s, 50°C annealing for 30s, 72°C extension for 80s, 35 cycles; 72°C Finally extend for 10 minutes. The PCR amplification reaction system is: 25μL PCR amplification system contains 12.5...

Embodiment 2

[0044] Construction of Expression Vector of Potato KNOX Transcription Factor StKNOX1 Gene

[0045] Add NcoI / SpeI restriction sites on both sides of the primers of the StKNOX1 gene amplified in Example 1, and use the constructed pMD-18T vector as a template for PCR amplification, and clone the amplified product into pMD-18T Vector, the specific steps are the same as the cloning method in Example 1. After the positive strain is obtained, the plasmid DNA is advanced, and the plasmid DNA with the full length of the target gene is digested with NcoI / SpeI. At the same time, the plant expression vector pcambia1302 is digested with NcoI / SpeI. The respective digested products are subjected to 1% agarose gel. After electrophoresis, the target band was recovered with a gel recovery kit (purchased from Tiangen), and the target gene fragment and pcambia1302 vector fragment were mixed in a ratio of 1:2, and T4 DNA ligase (purchased from Promga) 1U, 1 ×Reaction buffer, sterile water supplement...

Embodiment 3

[0047] The recombinant plasmid prepared in Example 2 was transformed into Agrobacterium GV3101 by the heat shock transformation method, and the positive spots were screened with LB solid resistance plates containing 100 mg / L rifampicin (Rif) and 50 mg / L kana (Km). Pick several positive spots in LB liquid medium containing 100mg / L of rifampicin (Rif) and 50mg / L of kana (Km) and incubate overnight at 180r / min at 28°C, and take 1uL as a template for PCR of recombinant plasmids The strains confirmed to be positive are stored for subsequent genetic transformation.

[0048] 3. Genetic transformation of potato KNOX transcription factor StKNOX1 gene

[0049] Pick a single colony and inoculate it with 50mg·L -1 Kan's 50ml LB liquid medium was cultured in a constant temperature shaker at 28°C, 180rpm shaking for 24h, and then used for dip transformation. Cut the sterile tube potato into slices about 1 to 2 mm, and immerse in the Agrobacterium solution for 15 minutes. After taking it out, t...

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Abstract

The invention provides a potato KNOX transcription factor StKNOX1 gene, an encoded protein and an application of the potato KNOX transcription factor StKNOX1 gene in potato planting or potato breeding, and belongs to the technical field of gene engineering. The nucleotide sequence of the potato KNOX transcription factor StKNOX1 gene provided by the invention is as shown in SEQ ID No. 1. The StKNOX1 gene is transferred into a potato plant and is obtained by analyzing the physiological properties of the potato with the transferred StKNOX1 gene, the expression quantity of the StKNOX1 gene in a transgenic positive strain is remarkably higher than that of a control group, and the plant height, the single-plant tuberization number and the single-plant yield of the three positive strains are remarkably higher than those of the control group; meanwhile, the transgenic lines show the characters of increasing the branch number, increasing the length-width ratio of tubers, changing the shapes ofthe tubers and the like. Therefore, the invention provides the application of the StKNOX1 gene in potato planting or potato breeding.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a potato KNOX transcription factor StKNOX1 gene, coding protein and applications thereof. Background technique [0002] Potato (Solanum tuberosum) is the fourth largest food crop after rice, wheat, and corn. It occupies an important position in the production of food, vegetables, feed and processing materials in my country. The tuber of the potato is its reproduction, storage and economic organ. The tuber has a high content of five basic nutrients and is a model plant for studying the formation mechanism of the storage organ. [0003] The size and shape of potato tubers are very important agronomic traits that directly affect their quality, so they are also an important screening target in the selection and breeding of potato varieties. Under normal growth conditions, the mature tubers of each variety have a definite shape, which can be used as an important basis...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/11C07K14/415C12N15/84A01H5/04A01H5/00A01H6/82
CPCC07K14/415C12N15/8261C12N15/8297C12N15/8291
Inventor 甘晓燕宋玉霞巩檑张丽聂峰杰石磊陈虞超杨文静刘璇
Owner 宁夏农林科学院农业生物技术研究中心
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