Poncirus trifoliata cold resistant gene PtrTZF1 and application thereof to plant cold resistant heredity improvement

A gene and plant technology, applied in the direction of plant gene improvement, application, plant peptides, etc., can solve problems such as TZF that has not yet been seen, achieve the effect of reducing agricultural production costs and achieving environmental friendliness

Active Publication Date: 2019-04-02
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the involvement of TZF in the low temperature response of citrus in the existing technology.

Method used

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  • Poncirus trifoliata cold resistant gene PtrTZF1 and application thereof to plant cold resistant heredity improvement
  • Poncirus trifoliata cold resistant gene PtrTZF1 and application thereof to plant cold resistant heredity improvement
  • Poncirus trifoliata cold resistant gene PtrTZF1 and application thereof to plant cold resistant heredity improvement

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Cloning of the full-length cDNA of the PtrTZF1 gene of Hovenia trifoliate

[0038] The cDNA of Hovenia trifoliate was used as a template and high-fidelity enzymes were used for amplification. The amplification system is shown in Table 1, and the amplification program is shown in Table 2. The sequences of the amplification primers are: 5'-CACATAAAAAGGCCCTCACC-3' and 5'-GAGCAGGGCCCTCATTTATCC-3'.

[0039] AxyPrep-96 DNA Gel Recovery Kit (Axygene, USA) was used to purify and recover the amplified product, and the purified product was compared with 18-T vector (TaKaRa, Japan) was used for ligation, and the ligation system was shown in Table 3. After incubation at 16°C for 30 min, it was transformed into Escherichia coli competent Trans5α.

[0040] Table 1 Gene amplification system

[0041]

[0042]

[0043] Table 2 Gene amplification PCR program

[0044]

[0045] 12-16 hours after transformation, pick a single clone on the plate and place it in a 1.5mL...

Embodiment 2

[0052] Example 2: Analysis of the expression of the PtrTZF1 gene under different stress conditions

[0053] The expression pattern of PtrTZF1 gene was analyzed by real-time fluorescent quantitative PCR (qRT-PCR), and the quantitative reagent was QuantiNova TM SYBRGreen PC (QIAGEN, Germany), the method refers to the instruction manual, and the reaction system is shown in Table 5.

[0054] Table 5 Quantitative PCR reaction system

[0055]

[0056] Primer 5.0 was used to design PtrTZF1 real-time quantitative primers (forward primer: 5'-GTTGCAATCTCCAACCGGGC-3'; reverse primer: 5'-GACTGGTCACTGCTCCACGA-3'), and citrus Actin was used as an internal reference gene (forward primer: 5'-CATCCCTCAGCACCTTCC- 3'; reverse primer: 5'-CCAACCTTAGCACTTCTCC-3'). The reaction procedure is shown in Table 6. After the reaction, use 2 -ΔΔCt The algorithm calculates gene expression.

[0057] Table 6 Quantitative PCR reaction program

[0058]

[0059]

[0060] The results showed that th...

Embodiment 3

[0061] Embodiment 3: Construction of PtrTZF1 gene overexpression vector

[0062] Primers were designed to amplify the full-length PtrTZF1 gene and insert it into the middle of the two restriction sites Xba I and Sma I on the pBI121 vector. The primers were designed as follows, using wild-type Citrus citrus cDNA as a template.

[0063] PtrTZF1-pBI121-F:5'-GC TCTAGA ATGGAAGGTGAACTCCCCAA-3'

[0064] PtrTZF1-pBI121-R:5'-TCC CCCGGG TTATGCCACCATCTGCTCCT-3'

[0065] Amplified fragment recovery, pMD18-T vector connection and transformation, positive clone detection and sample delivery for sequencing. Then PtrTZF1 was excised from the T vector and connected to the pBI121 vector by T4 ligase (for the schematic diagram of the vector, see figure 2 ), constructed pBI121-PtrTZF1, and transformed into GV3101 for the following examples.

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Abstract

The invention belongs to the field of plant genetic engineering, and discloses a poncirus trifoliata cold resistant gene PtrTZF1 and an application thereof to plant cold resistant heredity improvement. The PtrTZF1 gene is C3H type zinc finger protein separated and cloned from cold resistant poncirus trifoliata, and the amino acid sequence of the PtrTZF1 gene is shown as SEQID NO.2. The gene is subjected to construction of overexpression and an RNAi carrier, through agrobacterium tumefaciens mediated heredity transformation, the gene is guided into tobacco, lemons and poncirus trifoliate respectively, through biological function verification, the obtained genetic modification plant can indicate that the cloned PtrTZF1 gene has the function of improving cold resistance of plants. The gene isfound so that a new genetic resource is provided for designing and breeding of plant inverse resisting molecules, and a new heredity resource is provided for implementation of green agriculture and water-saving agriculture. The development and the utilization of the heredity resource are favorable for reduction of agricultural production cost and realization of purpose of being environmental-friendly.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering. It specifically involves the isolation and cloning of a C3H-type zinc finger protein PtrTZF1 from Poncirus trifoliata, and also involves the application of this gene in the genetic improvement of plant cold resistance. The gene is overexpressed in tobacco and lemon, and the obtained transgenic plants are resistant to The coldness is obviously improved. Background technique [0002] Low temperature is one of the important factors affecting crop yield and limiting the geographical distribution of plants. Low temperatures can damage cellular structures, affecting their critical physiological functions. Low temperature stress causes osmotic stress, which will lead to loss of turgor pressure, damage to membrane stability, inactivation or denaturation of proteins, accumulation of reactive oxygen species (Reactive Oxygen Species, ROS) and oxidative damage, resulting in inhibition of photosynth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/78A01H6/82
CPCC07K14/415C12N15/8273
Inventor 刘继红王敏戴文珊
Owner HUAZHONG AGRI UNIV
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