Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (10): 159-163.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0042
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ZONG Mei(), HAN Shuo, GUO Ning, DUAN Meng-meng, LIU Fan, WANG Gui-xiang()
Received:
2022-01-10
Online:
2022-10-26
Published:
2022-11-11
Contact:
WANG Gui-xiang
E-mail:zongmei@nercv.org;wangguixiang@nercv.org
ZONG Mei, HAN Shuo, GUO Ning, DUAN Meng-meng, LIU Fan, WANG Gui-xiang. Production of Marker-free Mutants of Brassica campestris Mediated by CRISPR/Cas9 Through Vacuum Infiltration[J]. Biotechnology Bulletin, 2022, 38(10): 159-163.
引物名称 Primer name | 序列 Sequence(5'-3') | 目的 Purpose |
---|---|---|
nCas9-IDF | CATACCTCCCAGAACACAAATAAGC | 扩增nCas9片段 |
nCas9-IDR | ACTGAAGGGCAATAGTGAAGAATGT | |
gRNA-IDF | TGTCCCAGGATTAGAATGATTAGGC | 扩增gRNA片段 |
RNA-IDR | CCCCAGAAATTGAACGCCGAAGAAC | |
PDS-1 | ATGCAACTGATCAATGCGGT | 扩增含有靶点的PDS基因片段 |
PDS-356 | CGGGAATATCGCAGCTAAAG | |
PDS-F | ATTGAACGAGAAGAAGCAAGCCT | 载体构建 |
PDS-R | AAACAGGCTTGCTTCTTCTCGTT |
Table 1 Primers used in this study
引物名称 Primer name | 序列 Sequence(5'-3') | 目的 Purpose |
---|---|---|
nCas9-IDF | CATACCTCCCAGAACACAAATAAGC | 扩增nCas9片段 |
nCas9-IDR | ACTGAAGGGCAATAGTGAAGAATGT | |
gRNA-IDF | TGTCCCAGGATTAGAATGATTAGGC | 扩增gRNA片段 |
RNA-IDR | CCCCAGAAATTGAACGCCGAAGAAC | |
PDS-1 | ATGCAACTGATCAATGCGGT | 扩增含有靶点的PDS基因片段 |
PDS-356 | CGGGAATATCGCAGCTAAAG | |
PDS-F | ATTGAACGAGAAGAAGCAAGCCT | 载体构建 |
PDS-R | AAACAGGCTTGCTTCTTCTCGTT |
Fig. 1 CRISPR/Cas9-mediated gene editing and vacuum infiltration in planta transformation to creates PDS mutants in B. campestris (a):DNA sequence of the first four exons(highlighted in different colors)of PDS of ‘49 Caixin’. The sgRNA at the end of the first exon is underlined and the PAM region CCC(complementary sequence is GGG)is shown in red letter.(b):pBSE401 vector was used in vacuum infiltration transformation and the transcribed sgRNA was designed to have 19-base matches with the target. The vector carries Bar selection marker.(c):Only plant #1 showed positive in Bar strip detection. And PDS gene sequencing of plant #1 showed sequence variation in the target region.(d):Two of the 2 032 seedlings(plant #2 and plant #3)were weak albino,which was consistent with the phenotypes of PDS mutants. The picture shows plant #2.(e):Sanger sequencing of plant #2 and plant #3 revealed the targeted mutagenesis of PDS gene in 2 plants compared with the wild type
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