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Soja bean HKT protein and coding gene thereof and application

A technology that encodes genes and soybeans, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problem of insufficient research on protein function, and achieve the effect of increasing yield and enhancing tolerance

Inactive Publication Date: 2009-06-17
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

HKT-like proteins were discovered for the first time in wheat, but there are few functional studies of this kind of proteins in plants

Method used

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  • Soja bean HKT protein and coding gene thereof and application
  • Soja bean HKT protein and coding gene thereof and application
  • Soja bean HKT protein and coding gene thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1, cDNA cloning and identification of soybean GmSKC1 and its coding gene

[0027] Using the soybean salt-tolerant variety Lee68 (commonly known and public, Abel & MacKenzie, 1964, Crop Science 14, 157-161) as a material, the reported rice HKT protein sequence (OsHKT8, accession number BAB93392) was used as a material by means of bioinformatics. Seed sequence, search soybean EST database, find a highly homologous EST fragment, the gene named GmSKC1. The cDNA of young soybean leaves was used as a template for PCR amplification, and a band with the expected size was amplified. Then, the PCR products were tapped and purified, ligated and transformed, and positive clones were picked for sequencing. Sequencing results showed that the partial sequence of the soybean GmSKC1 gene had been cloned, and its 5' and 3' ends were found to be incomplete. The 3' end was extended and cloned and sequenced. After sequence analysis, it was found that the 3' end sequence was complet...

Embodiment 2

[0031] Example 2, Expression characteristics of soybean GmSKC1 gene under high-salt stress

[0032] Soybean salt-tolerant variety Lee68 was treated with NaCl stress to analyze the expression of soybean GmSKC1 under salt stress. In order to analyze the expression trend of GmSKC1 under salt stress by RT-PCR, the soybean variety Lee68 was planted in hydroponics (Hoagland nutrient solution). The roots, stems and leaves of 6 periods including 0h, 6h, 12h, 24h, 36h and 48h after salt treatment were taken, and stored at -80°C after quick-freezing in liquid nitrogen.

[0033] The extraction of total RNA was the same as in Example 1. Using soybean constitutively expressed Actin gene (GenBankaccession No.V00450) as an internal reference, RT-PCR analysis was performed using total RNA from different soybean tissues and different tissues induced by salt for different time periods as templates. RT-PCR analysis showed that ( figure 1 ), under 150mM NaCI stress, the expression of GmSKC1 in...

Embodiment 3

[0034]Example 3, Functional Identification of GmSKC1 Encoded Protein

[0035] Using Invitrogen's Technology with Clonase TM II kit, insert GmSKC1 forward into the expression vector pMDC83 (Sokolov et al, 2005, PNAS, 103; 9732-9737) ( figure 2 ), the pMDC83-GmSKC1 plant overexpression vector was obtained, and pMDC83-GmSKC1 was transformed into Agrobacterium tumefaciens strain EHA105 (Avsian-Kretchmer et al, 2004, Plant Physiology, 135:1685-1696) by freeze-thaw method. pMDC83-GmSKC1 transformed tobacco through the mediation of Agrobacterium EHA105, PCR detection results showed that 62 positive plants were obtained, combined with RT-PCR analysis results, 3 positive transgenic lines (Line16, Line27, Line31) were selected, and Line16, Line27 and Seeds of Line31 were planted into MS medium, and wild-type tobacco (WT) and T 1 After the generation of transgenic lines (Line16, Line27, Line31) germinated and grew into seedlings (about 15 days), they were transferred to 1 / 2MS basic...

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Abstract

The invention discloses a soybean HKT-like protein and the coding gene and application thereof, which belongs to the technical field of biology. The soybean HKT-like protein is named as GmSKC1 protein which has an ammonia sequence shown by the SEQ ID NO.2 in a sequence list. The coding gene of the protein is a DNA sequence shown by the SEQ ID NO.1 of the GmSKC1 gene. The soybean HKT-like protein and the coding gene thereof can be used for cultivating salt tolerant plant species, in particular salt tolerant soybean species. The expression of GmSKC1 is inducted by high salt. Under the stress of 150mM NaCl, GmSKC1 is strongly inducted in the soybean tissues such as root and leaves; and the expression of GmSKC1 in the soybean stem has a basically invariable trend. Compared with the non-transgene tobacco, the tobacco with the excessive expression of GmSKC1 remarkably improves the salt tolerance, thereby demonstrating that the GmSKC1 gene has an important action on improving the salt tolerance of plants.

Description

technical field [0001] The invention relates to the field of plant genetic engineering. It specifically relates to the high-affinity potassium ion transporter (high-affinity K transporter, HKT) related to salt tolerance and its coding gene and application, and involves the HKT protein GmSKC1 related to salt tolerance derived from soybean and its Encoding gene and its application in breeding salt-tolerant plant varieties. Background technique [0002] Soil salinization and secondary salinization are one of the important factors affecting agricultural production and ecological environment. At least 20% of the cultivated land in the world is salinized, especially in arid and semi-arid areas, and the secondary salinization of soil is becoming more and more serious. There are about 40 million hm of salinized and secondary salinized land in my country 2 The above have become the main environmental factors restricting the development of agriculture. Breeding salt-tolerant varie...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N1/21C12N15/11A01H5/00
Inventor 喻德跃陈华涛
Owner NANJING AGRICULTURAL UNIVERSITY
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