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Elytrigia elongata HKT type transporter, namely EeHKT1; 4, as well as coding gene and application thereof

A long-eared wheatgrass and transport protein technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of large investment, low benefit, aggravated soil secondary salinization, etc., to improve adaptability and improve tolerance. The effect of the ability of the salt environment

Inactive Publication Date: 2016-10-26
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, traditional measures require large investment and low benefits, and the addition of a large number of chemical substances will aggravate the secondary salinization of the soil, which forces people to consider other measures to improve the use of saline soil

Method used

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  • Elytrigia elongata HKT type transporter, namely EeHKT1; 4, as well as coding gene and application thereof
  • Elytrigia elongata HKT type transporter, namely EeHKT1; 4, as well as coding gene and application thereof
  • Elytrigia elongata HKT type transporter, namely EeHKT1; 4, as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 cDNA Cloning and Identification of Echinopsis elongatum and Its Encoding Gene

[0027] According to the known partial sequence of E. elongatum EeHKT1; 4 gene, the length is 614bp, the 5'- and 3'-end primers were designed, and the 5'- and 3'-end primers were cloned from E. elongatum by RT-PCR and RACE methods The end sequences are 580bp and 1117bp in length, respectively. Finally, the above three fragments were spliced ​​to obtain EeHKT1; the full-length cDNA of the four genes is 1977bp, including an open reading frame (ORF) of 1722bp, a 5'untranslated region (UTR) of 28bp and a 3'-UTR of 227bp, see figure 1 . It encodes 573 amino acids, the estimated molecular weight is 62.92kDa, and the isoelectric point is 9.73. Name it EeHKT1;4. EeHKT1; 4 structure of the transmembrane domain figure 1 .

[0028] The specific operation is as follows:

[0029] (1) Primer information:

[0030] ① 5' end primer:

[0031] UPM:

[0032] Long (0.4μM):

[0033] 5'–CTAATACG...

Embodiment 2

[0048] Example 2 E. elongatum EeHKT1; Expression characteristics of 4 genes under salt stress

[0049] In order to analyze the changes of salt tolerance of E. elongatum, 4-week-old E. elongatum seedlings were treated with different concentrations of NaCl (0, 25, 50, 100, 150 and 200 mM NaCl) for 48 h, and then the tissues (leaf, leaf sheath and Root) in the EeHKT1; 4 gene expression level analysis, the results are shown in image 3 . The specific method is: using the Actin gene as an internal reference,

[0050] HKT1; 4 gene expression primer sequences are 5'-CCGATGATGAGACGAGCAAG-3' and 5'-ATGGCGAGGACGACGAA-3'.

[0051] Actin gene primer sequences are 5'-CTTGACTATGAACAAGAGCTGGAAA-3' and 5'-TGAAAGATGGCTGGAAAAGGA-3'.

[0052] The operation method is as follows:

[0053] (1) Total RNA extraction: Genomic total RNA of E. elongatum was extracted according to the operation steps of the EZ-10 RNA Mini-Preps kit.

[0054] (2) Synthesis of the first strand of cDNA:

[0055] ①DNA ...

Embodiment 3

[0059] Example 3 E. elongatum EeHKT1; 4 gene function identification

[0060] Utilize the technical method related to Clontech Infusion kit, EeHKT1; 4 gene is forward inserted between the Xba I and Sma I enzyme cuts of plant expression vector pBI121 ( Figure 4 ) to obtain the plant overexpression vector pBI121-35S-EeHKT1; 4-Nos. Using CaCl 2 The pBI121-35S-EeHKT1; 4-Nos plasmid was introduced into Agrobacterium EHA105 by the freeze-thaw method, and the Agrobacterium liquid containing the pBI121-35S-EeHKT1; 4-Nos plant expression vector plasmid was differentiated by the leaf disc infection method. Infect the leaf disk with callus, shake it for 7 minutes, treat it in the dark for 2 days, place it on MS medium containing 50mg / L kanamycin (Kan) and 500mg / L carbenicillin (Carb) for culture Identification to obtain transgenic tobacco plants.

[0061] The transgenic tobacco and the wild-type tobacco were respectively stressed in 0, 50, 100 and 200 mM NaCl Hoagland nutrient soluti...

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Abstract

The invention discloses an elytrigia elongata HKT type transporter, namely EeHKT1; 4, as well as a coding gene and application thereof. The amino acid sequence of the transporter is shown as SEQ ID NO. 2, and the nucleotide sequence of the coding gene of the transporter is shown as SEQ ID NO. 1. The elytrigia elongata HKT type transporter, namely EeHKT1; 4, and the coding gene thereof can be used for improving the salt resistance of crops or fine pasture. Under salt stress, the EeHKT1; 4 gene is mainly expressed in roots and leaf sheaths, and the expression quantity in leaves is very low and is in the basically unchanged trend. The EeHKT1; 4 gene is over-expressed in tobacco; compared with the salt resistance of wild-type tobacco, the salt resistance of transgenic tobacco under salt stress is significantly improved, which indicates that the EeHKT1; 4 gene has important application prospect in genetic salt resistance and stress resistance improvement of plants.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a HKT-like transporter of Echinopsis elongatum and its coding gene and application. Background technique [0002] Due to global warming, increasing world population, increasing industrial pollution, development of irrigated agriculture, improper use of chemical fertilizers and other factors, soil salinization is becoming more and more serious, which not only makes soil desertification and ecological environment worse, but also has become a hindrance to crop growth. The main factor of growth and high yield and quality. At present, soil salinization in my country is becoming more and more serious, which has seriously affected the production of crops and led to a decline in agricultural production. This serious problem needs to be solved urgently. The main way to solve the problem in the existing technology is still the traditional method of improving saline soil...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8273
Inventor 孟林郭强张琳毛培春田小霞
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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