Application of soybean nucleoprotein gene GmNup96 in regulating of development of plant root nodules
A nucleoporin and soybean nodule technology, applied in the field of plant genetic engineering, can solve problems such as changing the regulation of root nodule development and activity
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Embodiment 1
[0049] Example 1 Obtaining of soybean nucleoporin gene GmNup96 and its expression pattern
[0050] Utilize a pair of primers GmNup96.XhoI.F and GmNup96.EcoRI.R (SEQ ID NO:3-4) from soybean Tianlong No. 1 cDNA PCR amplification (Table 1) and digestion (Table 2) ligation (Table 3) to The entry vector Fu28-MCS-GFP, whose backbone is a GEC vector ( figure 2 ). The sequence of the GmNup96 coding region with a total length of 3069bp was obtained by sequencing, such as figure 1 Shown, its nucleotide sequence is shown in SEQ ID NO:1, and the corresponding amino acid sequence is shown in SEQ ID NO:2.
[0051] Table 1 PCR amplification system
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[0053] PCR reaction program: 94°C for 5min; 98°C for 10sec, 55°C for 15sec, 72°C for 3min, a total of 30 cycles; 72°C for 10min.
[0054] Table 2 enzyme digestion system
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[0056] Table 3 Connection reaction system
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[0058] The transformation method of Escherichia coli DH5α competent cells includes: (1) ...
Embodiment 2
[0060] Example 2 Subcellular Localization of Soybean Nucleoporin GmNup96
[0061] The vector Fu28-GmNup96-GFP obtained in Example 1 and another entry vector Fu76-35S were used to recombine the 35S promoter and GmNup96-GFP gene into the plant expression vector BDV2 by using the GateWay recombination cloning method (Invitrogen) through LR reaction ( Figure 4 ) and propagated in Escherichia coli DH5α. Through the Agrobacterium-mediated transformation method, the 35S:GmNup96-GFP gene carried by BDV2 was transformed into Arabidopsis thaliana to obtain Arabidopsis transformed plants. The results showed that the soybean nucleoporin GmNup96 was localized on the nuclear membrane ( Figure 5 ).
Embodiment 3
[0062] Example 3 Obtaining of Soybean Nucleoporin Gene GmNup96-RNAi Transgenic Plants
[0063] A pair of primers GmNup96RNAi-F and GmNup96RNAi-R (SEQ ID NO: 5-6) was used to perform PCR amplification using the vector Fu28-GmNup96-GFP obtained in Example 1 as a template.
[0064] The target fragment was connected to the pGWCm entry vector by the method of T-A cloning ( Image 6 ) and sequenced. Using the method of GateWay recombinant cloning (Invitrogen), the entry vector pGWC-GmNup96-RNAi was cloned into the plant RNAi expression vector pB7GWIWG2 ( Figure 7 ), completed the transformation of Agrobacterium EHA105, identified positive strains, and stored at -80°C. Then, the GmNup96-RNAi vector was transformed into soybean Tianlong No. 1 variety to obtain GmNup96-RNAi transgenic plants, which were positive plants through molecular detection ( Figure 8 ). We go further to T 1 The leaves of the generation plants were sampled, the total RNA was extracted and reverse-transcrib...
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