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Eucalyptus TPS gene, RNA interference vector and application thereof

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

Inactive Publication Date: 2015-10-28
CHINA EUCALYPT RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Eucalyptus TPS gene, RNA interference vector and application thereof
  • Eucalyptus TPS gene, RNA interference vector and application thereof
  • Eucalyptus TPS gene, RNA interference vector and application thereof

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Experimental program
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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 various plants such as tobacco (GenBank accession No.L04680), fir (GenBank accession No.U87910), sage, mint (GenBank accession No.L13459), and the conserved regions as Template, designed primer pair TPS-F1 and TPS-R1 as described in SEQ ID NO1 and SEQ ID NO2; (2) extract Eucalyptus leaf total RNA, 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 NO1 and SEQ ID NO2 as primers for RT-PCR amplification, the target ...

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 Kpn I 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 pART27-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 pART27-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 5m...

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Abstract

According to the invention, terpene synthase (TPS) gene is obtained through PCR and RACE technologies and sequence is as shown in SEQ ID NO.15. Sequences as shown in SEQ ID NO. 23 and SEQ ID NO.26 are respectively selected as a forward fragment and a reverse fragment to construct a RNA interference vector Part27-TPS-RNAi for reducing content of eucalyptus allelochemicals. The vector is transferred into eucalyptus so as to successfully obtain a plant with low content of allelochemicals. The invention relates to gene engineering for reducing content of eucalyptus allelochemicals and provides an important allelochemical synthase gene, the RNA interference expression vector and its application. Thus, a new idea is provided for applying RNA interference and transgenic technology to forest tree molecular breeding. The expected goal of obtaining a new eucalyptus material with low content of allelochemicals by the RNA interference principle is achieved; successful living examples are provided for the RNA interference-mediated eucalyptus molecular breeding and gene engineering; and the blank of RNA interference-mediated allelochemicals reduction in the field of study is filled.

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 Applications(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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