Soybean GmHKT protein, and encoding gene and application thereof
A gene and protein technology, applied in the application field of cultivating salt-tolerant plant germplasm, can solve problems such as yield reduction and quality decline, salinization and secondary salinization, and soil effective components reducing the degree of salinization.
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Embodiment 1
[0027] Example 1, cDNA cloning and identification of soybean GmHKT2 and its coding gene
[0028] Using the soybean salt-tolerant variety Lee68 (Chen et al., Aus J Agric Res, 2008, 59:1086–1091) as the material, the reported GmHKT1 protein sequence was used as the seed sequence through the method of bioinformatics, and the BLAST soybean genome database , a highly homologous sequence was identified on soybean chromosome 7 and named GmHKT2. The full-length ORF of GmHKT2 gene is 1440bp, encoding 479 amino acids.
[0029] Primers were designed according to the GmHKT2 gene sequence: GmHKT2 ORF forward primer: 5'-ATGGGTTTAGTTTTTCTCC-3', GmHKT2 ORF reverse primer: 5'-TTAGGAGAGGTTCCAGGCTT-3', and the GmHKT2 gene was amplified from soybean total RNA by RT-PCR. The leaves of soybean salt-tolerant variety Lee68 were taken, and total RNA was extracted with QiaGen kit. 2 μl of total RNA was reverse-transcribed using a reverse transcription kit (TOYOBO Company) according to the method of t...
Embodiment 2
[0030] Expression characteristics of embodiment 2, GmHKT2 gene under high-salt stress
[0031] The NaCl stress treatment of soybean salt-tolerant variety Lee68 was used to analyze the expression of soybean GmHKT2 under salt stress. In order to analyze the expression of GmHKT2 under salt stress by Real-time PCR, the seeds of soybean variety Lee68 were germinated for 4 days, and then transferred to the standard Hoagland nutrient solution for hydroponic cultivation. After the first cluster of compound leaves of soybean plants grew , use the standard Hoagland nutrient solution containing 150mM NaCl for stress treatment, take samples of roots, stems and leaves at 0, 6, 12, 24, 36 and 48h after salt treatment, and store them at -80°C after quick freezing in liquid nitrogen spare. The extraction of total RNA was the same as in Example 1. The soybean constitutively expressed β-tublin gene (Genbank No.NM_001252709.1) was used as the internal reference gene (5'-ATCACTTGATCTCCGCAACC-3'...
Embodiment 3
[0032] Example 3, Functional Identification of GmHKT2 Encoded Protein
[0033] Using Invitrogen's Technology with Clonase TM II kit, GmHKT2 is forward inserted into the expression vector pMDC83 (Invitrogen company) ( figure 2 ) to obtain 2×35S-GmHKT2-Nos plant overexpression vector, and transfer 2×35S-GmHKT2-Nos into Agrobacterium tumefaciens EHA105 strain (preserved in our laboratory) by freeze-thaw method. Using Agrobacterium EHA105 to introduce 2×35S-GmHKT2-Nos into tobacco, the test results showed that 38 positive plants were obtained, combined with the results of Real-time PCR analysis, two positive transgenic lines (Line3, Line12) were selected for functional verification , the seeds of Line3 and Line12 were planted in MS medium, and the non-transgenic tobacco (CK) and T 2 After the first generation of transgenic lines (Line3, Line12) germinated and grew into seedlings (about 2 weeks), they were transferred to 1 / 2MS basic medium containing 200mM NaCl for salt treatmen...
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