Method for obtaining salt-tolerant transgenic soybean material

A transgenic soybean and gene technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems that have not been seen before, and achieve the improvement of salt tolerance, herbicide resistance and broad application prospects. Effect

Inactive Publication Date: 2017-08-18
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These studies show that, AgGlpF The gene is a kind of salt tolerance gene with more outstanding characters, but there is no future AgGlpF Report on Gene Transfer into Soybean

Method used

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  • Method for obtaining salt-tolerant transgenic soybean material
  • Method for obtaining salt-tolerant transgenic soybean material
  • Method for obtaining salt-tolerant transgenic soybean material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Plant expression vector construction.

[0028] According to published on NCBI AgGlpF The nucleotide sequence of the gene (GenBank accession number KJ679500), artificially synthesized according to plant codon preference AgGlpF Gene. According to the molecular characteristics of multiple cloning sites of plant binary expression vectors, respectively in AgGlpF Introducing endonuclease upstream of the gene wxya Restriction site, downstream introduction of endonuclease SpeI Restriction sites.

[0029] Amplified by PCR AgGlpF The full-length sequence of the gene (861bp) was performed by double cutting and double joining wxya with SpeI After digestion, the AgGlpF The gene was connected to the pYL vector to obtain the expression vector pYL-AgGlpF ( figure 1 A), the constructed plasmid was sequenced and detected by enzyme digestion ( figure 1 B), the verified correct plant expression vector was transformed into Agrobacterium EHA101 for the next step of ...

Embodiment 2

[0030] Example 2 Agrobacterium-mediated genetic transformation of soybean.

[0031] In this study, selection with the target gene AgGlpF The Agrobacterium strain EHA101 was used to genetically transform the soybean recipient variety P3 using the genetic transformation method of soybean cotyledon nodes mediated by Agrobacterium tumefaciens.

[0032] The process of Agrobacterium-mediated soybean cotyledon node transformation method is shown in figure 2 , the basic process is as follows:

[0033] (1) After culturing Agrobacterium at 28°C for 16 hours, collect the cells and transfer them to YEP liquid medium until the OD600 value is 0.5-0.7 for later use;

[0034] (2) Select mature seeds of soybean recipient variety P3 and sterilize them with chlorine gas for 16 hours. After sterilization, put the seeds into the GM germination medium (medium formula refers to OLHOFT, etc.) and germinate in weak light for 16 hours;

[0035] (3) Cut the cotyledon nodes, divide the soybean seeds...

Embodiment 3

[0041] Example 3 Detection of glufosinate resistance in transgenic plants.

[0042]Since glufosinate is used as a screening marker in the plant expression vector, the method of smearing leaves with glufosinate can be used to quickly detect transgenic positive seedlings at the seedling stage of T0 transgenic materials.

[0043] Specific method: Dip a proper amount of glufosinate solution with a concentration of 135mg / L with a cotton swab, gently wipe half of the leaf, and smear water on the same leaf as a control, and observe the growth of the leaves after 3 to 5 days under normal light conditions. withered, it is not resistant to glufosinate ( image 3 ).

[0044] In this study, we transplanted 49 regenerated plants that were initially positive by Bar test strips to flower pots in the greenhouse for cultivation. Using the glufosinate resistance detection method, we finally obtained 36 transgenic materials of the T0 generation.

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Abstract

The invention belongs to the technical field of plant genetic engineering and provides a method for obtaining a salt-tolerant soybean new material by utilizing a transgenic technology. The method comprises the following steps: (1) introducing a plant expression vector pYL-AgGlpF containing a gene AgGlpF into a receptor soybean genome by utilizing agrobacterium tumefaciens-mediated soybean cotyledonary node genetic transformation method; and (2) carrying out molecular screening and salt tolerance identification, so that a transgene AgGlpF salt-tolerant soybean material is obtained. The method provided by the invention has the advantages that a gene AgGlpF is transferred into the soybean genome, and salt tolerance of soybeans can be obviously improved, so that the method provided by the invention has important economic values and broad application prospects on agricultural production.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for obtaining new salt-tolerant soybean materials by using transgenic technology. Background technique [0002] Soybean has always been an important food crop and oil crop in my country. However, in the past 20 years, affected by the impact of imported soybeans and the adjustment of planting structure caused by domestic comparative benefits, the planting area of ​​soybean production in my country has dropped sharply. Since 1996, soybeans have been net exported Since China became a net importer of soybeans, soybean imports have increased year by year, reaching 81.69 million tons in 2015, accounting for 87% of total domestic soybean consumption. With the proposal and implementation of my country's agricultural supply-side structural reform, the domestic soybean planting industry has ushered in a good opportunity. [0003] Soil salinization is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H1/02A01H5/00
CPCA01H1/02C12N15/8205C12N15/8273C12N15/8277
Inventor 张玲李飞武董英山张原宇谭喜昌马虹
Owner JILIN ACAD OF AGRI SCI
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