FAD2 family gene bioinformatics analysis
Annotation of FAD2 family genes
A conserved structure was identified based on the National Center for Biotechnology Information (NCBI) database, the soybean database online website soybase (https://soybase.org/) and the Pram (http://pfam.xfam.org/) database. Gene location on chromosomes and the conserved domains in seven genes in the soybean FAD family were identified using Hmmer 3.0 software.
FAD2 gene evolutionary tree analysis and sequence alignment
The online program Clustal X (version 3.0) was used to align the sequences of the soybean FAD2 family, and a phylogenetic tree was constructed using MEGA 5.0 software for all functionally defined FAD2 genes in soybean species and between species to classify genes. The neighbour-joining (NJ) method was used to analyse the results.
Analysis of FAD2 gene protein sequences
The online website MEME (http://meme.nbcr.net/meme) was used to predict the structure and classification of other protein sequences in addition to the conserved sequences in the gene family. The protein sequences of all the genes in the family were entered in the MEME website, and the parameters were all set to the default parameters.
Analysis of gene expression patterns
Experimental materials
Plant Materials
The soybean genotypes "Jike Soybean 20", "Ji Midou 3", "Jike Fresh Bean 1", and "Jike Midou No. 1", among which the "Jike Fresh Bean 1" oleic acid content is relatively low, while the other genotypes have relatively high oleic acid content, were selected and provided by the Plant Biotechnology Center of Jilin Agricultural University.
Required reagents
RNA extraction kit, real-time PCR kit, DNA standard molecular weight marker, and DL2000 kits were from TaKaRa. Fluorescent quantitative primers were synthesized by Changchun Kumei Biotechnology Co(Table S1)., Ltd. The plant RNA extraction kits were purchased from Omega. Sodium chloride was purchased from Thermo Fisher Company, and the other major reagents were obtained domestically and were of pure analytical grade.
Instruments and equipment
A fluorescence quantitative polymerase chain reaction kit (purchased from the United States), cryogenic freezing centrifuge at 4 °C, cryogenic − 80 °C refrigerator, water bath heating pot, and indoor plant culture room were utilized.
Low Temperature Stress Treatment
Clean and full "Jike Soybean 20" soybean seeds were chosen and planted in small plastic buckets with a puncture in the bottom to allow for easy draining. The planted seeds were placed in an artificial climate chamber that simulated the growth environment under natural conditions and were allowed to grow for approximately 2 weeks after germination. When the first three leaves were present, a series of abiotic stress treatments were carried out on the seedlings. Abiotic stress treatment of plants were conducted using the following methods.
Low temperature stress: 20 days after germination, the seedlings were placed in a refrigerator at 4 °C for cold treatment. After treatment for 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h, root, stem, leaf and germinated seed parts were taken. The control plants treated as described above were watered every other day. In the treatment process, replicates were prepared for each treatment group to reduce the error generated during the experiment. After the samples were collected, they were placed in a -80 °C refrigerator for storage.
Determination of oleic acid content and relative expression of FAD2 genes at different stages of grain development
The soybean test strains ”Jike Soybean 20”, “Ji Midou 3”, “Jike Fresh Bean 1” and “Jike Midou No. 1” were sown in the experimental field of Jilin Agricultural University, planted at the end of April, harvested at the end of September, and observed for the flowering period; the first sampling was performed on the 25th day after flowering. Every 5 days, the young pods in development were taken, placed in liquid nitrogen immediately, and then stored in a refrigerator at -80 °C. A total of 5 young pods were taken, and 3 replicate samples were used. A DS2500 grain analyser was used to measure the change in oleic acid content at different periods of grain development. Primer5.0 software was used to design fluorescent quantitative primers (Table S1) to quantify the genes in the soybean FAD2 family. The expression levels in pods at different stages of development were determined.
Real-time PCR
In this experiment, cDNA of the internal reference gene β-actin and the target gene was amplified by a two-step method using a fluorescence quantitative analyser, and three replicate experiments were performed for each set of samples. Following the instructions in the Fluorescence Quantitation Kit, the qRT-PCR reaction mixture was prepared as follows (operating on an ice box). To reduce error caused by the pipetting gun during the loading and ensure accuracy, the reaction mixture was uniformly mixed with a pipette and then dispensed into a sterile PCR tube, and finally, reverse transcription was carried out. The cDNA sample was added, centrifuged and placed in the qRT-PCR thermocycler (Table 1–7).
Table 1-7
Name | System |
2 × qPCR Mix | 10 µL |
Primer1 | 2 µL |
Primer2 | 2 µL |
RNase-free H2O | 4 µL |
cDNA | 2 µL |
The amplification conditions were predenaturation at 95 °C for 10 min, denaturation at 95 °C for 10 s, annealing at 55 °C for 20 s, and extension at 72 °C for 15 s; set to 55 cycles, and the relative expression of the target gene was calculated using the 2−ΔΔCt method.
Determination of oleic acid content in soybean seeds with the near-infrared grain analysis
In this experiment, an NIRS DS2500 grain analyser was used to select the clean grain to be tested, which was put into a measuring cup. The seed volume needed to reach the bottom of the cup and cover the infrared scanning area (If the seed quantity is small, the seed can be ground into powder before being placed in the measuring cup). The sample was placed into the sample tank of the near-infrared grain analyser, according to the previously established laboratory procedure for acquisition of spectra to determine soybean oleic acid content. Analysis of each sample was repeated 3 times, and the measurement results were determined by the software Operator and automatically saved in the computer. The error between the results measured by the near-infrared grain analyser and the gas chromatography method was previously verified to be between 1% and 1.5%, which is within the acceptable range, indicating that the results of the near-infrared detection are authentic and can be widely applied to fatty acids.
Data analysis
All data in this experiment were analyzed using SPSS2.0 software. Each group of experiments was set up to three replicates to reduce experimental errors. ANOVA was used to analyze the correlation between the difference in FAD2 genes expression and changes in oleic acid content among different soybean varieties. Correlation coefficient.