Biotechnology Bulletin ›› 2025, Vol. 41 ›› Issue (3): 83-89.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0813
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XUE Rui-ying1,2(
), LIU Yong-ju1,2, JIANG Yan-yan2,3, PENG Xin-ya1,2, CAO Dong1,3, LI Yun2(), LIU Bao-long1,2,3(), BAO Xue-mei1()
Received:2024-08-23
Online:2025-03-26
Published:2025-03-20
Contact:
LI Yun, LIU Bao-long, BAO Xue-mei
E-mail:xry198@foxmail.com;liyun@nwipb.cas.cn;baoxuemei1991@163.com
XUE Rui-ying, LIU Yong-ju, JIANG Yan-yan, PENG Xin-ya, CAO Dong, LI Yun, LIU Bao-long, BAO Xue-mei. Reducing the Expression of GBSSI Gene in Barley via the Editing in the 5′UTR Region[J]. Biotechnology Bulletin, 2025, 41(3): 83-89.
基因名称 Gene name | 引物序列 Primer sequence（5′-3′） | 备注 Remark |
|---|---|---|
| HvUTR-cta-F | GCTGCCTCTCGCACGGTC | 载体构建 Vector construction |
| HvUTR-aac-R | GCAAAGGAACCTCAGGGC | |
| BS8 | CAACTGCCGATGATCTGTCGTGTAG | 阳性克隆检测 Detection of positive clones |
| BS9 | CCGACGCGGAAATTCGTCAAGC | |
| BS11 | GGTAGAACTGATGTGAGAGATGTAGAAG | |
| BS32 | TGGTCTTCCCTTGGGGGACCGAA | |
| UTR-TOM-F | ggagtgagtacggtgtgcGCTGCCTCTCGCACGGTC | 高通量测序 High-throughput sequencing |
| UTR-TOM-R | gagttggatgctggatggCGGGACTCCAAGGAAACG | |
| WXQF1 | CCAGTCCAATGGCATCTACA | RT-qPCR |
| WXQR1 | GGCTCACCGTCAGCACCT | |
| Tubulin-F | CAAGGAGGTGGACGAGCAGATG | RT-qPCR |
| Tubulin-R | GACTTGACGTTGTTGGGGATCCA |
Table 1 The primer sequences
基因名称 Gene name | 引物序列 Primer sequence（5′-3′） | 备注 Remark |
|---|---|---|
| HvUTR-cta-F | GCTGCCTCTCGCACGGTC | 载体构建 Vector construction |
| HvUTR-aac-R | GCAAAGGAACCTCAGGGC | |
| BS8 | CAACTGCCGATGATCTGTCGTGTAG | 阳性克隆检测 Detection of positive clones |
| BS9 | CCGACGCGGAAATTCGTCAAGC | |
| BS11 | GGTAGAACTGATGTGAGAGATGTAGAAG | |
| BS32 | TGGTCTTCCCTTGGGGGACCGAA | |
| UTR-TOM-F | ggagtgagtacggtgtgcGCTGCCTCTCGCACGGTC | 高通量测序 High-throughput sequencing |
| UTR-TOM-R | gagttggatgctggatggCGGGACTCCAAGGAAACG | |
| WXQF1 | CCAGTCCAATGGCATCTACA | RT-qPCR |
| WXQR1 | GGCTCACCGTCAGCACCT | |
| Tubulin-F | CAAGGAGGTGGACGAGCAGATG | RT-qPCR |
| Tubulin-R | GACTTGACGTTGTTGGGGATCCA |
Fig. 1 BSMV vector and identification of positive strainsA: Target position. B: Schematic diagram of BSMV-α, BSMV-β, and BSMV-γ-sgUTR carriers. C: Electrophoretic map of colony PCR; lane 1-3: BSMV-α; lane 4-6: BSMV-β; lane 7-9: BSMV-γ-sgUTR
| Edit type | Sequence （5′-3′） | UTR-1 | UTR-2 | UTR-3 | UTR-4 | UTR-5 | UTR-6 | UTR-7 |
|---|---|---|---|---|---|---|---|---|
| WT | G | 7.01 | 25.55 | 98.87 | 96.89 | 19.5 | 98.26 | 98.94 |
| +A | G | 65.26 | 48.83 | / | / | 48.72 | / | / |
| +T | G | 13.42 | 7.56 | / | / | 11.48 | / | / |
| +AA | G | 1.44 | / | / | / | 1.96 | / | / |
| A→G | G | / | 2.70 | 1.13 | 1.53 | 4.20 | / | / |
| -GGT | G | / | 1.32 | / | / | / | / | / |
| +C | G | / | 2.46 | / | / | 2.62 | / | / |
| -T | G | 1.49 | 2.19 | / | / | 4.36 | / | / |
| -AT | G | 4.29 | 1.08 | / | / | 1.31 | / | / |
| -TAT | G | 1.39 | 3.72 | / | / | / | / | 1.06 |
| -GC | G | 3.87 | / | / | / | / | / | / |
| -GT | G | / | 1.62 | / | / | 2.26 | / | / |
| -GGTATT | G | 1.83 | / | / | / | / | / | / |
| -ATT | G | / | 3.02 | / | / | 1.71 | / | / |
| T→G | G | / | / | / | / | 1.88 | / | / |
| T→C | G | / | / | / | 1.58 | / | 1.74 | / |
| Edit type | Sequence （5′-3′） | UTR-8 | UTR-9 | UTR-10 | UTR-11 | UTR-12 | UTR-13 | UTR-14 |
| WT | G | 98.81 | 98.11 | 19.77 | 12.81 | 11.48 | 7.93 | 92.81 |
| +A | G | / | / | 52.05 | 56.66 | 49.91 | 40.35 | 4.00 |
| +T | G | / | / | 7.68 | 13.62 | 17.86 | 25.89 | 1.23 |
| +C | G | / | / | 6.08 | 3.32 | / | 4.78 | / |
| +AT | G | / | / | / | 4.68 | / | 2.47 | / |
| +G | G | / | / | / | 3.03 | 6.27 | 5.23 | / |
| -GT | G | / | / | 1.19 | / | / | / | / |
| -CG | G | 1.19 | / | 3.08 | / | / | / | / |
| -TAT | G | / | / | 3.24 | / | / | / | / |
| T→C | G | / | / | 1.59 | / | / | 6.12 | / |
| T→G | G | / | / | 4.18 | / | 7.60 | / | / |
| +GG | G | / | / | / | 5.89 | / | / | / |
| T→A | G | / | / | 1.14 | / | / | 7.23 | / |
| A→G | G | / | 1.89 | / | / | 6.87 | / | 1.96 |
Table 2 M0 generation editing efficiency and mutation types in BSMV:UTR plants
| Edit type | Sequence （5′-3′） | UTR-1 | UTR-2 | UTR-3 | UTR-4 | UTR-5 | UTR-6 | UTR-7 |
|---|---|---|---|---|---|---|---|---|
| WT | G | 7.01 | 25.55 | 98.87 | 96.89 | 19.5 | 98.26 | 98.94 |
| +A | G | 65.26 | 48.83 | / | / | 48.72 | / | / |
| +T | G | 13.42 | 7.56 | / | / | 11.48 | / | / |
| +AA | G | 1.44 | / | / | / | 1.96 | / | / |
| A→G | G | / | 2.70 | 1.13 | 1.53 | 4.20 | / | / |
| -GGT | G | / | 1.32 | / | / | / | / | / |
| +C | G | / | 2.46 | / | / | 2.62 | / | / |
| -T | G | 1.49 | 2.19 | / | / | 4.36 | / | / |
| -AT | G | 4.29 | 1.08 | / | / | 1.31 | / | / |
| -TAT | G | 1.39 | 3.72 | / | / | / | / | 1.06 |
| -GC | G | 3.87 | / | / | / | / | / | / |
| -GT | G | / | 1.62 | / | / | 2.26 | / | / |
| -GGTATT | G | 1.83 | / | / | / | / | / | / |
| -ATT | G | / | 3.02 | / | / | 1.71 | / | / |
| T→G | G | / | / | / | / | 1.88 | / | / |
| T→C | G | / | / | / | 1.58 | / | 1.74 | / |
| Edit type | Sequence （5′-3′） | UTR-8 | UTR-9 | UTR-10 | UTR-11 | UTR-12 | UTR-13 | UTR-14 |
| WT | G | 98.81 | 98.11 | 19.77 | 12.81 | 11.48 | 7.93 | 92.81 |
| +A | G | / | / | 52.05 | 56.66 | 49.91 | 40.35 | 4.00 |
| +T | G | / | / | 7.68 | 13.62 | 17.86 | 25.89 | 1.23 |
| +C | G | / | / | 6.08 | 3.32 | / | 4.78 | / |
| +AT | G | / | / | / | 4.68 | / | 2.47 | / |
| +G | G | / | / | / | 3.03 | 6.27 | 5.23 | / |
| -GT | G | / | / | 1.19 | / | / | / | / |
| -CG | G | 1.19 | / | 3.08 | / | / | / | / |
| -TAT | G | / | / | 3.24 | / | / | / | / |
| T→C | G | / | / | 1.59 | / | / | 6.12 | / |
| T→G | G | / | / | 4.18 | / | 7.60 | / | / |
| +GG | G | / | / | / | 5.89 | / | / | / |
| T→A | G | / | / | 1.14 | / | / | 7.23 | / |
| A→G | G | / | 1.89 | / | / | 6.87 | / | 1.96 |
Fig. 2 Editing efficiency and mutation types of different tillers in UTR plants
Fig. 3 Relative expressions of GBSSI gene RNA in M1 plantsDifferent lowercase letters indicate significant difference among different treatments (P<0.05)
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