Variable aegilops tauschii phenylalanine ammonia-lyase gene and application thereof

A kind of technology of grass phenylalanine ammonia lyase amino acid, grass phenylalanine ammonia lyase, applied in the field of volatile goat grass phenylalanine ammonia lyase nucleic acid sequence, can solve problems such as unreported PAL gene, and achieve The effect of high resistance to cereal cyst nematodes

Active Publication Date: 2019-05-17
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the biological functions of PAL have been continuously studied and reported, so far, i...

Method used

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  • Variable aegilops tauschii phenylalanine ammonia-lyase gene and application thereof
  • Variable aegilops tauschii phenylalanine ammonia-lyase gene and application thereof
  • Variable aegilops tauschii phenylalanine ammonia-lyase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Amplification of the full-length coding sequence of A. vulgaris PAL and extraction and reverse transcription of plant total RNA:

[0032] (1) Soak the seeds of No. 1 goat grass in water, put them in the refrigerator at 4°C for 2 days, spread the seeds evenly on the continuously moist filter paper and put them in a petri dish with a diameter of 5 cm. Seedlings were germinated under a photoperiod of / 8h.

[0033] (2) The roots of the seedlings 20 days after seeding were cut, ground in liquid nitrogen, and total RNA was extracted according to the Trizol reagent protocol.

[0034] (3) Synthesize cDNA by reverse transcription according to TOYOBO reverse transcription reagent method

[0035] Amplification of the full-length coding sequence and silent fragment of A. vulgaris PAL:

[0036] Using the reverse transcription product cDNA as a template, two pairs of different primers were used to amplify the full-length coding sequence of AeVPAL and the silencing fragment of AeVPA...

Embodiment 2

[0045] AeVPAL VIGS vector construction and VIGS (virus-induced gene silencing) inoculation:

[0046] (1) BSMV-α, BSMV-β, BSMV-γ:GFP plasmids were stored in a -20°C refrigerator for later use.

[0047] (BSMV-α, BSMV-β, BSMV-γ:GFP plasmids were obtained from the Institute of Genetics and Development, Chinese Academy of Sciences.)

[0048] (2) Digestion of target fragment and BSMV vector:

[0049] Restriction target fragment (silent fragment with restriction site): the restriction site is NheI site.

[0050]

[0051] Restriction digestion of BSMV-γ:GFP plasmid:

[0052]

[0053] The operation steps are: the temperature is 37°C, after 5 hours of enzyme digestion, the gel is run to recover the product. The recovery steps are the operating steps in the instructions of the agarose gel electrophoresis kit. Enzyme digestion is single enzyme digestion, the same as below. Digestion cuts the plasmid and linearizes it for subsequent ligation.

[0054] The recovery steps can be ...

Embodiment 3

[0083] Construction of the PAL gene binary vector of A. vulgaris No. 1 and its genetic transformation to wheat:

[0084] (1) AeVPAL overexpression vector construction:

[0085] The full-length coding sequence of AeVPAL was connected into the pLGY02 vector by exchange reaction, and transformed into Agrobacterium EA105, and the positive Agrobacterium was selected for subsequent transformation. The exchange reaction was performed referring to the instructions of EasyGeno Rapid Recombination Cloning Kit from Tiangen Company. The pLGY02 vector was obtained from the Institute of Crops, Shandong Academy of Agricultural Sciences.

[0086] (2) Genetically transformed wheat:

[0087] Preparation of Agrobacterium suspension: shake the bacteria one day before the test, culture at 160r, 28°C; after the wheat ears are ready, start to prepare the Agrobacterium suspension; take 1ml of the bacteria solution in a 1.5ml centrifuge tube, add 1.4μL of acetosyringone (0.1M) and mix well.

[008...

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Abstract

The invention discloses a variable aegilops tauschii phenylalanine ammonia-lyase nucleic acid sequence and application thereof. The variable aegilops tauschii phenylalanine ammonia-lyase gene nucleicacid sequence is shown as SEQ ID NO.1. The variable aegilops tauschii phenylalanine ammonia-lyase gene encodes a phenylalanine ammonia-lyase, has high resistance to cereal cyst nematodes, and has important reference and application values for breeding wheat resistance to cereal cyst nematodes.

Description

technical field [0001] The present invention relates to the field of phenylalanine ammonia-lyase, in particular to a nucleic acid sequence of A. vulgaris phenylalanine ammonia-lyase and its application. Background technique [0002] PAL (Phenylalanine Ammonia-Lyase, phenylalanine ammonia-lyase) is the first enzyme in the phenylpropane metabolic pathway, and it is also a key enzyme, which catalyzes the conversion of L-phenylalanine into trans-cinnamic acid. Trans-cinnamic acid is then transformed into secondary metabolites such as anthocyanins, lignin, flavonoids, and phytoalexins through different downstream metabolic branches, some of which have been reported to play a role in plant disease resistance. [0003] PALs in plants are homologous isoform proteins encoded by multi-gene families, which have their own expression characteristics and functions. In 1961, Conn and Koukol first discovered and purified PAL in barley seedlings. Since then, the research on PAL has graduall...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/82A01H5/00A01H6/46
Inventor 张海莉黄秋兰龙海邓光兵梁俊俊余懋群
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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