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Application of barley HvHKT1gene

A gene and barley technology, applied in the field of plant functional genomics, can solve the problems of normal growth and development of transgenic plants, improvement of salt tolerance, little knowledge of environmental stress tolerance genes, and far-flung requirements, so as to reduce sodium ions. The accumulation, obvious practicability and application prospects, the effect of increasing production

Inactive Publication Date: 2013-09-04
ZHEJIANG UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

There are abundant allelic variations in tolerance-related genes of wild barley. However, the research on annual wild barley resources on the Qinghai-Tibet Plateau in my country is generally far behind the requirements for excavating and utilizing these resources, and little is known about the environmental stress tolerance genes contained in them. It still needs to be further protected, excavated and utilized
The HvHKT1 gene exists in wild barley, and the encoded protein sequence has only 47.4% similarity with the Arabidopsis AtHKT1 sequence. The function of the HvHKT1 gene has not yet been clarified, and related transgenic research has not yet been carried out. It is unknown whether the saltiness can be improved

Method used

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  • Application of barley HvHKT1gene
  • Application of barley HvHKT1gene
  • Application of barley HvHKT1gene

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1, the cloning of HvHKT1 coding gene

[0023] Wild barley X16 seeds treated with 3% (v / v) H 2 o 2After surface disinfection for 20 minutes, rinse with distilled water three times, sow on moist double-layer filter paper, and germinate in a growth chamber at 20°C for 10 days (14 hours of light / 10 hours of darkness). Cut the young leaves, put them in a mortar and add liquid nitrogen to grind them, extract the total RNA of the tissue with reference to the Tiangen Plant RNA Extraction Kit (DP432), and reverse transcribe to obtain the first-strand cDNA (Takara Reverse Transcription Kit RR037Q), using this cDNA as a template, use

[0024] Primer KTI-F1 (5'-attTCTAGAatgcctgaactcgaaagccc-3')

[0025] PCR amplification was performed with primer KTI-R1 (5'-attGGATCCctaatttgcagccacctctgg-3'), and the kit was from Quanshijin Company (AP131).

[0026] The 50μL reaction system is:

[0027] Transgen HiFi system

[0028] The reaction program was: 95°C for 5 min...

Embodiment 2

[0029] Example 2. Application of HvHKT1 gene to enhance plant salt tolerance

[0030] 1. Construction of overexpression vector in Arabidopsis

[0031] The coding sequence of HvHKT1 was cloned into pCambia 1301 vector and placed under the drive of 35S promoter. The specific method is as follows: the fragment amplified in Example 1 and the pCambia 1301 plasmid vector were double digested with BamHI and XbaI, and the gene fragment was inserted between the BamHI and XbaI enzyme recognition sites of the vector using T4 DNA ligase to obtain p1301-HvHKT1 recombinant vector. After the recombinant vector was sequenced and identified by multi-enzyme digestion, it was shown that the HvHKT1 gene was correctly connected to the pCambia 1301 vector.

[0032] 2. Transformation of Arabidopsis thaliana with p1301-HvHKT1

[0033] The recombinant vector p1301-HvHKT1 was transformed into Agrobacterium GV3101 competent by the freeze-thaw method, and a positive single colony was picked on the LB ...

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Abstract

The invention relates to plant function genomics, in particular to the application of a barley HvHKTI gene in building transgenic plants, wherein after the separation and cloning of a HvHKT1gene coding sequence (coding sequence), the coding sequence is connected with a promotor of a mosaic virus of a cauliflower, and then arabidopsis is converted; and finally evaluation is conducted for salt endurance of the transgenic arabidopsis and the wild type Arabidopsis. According to the invention, the salt endurance of the transgenic plants is improved, and the barley HvHKT1 gene has a nucleotide sequence shown by SEQ ID NO:1.

Description

technical field [0001] The present invention relates to plant functional genomics. Specifically, after isolating and cloning the barley HvHKT1 gene coding sequence (coding sequence), it is connected with the cauliflower mosaic virus promoter (CaMV35S), and then transformed into Arabidopsis, and the transgenic Arabidopsis and wild-type Arabidopsis Mustard was evaluated for salt tolerance. Background technique [0002] Currently, about 800 million hectares of land worldwide are salinized, including nearly 20% of arable land and 50% of irrigated land (FAO, 2009; http: / / www.fao.org / ag / agl / agll / spush ). In my country, the salinized cultivated land has reached 36 million hectares, and due to the further aggravation of environmental pollution and the continuous deterioration of agricultural irrigation water quality, the threat of salt damage to agricultural production is becoming more and more serious. Therefore, improving the salt resistance ability of crops has become an impor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/82A01H5/00
Inventor 韩勇尹舒雅吴学龙沈秋芳张国平
Owner ZHEJIANG UNIV
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