Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (10): 1931-1939.doi: 10.3864/j.issn.0578-1752.2020.10.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Analysis of Foreign Gene Copy Number in Transgenic Wheat by Optimized Digital PCR

JU PengJu1,NING Lei1,GE LinHao2,XU ChengJie1,SHI HuaWei1,LIANG KaiGe3,MA Liang4,LIU TaoRan2,CHEN Ming2(),SUN DaiZhen1()   

  1. 1 College of Agriculture, Shanxi Agricultural University, Taigu 030800, Shanxi
    2 Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081
    3 Minzu University of China, Beijing 100081
    4 Shijiazhuang Academy of Agricultural and Forestry Sciences, Shijiazhuang 050047)
  • Received:2019-09-26 Accepted:2019-12-19 Online:2020-05-16 Published:2020-05-22
  • Contact: Ming CHEN,DaiZhen SUN E-mail:chenming02@caas.cn;sdz64@126.com

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Abstract:

【Objective】In order to explore the analysis technology of wheat gene copy number based on digital PCR, and improve the analysis efficiency of target gene copy number, and promote the research of wheat genome and gene engineering.【Method】Using the transgenic wheat with high resistance to wheat yellow mosaic disease transformed by nib8 gene created by the wheat stress resistance molecular breeding group in the Crop Science Institute of Chinese Academy of Agricultural Sciences as the experimental material, and used the single copy homozygous endogenous gene PIN-D1b (grain hardness gene) in wheat D genome as the internal reference gene, and four pairs of specific primers and corresponding probes were designed according to the sequence of the transformed disease resistance gene nib8, and the optimal concentration of probe and primer, the optimal annealing temperature of the system, to find the most appropriate template concentration were determined through experiments. Then, the copy number of nib8 genes in the nib8 transgenic wheat was detected by digital PCR; the accuracy of the above-mentioned copy number assay results were verified through using methods including the real time PCR and Southern blot; the copy number of the Wx012 (waxy gene) and SSII (starch synthesis gene) were detected by using the PINb-D1b as reference gene, and copy number of nib8 were detected by using the Wx012 and SSII as reference genes in the nib8 transgenic wheat, to verify the accuracy of the results using the PINb-D1b as reference gene; the specific primers were designed in different regions of the nib8 gene to compare the difference of the copy number analysis results of the nib8 transgenic wheat. Finally, a high-throughput assay method based on digital PCR was established to detect the copy number of target gene in wheat genome. 【Result】Finally, the detection system of target gene copy number in wheat genome based on digital PCR was determined. The optimal final concentration of primer and probe was 500 and 250 nmol·L -1, respectively. The optimal annealing temperature was 59℃ and the optimal amount of DNA template was 40 ng. PINb-D1b gene was used as the internal reference gene to detect the copy number of nib8 gene in the nib8 transgenic wheat. The results showed that the copy number of nib8 gene in the 12th, 16th, 17th, 23rd, 29th and 30th lines was 7, 1, 1, 1, 1, 1, 7, respectively. At the same time, the results were consistent with the real-time PCR and Southern blot results. In the nib8 transgenic lines, the PINb-D1b was used as the internal reference gene to detect copy number of the Wx012 and SSII, and we found that the results of copy number of nib8 gene in nib8 transgenic wheat by using these three kinds of internal reference gene analysis were consistent, which indicated that these three internal reference genes were all suitable for digital PCR method; the primers were designed in the upstream, middle and downstream regions of nib8 gene, and the results of copy number using different primer were consistent. 【Conclusion】The method and reaction system to detect target gene copy number in wheat genome based on digital PCR method were optimized, and the detection system to detect target gene copy number in wheat genome based on digital PCR method was established. The results of digital PCR analysis are stable and reliable, and the detection efficiency is significantly improved, which has a certain application prospect.

Key words: wheat, copy number of genes, the digital PCR, the reference genes

Table 1

Sequences of probes and primers used in digital PCR"

序号No． 引物名称Primer name 引物序列Primer sequence (5'-3')
1 Nib8-F CTCAATGCTGCTATCACCATAGTG
Nib8-R CACTCTTACT TGGCAGGAAT CTCA
Nib8-P ATTGCAGCTAGGATGTCAGAGCCGTGTT
2 PINb-D1b-F AGTTGGCGGCTGGTACAATG
PINb-D1b-R ACATCGCTCCATCACGTAATCC
PINb-D1b-P TCTCAACAATGTCCGCAGGAGCGGCC
3 Wx012-F GGTCGCAGGAACAGAGGTGT
Wx012-R GGTGTTCCTCCATTGCGAAA
Wx012-P CAAGGCGGCCGAAATAGGTTGCC
4 SSII-F CACCATCAGTGAAGGAATGAATG
SSII-R GGCGATATTTGGTACCTAATTGAAG
SSII-P TACCCGATCGACCGTTTTGCC

Table 2

PCR amplification reaction system"

组分component 用量dosage (μL)
Nib8-5-F 1 (500 nmol·L-1)
Nib8-5-R 1 (500 nmol·L-1)
Nib8-5-P 0.5 (250 nmol·L-1)
PINb-D1b-F 1 (500 nmol·L-1)
PINb-D1b-R 1 (500 nmol·L-1)
PINb-D1b-P 0.5 (250 nmol·L-1)
2 × ddPCR Super Mix 10
DNA 5 (20 ng·μL-1)
总计Total 20

Fig. 1

Specific amplification results of four pairs of primers"

Fig. 2

Digital PCR detection of nib8 (A) and internal reference genes (B) at different annealing temperatures The blue point was positive for nib8, the green point was positive for internal reference gene, and the gray point was negative. The red line was the fluorescence threshold, which was 56℃, 56.9℃, 58.1℃, 59℃, 59.6℃, 60℃ and the control from left to right"

Fig. 3

Digital PCR results of nib8 gene(A) and control gene (B) at different template concentrations The blue dots were positive for the internal reference gene, the green dots were positive for nib8, the gray dots were negative, and the red line was the fluorescence threshold. From left to right, the template and water were 20 (A01), 40 (B01), 160 (C01) and 320 ng (D01)"

Fig. 4

Specific detection of nib8 strains was performed Different transgenic lines, negative control and blank control were shown from left to right"

Table 3

The results of low limit determination by digital PCR were obtained by transplanting nib8 strain"

品系（基因）
Lines (gene)		 质量分数
Mass fraction
(%)		 用量
Dosage
(ng)		 检测拷贝数
Copy number
(个/μL)
12 100 10 7
16 100 10 5
17 100 10 1
23 100 10 1
29 100 10 1
30 100 10 7

Table 4

Comparison between fluorescence quantitative method such as probe method and dye method, and digital PCR method"

株系
Strain		 染料法qPCR
Dye method qPCR		 探针法qPCR
Probe method qPCR		 数字PCR
Digital PCR
12 7 8 7
16 1 1 1
17 1 1 1
23 1 1 1
29 1 1 1
30 6 7 7

Fig. 5

Southern blot results of nib8 transgenic wheat line 16"

Fig. 6

Copy number analysis of internal reference genes A1 and B1 are the copy numbers of Wx012 when pinb-d1b is the internal reference gene.C1 and D1 are the copy numbers of SSII when Wx012 is the internal reference gene; E1 and F1 are negative controls and blank controls"

Fig.7

Copy number analysis of transferred nib8 lines Copy number analysis of transferred nib8 line no. 12 with Wx012 and SSII as internal parameters for A1 and B1; The copy number analysis of C1 and D1 with Wx012 and SSII as internal parameters, respectively; E1 and F1 are negative controls and blank controls"

Fig.8

Results of designing primers at different positions of nib8 gene (take 12 strains as an example) A and B were used to detect the internal reference gene and target gene of the no.12 transferred nib8 line by digital PCR with primers of different regions of nib8; C: Copy number detection of No. 12 to nib8 line"

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