A kind of salt tolerance gene and its recombinant vector
A technology of salt tolerance gene and recombinant vector, which is applied in the fields of molecular biology and biology, and can solve the problems of complex salt tolerance mechanism research and other problems.
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Embodiment 1
[0027] 1. Cloning of a salt-tolerant gene: (Suaeda salsa for short S.sa)
[0028] Harvest the seeds from the whole plant of Suaeda salsa (taken from along Donghai Road, Beitangkou, Binhai New District, Tianjin), sow the seeds respectively in the medium of 1 / 2MS and 1 / 2MS+100mmol / LNaCl solution, and put them in for five weeks Afterwards, the two plant seedlings obtained respectively were sent to Huada Company for RNA Sequncing, and the obtained data sequences were compared, and the gene sequence 660bp (SEQ ID No.1) with the largest difference was obtained through gene expression difference multiple screening, see figure 1 . Total RNA was extracted using Plant RNeasy Plant Mini Kit (Trans gene Code #EP101-01 50rxns), and cDNA was reverse transcribed using EasyScript Frist-Strand cDNA SynSgesis SuperMix (Trans gene Code #AE301-03 100rxns). According to SEQ ID No.1, primers SEQ ID No.3F: 5'-CTGCATGGGCTTGGCTTG-3' and SEQ ID No.4R: 5'-GCTCCCAGACATCAATGGTAAG-3' were designed to obta...
Embodiment 2
[0059] Embodiment 2 transforms Arabidopsis
[0060] (1) Transform Arabidopsis.
[0061] Specific steps for transforming Arabidopsis:
[0062] ①Activation and expanded culture of the positive clone colonies obtained in Example 1
[0063] Activation: Pick the preserved positive clone colonies and place them in 3mL YEB liquid medium (add Gen, Rift, Sp antibiotics to make the concentration respectively 30mg / L, 25mg / L, 50mg / L) and cultivate for about 15 hours (to OD 600 = about 0.8), 180rpm, 28°C.
[0064] Expansion culture of positive clones: add appropriate amount of antibiotics (Gen, Rift, Sp antibiotics, concentrations are 30mg / L, 25mg / L, 50mg / L) in fresh 10ml YEB liquid medium, and then inoculate appropriate amount of positive clones Bacterial solution was cultured in YEB liquid medium, 180rpm, and cultivated to OD at 28°C 600 = 0.6.
[0065] ②
[0066] Centrifuge the bacterial solution (3,000rpm, 15°C, 10min), discard the supernatant, and resuspend the bacterial cells w...
Embodiment 3
[0073] Embodiment 3 transforms poplar
[0074] The poplar used for the transformation of positive clones was Populus tremula × P. alba INRAclone N717 1-B4 (hereinafter referred to as 717 poplar) tissue culture seedlings.
[0075] (1) Place the axillary buds or terminal buds of 717 poplar on the basic medium for subculture propagation, and cultivate for 6 weeks to obtain tissue cultured seedlings; cut off the 1 cm stem section of the tissue cultured seedlings without axillary buds, scratch the wound and store at 24°C Pre-cultivate for 3 days in the dark;
[0076] (2) the positive clone bacterial liquid (OD) obtained by the selected embodiment 1 600 =0.8) Centrifuge at room temperature and 4000rpm for 10min, discard the supernatant, and use an equal volume of M solution (M solution: MS salt 4.4g, sucrose 30g, auxin NAA 1.86mg, cytokinin 2ip 1.02mg, acetyl Syringone As 19.86mg, dilute to 1L, pH=5.7) resuspended, 24°C, 100rpm activated for 1h to obtain the infection solution; pu...
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