Application of an Isolated Soybean Gene in Improving Resistance to Soybean Cyst Nematode
A soybean cyst nematode and gene technology, applied in the fields of biology and genetic engineering, can solve problems such as lack of excellent germplasm resources, long cycle, and difficulty in achieving expected goals
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Embodiment 1
[0027] Embodiment 1: GmNIG1 full-length gene cloning
[0028]According to the kit instructions, total RNA was extracted from soybean Williams 82 plant roots using Kangwei RNA extraction kit, and the quality of RNA samples was detected by agarose gel electrophoresis, and the concentration was determined by Thermo spectrophotometer. According to the kit instructions, the total RNA was converted into cDNA using HiScriptII QRT SuperMix from Vazyme. Find the corresponding gene CDS coding sequence from the phytozome website (https: / / phytozome.jgi.doe.gov / pz / portal.html) according to the gene number, and design primers to clone the full-length CDS sequence 474bp of GmNIG1 using the above cDNA as a substrate ( The nucleotide sequence encoding the protein shown in SEQ ID NO.2) was constructed on the pMD18-T vector. The primer sequences used for amplification are:
[0029] GmNIG1-F: 5'-TGgtcgacCTTATCATCACCATCATGGGTGTT-3'
[0030] GmNIG1-R: 5'-AGtctagaGTTGTAATCAGGATTGGCCAAAA-3'
Embodiment 2
[0031] Example 2: qRT-PCR analysis of the expression level of GmNIG1 infected by nematodes
[0032] The expression of GmNIG1 gene was analyzed by qRT-PCR, and three soybean varieties Williams 82, PI88788 and PI548402 infected with soybean cyst nematode Race 3 for 3 and 5 days and the control group (grown 3 without nematode infection) were extracted. The RNA of the root tissue of the soybean Williams 82, PI88788 and PI548402) of 1 day and 5 days, the reverse transcription cDNA is used as a template, and the expression of GmNIG1 is analyzed with the SKIP16 gene as an internal reference. The specific implementation steps are as follows:
[0033] 1) Select about 20 soybeans of the same size Williams 82, PI88788 and PI548402 respectively, and grow them on germination paper for 4 days;
[0034] 2) Select seedlings with the same root length, inoculate about 250 soybean cyst nematode Race 3 second-instar larvae at the root tip 1cm, each treatment is repeated three times, and after ino...
Embodiment 3
[0040] Example 3: Construction of GmNIG1 promoter GUS expression vector and staining of transgenic soybean root tissue
[0041] 1) Find the 1997bp DNA sequence of the upstream promoter fragment of the GmNIG1 gene from the phytozome website (https: / / phytozome.jgi.doe.gov / pz / portal.html), and design primers to amplify the promoter sequence. The primer sequence used is :
[0042] GmNIG1p-F: 5'-TGaagcttGTTCTTGTCACTACAATGAGTGCCA-3'
[0043] GmNIG1p-R: 5'-ACgtcgacGATGGTGATGATAAGGGGAAATTAAC-3'
[0044] 2) The whole genome of soybean was extracted by CTAB method as a template, and the promoter of GmNIG1 gene was cloned by KOD FX high-fidelity enzyme, connected to pMD18-T vector, and transformed into Escherichia coli DH5α;
[0045] 3) Use Hind III and Sal I to digest the target fragment and the pSM101-GUS binary expression vector, and transfer it into Escherichia coli DH5α after ligation with T4 ligase. The positive expression vector pSM101-GmNIG1p-GUS ( figure 1 Middle A). Transf...
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