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Primers for cloning tea tree lob gene and its cloning method and application

A gene and tea tree technology, applied in the field of tea tree LOB gene, primers for cloning tea tree LOB gene and its cloning

Active Publication Date: 2020-07-17
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Studies have found that the LOB genes of plants such as Arabidopsis thaliana, sweet orange, and camelina have important effects on the formation and development of specific organs in the aboveground and underground parts of plants. The research on the relationship of the synthetic relationship of tea plant LOB has not been reported yet, so it is particularly important for the cloning and other molecular studies of the tea tree LOB gene

Method used

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  • Primers for cloning tea tree lob gene and its cloning method and application
  • Primers for cloning tea tree lob gene and its cloning method and application
  • Primers for cloning tea tree lob gene and its cloning method and application

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Effect test

Embodiment 1

[0040] Example 1: Extraction of Longjing Changye Total RNA

[0041] Take 2g of Longjing long leaf leaf tissue and grind it in liquid nitrogen, and extract Total RNA according to the instructions of the column-type small plant tissue extraction kit.

Embodiment 2

[0042] Embodiment 2: Amplification reaction of tea tree LOB gene conserved domain

[0043] The primers LOB-degenerate-F and LOB-degenerate-R for amplifying the conserved sequence of the tea tree LOB gene were designed according to the known amino acid and nucleotide sequences of the LOB genes of other species, and the tea tree LOB gene was amplified using Longjing longye cDNA as a template Sequences of conserved domains. The reaction program was 94°C for 3min, 94°C for 30s, 58°C for 30s, 72°C for 30s, 35Cycles, and 72°C for 10min.

[0044] The primers LOB-degenerate-F and LOB-degenerate-R designed to amplify the conserved sequence of the tea tree LOB gene according to the amino acid and nucleotide sequences of the known LOB genes of other species are as follows: The amino acid sequence of Arabidopsis LOB gene has the amino acid sequence described in SEQ ID No.3. This sequence was then used to search the entire Nr database for amino acid sequences similar to it using BLASTp (...

Embodiment 3

[0046] Embodiment 3: tea tree LOB gene 5' end amplification reaction

[0047] A. Total RNA dephosphorylation treatment

[0048] According to the instructions of TaKaRa 5'-Full Race Kit, use Alkaline Phosphatase (CIAP) to dephosphorylate the naked 5' phosphate group in TotalRNA. The dephosphorylation reaction solution system is tea tree Total RNA (1μg / μl) 1μl, RNase Inhibitor (40U / μl) 1μl, 10×Alkaline Phosphatase Buffer (Mgcl 2 Free) 5μl, Alkaline Phosphatase (Calf intestine) (16U / μl) 0.6μl, RNase Free dH 2 O 46.9 μl. After reacting at 50°C for 1 hour. Add 20 μl of 3M CH to the above reaction solution 3 COONa (PH5.2), 130 μl of RNase FreedH 2 O, mix well. Then add 200 μl of phenol / chloroform / isoamyl alcohol (25:24:1), mix well, centrifuge at 13,000×g for 5 minutes at room temperature, and transfer the upper aqueous phase to a new Microtube. Then add 200 μl of chloroform, mix well, centrifuge at 13,000×g for 5 minutes at room temperature, and transfer the upper aqueous ph...

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Abstract

The invention discloses a primer for cloning tea tree LOB gene, a cloning method and application thereof. According to the known amino acid and nucleotide sequences of LOB genes of other species, primers for amplifying the conserved sequence of the tea tree LOB gene were designed, and then the 5' and 3' ends of the tea tree LOB gene were amplified according to the amplified conserved sequence of the tea tree LOB gene Finally, the full-length sequence of the tea tree LOB gene was obtained for the first time, and the nucleotide sequence of the gene is SEQ ID No.8. By cloning the primer set and the cloning method of the tea tree LOB gene, it can rapidly and accurately clone the complete sequence of the tea tree LOB gene. The preliminary molecular identification results showed that LOB may play a negative regulatory role in the synthesis of catechins, which laid the foundation for the subsequent research on the exact function of tea tree LOB genes and the pathways involved in the regulation of catechin metabolism.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to tea tree LOB gene, primers for cloning the tea tree LOB gene, a cloning method and application thereof. Background technique [0002] The top growth of higher plants and the successive differentiation and development of many lateral organs are the most basic growth and development issues of higher plants, which are directly related to the composition of various parts of the plant, the size of the individual and the level of economic value, so it has become a plant in recent years. An important hotspot in functional genomics research. [0003] The shoot apical meristem (SAM, shoot apical meristem) of a plant is a group of cells located at the growth point, which starts from the beginning of the embryonic morphogenesis, maintains the meristem state and runs through the growth and development of the plant. The maintenance of shoot apical meristem requires a balance between stem cells i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/11C12N15/10C12Q1/6895
CPCC07K14/415C12N15/1096C12Q1/6895C12Q2600/158
Inventor 洪高洁何宇青李林颖孙宗涛李俊敏
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES