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Helianthus tuberosus L. Na<+>/H<+> reverse transport protein genes HtNHX1 and HtNHX2 and use thereof

An anti-transporter, Jerusalem artichoke technology, applied in the field of genetic engineering, can solve the problems of weak salt tolerance, low activity, and no improvement of the salt tolerance of Arabidopsis thaliana, and achieve the effect of improving the salt tolerance

Active Publication Date: 2014-02-26
安徽田间云生物科技有限公司
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Problems solved by technology

However, it was also reported that overexpression of AtNHX1 alone did not improve the salt tolerance of Arabidopsis (Yang et al., 2009)
Therefore, on the one hand, the NHX genes that have been cloned so far have low activity (relatively low Na + / H + translocation rate), salt tolerance is not strong (Xu et al., 2010), and there are multiple endogenous NHX genes in the plant itself, thus limiting the application of this gene to a large extent

Method used

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  • Helianthus tuberosus L. Na&lt;+&gt;/H&lt;+&gt; reverse transport protein genes HtNHX1 and HtNHX2 and use thereof
  • Helianthus tuberosus L. Na&lt;+&gt;/H&lt;+&gt; reverse transport protein genes HtNHX1 and HtNHX2 and use thereof
  • Helianthus tuberosus L. Na&lt;+&gt;/H&lt;+&gt; reverse transport protein genes HtNHX1 and HtNHX2 and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Cloning of Example 1 HtNHX1 and HtNHX2 Genes

[0033] (1) Using the cDNA of Jerusalem Artichoke No. 1 as a template, according to the plant tonoplast-type Na + / H + Design a pair of degenerate primers P1 and P2 for the homologous sequence of the antiporter gene;

[0034] (2) Using the cDNA of Jerusalem artichoke leaves treated with 200mM NaCl for 24h as a template, a fragment of about 400bp was amplified by RT-PCR,

[0035] (3) According to the sequencing results and the primers provided by Invitrogen's Gene Racer Kit, design two pairs of nested PCR primers: 5' reverse specific primer P3, nested primer P4 and 3' forward specific primer primer P5, nested primer P6 obtained about 1190bp 5' end fragment and about 765bp 3' end fragment by RLM-RACE method. The complete cDNA sequences of HtNHX1 and HtNXH2 were obtained after sequence splicing;

[0036] (4) By designing the full-length sequence primers P7 and P8, and using the cDNA of Jerusalem artichoke 1 as a template, P...

Embodiment 2

[0038] Expression characteristics of embodiment 2 HtNHX1 and HtNHX2 genes in Jerusalem artichoke leaves

[0039] (1) Extraction of total RNA; after culturing with 1 / 2 Hoagland nutrient solution for one week, treat with nutrient solution containing different NaCl concentrations for 24 hours, and then take about 0.1g of leaf samples to extract their RNA;

[0040] (2) Synthesize cDNA with a reverse transcription kit;

[0041] (3) RT-PCR and qRT-PCR; through the expression analysis of HtNHX1 and HtNHX2 in Jerusalem artichoke leaves, it was found that the expression of these two genes in leaves increased with the increase of NaCl concentration ( figure 1 ).

[0042] The primer sequences of the genes are designed in the following table:

[0043]

Embodiment 3

[0044] Example 3 Analysis of the subcellular localization of HtNHX1 and HtNHX2:

[0045] (1) According to the sequences of HtNHX1 and HtNHX2, we designed primers containing corresponding restriction sites (N-HtNHX F:

[0046] 5'-ATATAAGCTTAGATGCATGCTCGAGCGGCCGC-3' (SEQ ID NO. 21); N-HtNHX R:

[0047] 5'-TTATCCCGGGGTTTCCGGTGGTTTCTTCATC-3' (SEQ ID NO.22)), with HNHX1 or HNHX2 gene

[0048] The cDNA clone was used as a template to amplify the fragments of HNHX1 or HNHX2 without stop codon by PCR with high-fidelity enzyme KOD-Plus.

[0049] PCR products were recovered and purified by agarose gel electrophoresis and cloned into pEasy-Blunt vector. Positive clones were subjected to HindⅢ and

[0050] After SmaⅠ double enzyme digestion, it was connected to the intermediate vector pSAT6A-EGFP-N1 ( Figure 8 ), to obtain HtNHX1-GFP and

[0051] pSAT6A-EGFP-N1 vector for the HtNHX2-GFP gene. Respectively with the genes containing HtNHX1-GFP and HtNHX2-GFP

[0052] The pSAT6A-EGFP-...

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Abstract

The invention discloses helianthus tuberosus L. Na<+> / H<+> reverse transport protein genes HtNHX1 and HtNHX2 and a use thereof. The helianthus tuberosus L. Na<+> / H<+> reverse transport protein genes HtNHX1 and HtNHX2 are first cloned from Nan helianthus tuberosus L. I having extreme resistance to salt and barren soil, and cDNA sequences of the helianthus tuberosus L. Na<+> / H<+> reverse transport protein genes HtNHX1 and HtNHX2 are respectively shown in the formulas of SEQ ID NO.2 and SEQ ID NO.1. HtNHX1- and HtNHX2-overexpressed paddy rice materials are obtained by a transgenosis method. Compared with wild-type paddy rice materials, the HtNHX1- and HtNHX2-overexpressed paddy rice materials have bigger biomass after long-term and short-term NaCl stress. The trans-HtNHX2 paddy rice has stronger barren soil resistance, has bigger biomass in the low-K<+> content soil and can improve a paddy rice single-plant yield by more than 50%.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to Jerusalem artichoke Na + / H + Antiporter genes HtNHX1 and HtNHX2 and their applications. Background technique [0002] Jerusalem artichoke (Helianthus tuberosus L.), commonly known as Jerusalem artichoke, is a perennial herbaceous plant belonging to the genus Helianthus L. in the Compositae family (Kays and Nottingham, 2007). Jerusalem artichoke is an energy plant that is resistant to barrenness, drought, and disease and insect pests. It has extremely high promotion and application value and research value. In addition, planting Jerusalem artichoke on coastal and saline land can also take away The salinity in the soil thus plays a certain role in desalination and salt washing, and achieves the purpose of improving saline soil (Zhao Gengmao et al., 2005). [0003] Salinization hazard is one of the most common major limiting factors of modern agricultural production. A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12N5/10A01H5/00
Inventor 徐国华李青余玲刘兆普
Owner 安徽田间云生物科技有限公司
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