A kind of eucalyptus tps gene, rna interference carrier and application

A technology of RNA interference and carrier, which is applied in the field of RNA interference carrier, can solve the problems of reducing the allelopathic effect of eucalyptus, and achieve the effects of overcoming the long cycle, reducing the content of allelochemical substances, and high-efficiency allelopathic effect

Inactive Publication Date: 2018-06-12
CHINA EUCALYPT RES CENT
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  • Summary
  • Abstract
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Problems solved by technology

[0005] However, the key regulatory genes of a-terpineol and eucalyptol in eucalyptus are still unrecorded, and there is no record of using them to construct interference vectors and apply them to eucalyptus to reduce the allelopathic effect of eucalyptus, so it is necessary to carry out research in this direction further research

Method used

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  • A kind of eucalyptus tps gene, rna interference carrier and application
  • A kind of eucalyptus tps gene, rna interference carrier and application
  • A kind of eucalyptus tps gene, rna interference carrier and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1, the acquisition of full-length cDNA of Eucalyptus TPS gene

[0070] Step 1. Obtaining a fragment of the conserved region of the TPS gene of Eucalyptus: using the conserved functional region of the TPS gene of various plants as a template, a partial cDNA fragment of the TPS gene was obtained by RT-PCR.

[0071] (1) Comparative analysis of the TPS genes of tobacco (GenBank accession No.L04680), fir (GenBank accession No.U87910), sage, mint (GenBank accession No.L13459) and other plants, using the conserved region as a template , designed the primer pair TPS-F1 and TPS-R1 as described in SEQ ID NO 1 and SEQ ID NO 2; (2) extract the total RNA of eucalyptus leaves, utilize the reverse transcriptase RevertAid M that cDNA reverse transcription kit provides -MuLV Reverse Transcriptase and Oligo dT18 primers were reverse-transcribed into total cDNA; (3) Using the transcribed cDNA as a template and using SEQ ID NO 1 and SEQ ID NO 2 as primers for RT-PCR amplificatio...

Embodiment 2

[0081] Embodiment 2, reduce the construction of eucalyptus allelopathy RNA interference carrier

[0082] Step 1, design the RNA interference fragment primer of Eucalyptus TPS gene

[0083] According to the TPS gene sequence and the analysis of multiple cloning sites on the pKANNIBAL vector, Xho I and KpnI can be used to connect the forward fragment of the target gene to the vector; and Hind III and Xba I can be used to connect the reverse fragment to the vector. Therefore, the following primers were designed:

[0084] TPS-RNAi-F1:5'-CATG CTCGAG TGCCTTGACGGACTTCAA-3' (the underlined part is the Xho I restriction site)

[0085] TPS-RNAi-R1:5'-CTGA GGTACC ATGCTTGGGCTTGCTAAGA-3' (the underlined part is the Kpn I restriction site)

[0086] TPS-RNAi-F2:5'-CATG AAGCTT ATGCTTGGGCTTGCTAAGA-3' (the underlined part is the Hind III restriction site)

[0087] TPS-RNAi-R2:5'-CTGA TCTAGA TGCCTTGACGGACTTCAACGT-3' (the underlined part is the Xba I restriction site)

[0088] Step 2....

Embodiment 3

[0097] Example 3, RNA interference vector electroporation transformation of Agrobacterium tumefaciens EHA105

[0098] Since the heat shock transformation efficiency of the recombinant plasmid pART 27-TPS-RNAi into Agrobacterium EHA105 is low, the present invention adopts the electric shock transformation method to obtain the recombinant Agrobacterium containing the target gene. Specific steps are as follows:

[0099] Step 1. Mix 1 μg of plasmid pART 27-TPS-RNAi with 150 μl of Agrobacterium EHA105 competent cells, add them into a sterilized electric shock cup, and perform electric shock transformation at a voltage of 2.5KV.

[0100] Step 2, then add 800 μl of liquid medium to rinse the electric shock cup and transfer to a 1.5ml centrifuge tube, culture at 28°C, 220rpm for 2h.

[0101] Step 3. Spread the culture solution on a YEB solid plate (kanamycin 50 μg / ml; rifampicin 50 μg / ml), and culture at 28° C. for about 18 hours.

[0102] Step 4. Pick a single colony and put it in ...

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Abstract

The present invention obtains the terpene synthase (Terpene Synthase, TPS) gene by PCR and RACE technology, and the sequence is shown in SEQ ID NO15, and the sequences shown in SEQ ID NO23 and SEQ ID NO26 are selected as forward fragments and reverse fragments respectively The fragment constructs the RNA interference vector pART27‑TPS‑RNAi that reduces the content of eucalyptus allelochemicals. The vector was transformed into eucalyptus, and plants with low allelochemical substances were successfully obtained. The invention provides an important allelochemical synthetase gene, an RNA interference expression carrier and its application for the genetic engineering of reducing the allelochemical content of eucalyptus, and provides a new idea for the application of RNA interference and transgenic technology in tree molecular breeding. Achieved the expected goal of using the principle of RNA interference to obtain new materials of eucalyptus with low allelochemicals, provided a successful example for RNA interference-mediated eucalyptus molecular breeding and genetic engineering, and made up for the RNA interference-mediated reduction of allelochemicals in this field Gaps in research.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a eucalyptus TPS gene, an allelopathy-reducing RNA interference vector for eucalyptus genetic transformation constructed by using it, and its application on eucalyptus. Background technique [0002] Eucalyptus (Eucalyptus) has the characteristics of various types, fast growth, resistance to barrenness, good stem shape, and wide application. However, the large-scale introduction and planting of eucalyptus has also brought some controversial ecological and environmental problems, such as soil degradation, decline in land productivity, and weakening of biodiversity. Among them, allelopathy is an ecological phenomenon that has attracted much attention in the eucalyptus artificial wood ecosystem. Its manifestation is to inhibit the growth of microorganisms in other plants, understory vegetation and even soil, resulting in simple forest community structure, weak...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12N15/10C12N15/63C12N15/66A01H5/00
Inventor 陈鸿鹏谢耀坚
Owner CHINA EUCALYPT RES CENT
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