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Taxodium 'Zhongshansha' promoter ProThADH1 specifically expressed in floral organs and application of promoter

A promoter and flower organ technology, which is applied in the field of plant genetic engineering, can solve the problems such as unreported flowering regulation of chinensis plants, and achieve the effects of reducing the consumption of plant resources and improving the breeding process.

Active Publication Date: 2021-04-13
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are very few studies on the flowering genes of forest trees at home and abroad, and there is no report on the flowering regulation of baldwood plants.

Method used

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  • Taxodium 'Zhongshansha' promoter ProThADH1 specifically expressed in floral organs and application of promoter
  • Taxodium 'Zhongshansha' promoter ProThADH1 specifically expressed in floral organs and application of promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Extraction and Purification of Zhongshan Shanshan 406 Genomic DNA

[0020] Zhongshan Shanshan 406 was collected from the nursery of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province (35°50′N, 45°70′E) in June 2020, and the DNA was extracted using the Plant Genomic DNA Kit (Plant Genomic DNA Kit ), the operation steps are as follows:

[0021] (1) Take 1 mL of buffer solution GP1 into a 2 mL centrifuge tube and preheat at 65°C; at the same time, take 200 mg of Zhongshan Shanshan 406 cutting seedling leaves, wash them with deionized water, and grind them fully in liquid nitrogen;

[0022] (2) Quickly transfer the finely ground sample powder to the preheated 1mL GP1 buffer (add 10μL β-mercaptoethanol to the preheated 1mL buffer GP1 before adding the sample), vigorously invert and mix well, and then bathe in water at 65°C for 20min ;

[0023] (3) Add 1mL of chloroform, mix well, centrifuge at 12000rmp for 5min, transfer the upper aqueous pha...

Embodiment 2

[0033] Example 2: Cloning of the ProThADH1 promoter sequence of Zhongshan fir

[0034] The cloning of the ProThADH1 promoter sequence of Zhongshanshan fir was performed strictly according to the instructions of the Genome Walking kit of TAKARA Company. Primers were designed based on the DNA of Zhongshanshanshan 406 gene DNA as a template and the sequence of Zhongshanshanshanshan ThADH1 gene (GenBank ID: AWL83216) obtained earlier as a template. The sequence was designed using Oligo7 software to design gene-specific primers SP1 (5'-TAGTATAAATTTGCGAAGCGAATCTAAAAAACCTTTTTA-3'), SP2 (5'-GTGTTAGCTCTTATGATCGAACACTGTCAAATTTCTCAT-3'), SP3 (5'-CGTGAAAGACTATGCCGTGAATTAATGAATAACCGACGC-3'). The main precautions are as follows: the length of the primer is 18-30bp; the Tm value is 50-65°C, and the primer should meet the GC content of 40%-60%; the number of consecutive complementary bases of the primer itself should be less than 4 to avoid the accumulation of purine or pyrimidine; The 3' en...

Embodiment 3

[0044] Example 3: Construction of Plant Expression Vector of Zhongshan Shanshan ProThADH1 Promoter Sequence and Transformation of Vector Agrobacterium

[0045] Using Gateway directional cloning technology, the kit is BP / LR Clonase II enzyme Mix (Invitrogen, USA), and the target gene fragment is transferred to the target expression vector, including two parts of BP reaction and LR reaction. The target expression vector used in this example is the binary expression vector Pcambia1301. The vector contains the 35S promoter element and the GUS site, which is kana resistant. The vector structure is as follows: figure 1 shown.

[0046] (1) The purpose of the BP reaction is to transfer the gene fragment to the entry vector. Reaction system: 10-20ng of Zhongshan fir ThADH1 promoter sequence fragment, 1μL of Salt solution, 1μL of pCRTM8 / GW / TOPOTM vector (entry vector), add Nuclease- Free Water to a total volume of 6 μL, mix gently, react at 22°C for 60 minutes, and then transfer to ice...

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Abstract

The invention discloses a taxodium 'Zhongshansha' promoter ProThADH1 specifically expressed by floral organs and application of the promoter, and belongs to the technical field of plant genetic engineering. According to the invention, a 1717 bp promoter sequence of a taxodium 'Zhongshansha' ThADH1 gene is cloned through a Genome Walking technology and is as shown in SEQ ID NO.1, a Pcambia 1301 expression vector of the promoter sequence is constructed through a pathway cloning technology, arabidopsis thaliana genetic transformation is further conducted, and after GUS dyeing, it is found that the taxodium 'Zhongshansha' promoter ProThADH1 has the promoter activity and is specifically expressed in anthers, stamens and stigmas of arabidopsis thaliana. Therefore, by applying the promoter provided by the invention, the specific expression of the corresponding target gene in the floral organs of the plant can be induced in the plant genetic engineering, and the breeding process of excellent varieties is improved.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a Zhongshan fir promoter ProThADH1 specifically expressed in flower organs and its application. Background technique [0002] "Taxodium'Zhongshansha'" is a collective name for the excellent clones bred by the Institute of Botany, Chinese Academy of Sciences in Jiangsu Province from the hybrid combination of Taxodium, Sequoia and Mexican Taxodium. It has fast growth, water resistance, and salt-alkali tolerance. , Resistance to diseases and insect pests and other advantages. Due to its outstanding ability to withstand flooding, it has played an important role in the construction of wetland ecosystems in Yunnan Dianchi Lake, Anhui Chaohu Lake and the Three Gorges Reservoir area in recent years. At present, the breeding work of Zhongshan fir has been carried out for many years, but due to the large difference in the flowering time of bald cypress, b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/84C12N1/21C12N15/65C12N5/10A01H5/02A01H6/20A01H6/00C12R1/01
CPCC07K14/415C12N15/8231C12N15/65
Inventor 宣磊华建峰王芝权杨颖於朝广殷云龙
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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