Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (7): 62-69.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1592
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SHI Jia(), ZHU Xiu-mei, XUE Meng-yu, YU Chao, WEI Yi-ming, YANG Feng-huan, CHEN Hua-min()
Received:
2021-12-25
Online:
2022-07-26
Published:
2022-08-09
Contact:
CHEN Hua-min
E-mail:qwer1768004860@163.com;chenhuamin@caas.cn
SHI Jia, ZHU Xiu-mei, XUE Meng-yu, YU Chao, WEI Yi-ming, YANG Feng-huan, CHEN Hua-min. Optimization and Application of the Chromatin Immunoprecipitation Based on Rice Protoplast[J]. Biotechnology Bulletin, 2022, 38(7): 62-69.
家族Family | 基因Gene | ID | CCAAT 数目及位置CCAAT number and position |
---|---|---|---|
OsNRT1家族(低亲和力)OsNRT1 family(low affinity) | OsNRT1.1 | Os03g13274 | 2(-1404,-1055) |
OsNRT2家族(高亲和力)OsNRT2 family(high affinity) | OsNRT2.1 | Os02g02170 | 1(-175) |
OsNRT2.2 | Os02g02190 | 2(-276,-53) | |
OsNAR2.1 | Os02g38230 | 2(-631,-460) |
Table 1 Prediction of regulatory elements in the promoter region of NRTs genes
家族Family | 基因Gene | ID | CCAAT 数目及位置CCAAT number and position |
---|---|---|---|
OsNRT1家族(低亲和力)OsNRT1 family(low affinity) | OsNRT1.1 | Os03g13274 | 2(-1404,-1055) |
OsNRT2家族(高亲和力)OsNRT2 family(high affinity) | OsNRT2.1 | Os02g02170 | 1(-175) |
OsNRT2.2 | Os02g02190 | 2(-276,-53) | |
OsNAR2.1 | Os02g38230 | 2(-631,-460) |
基因名称 Gene name | 引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 片段大小及内容 Segment size and content |
---|---|---|---|
OsNRT1.1 | OsNRT1.1 Q-F2 | TGCTACGGTCTCATCTTCTCT | 199 bp 包含NRT1.1基因启动子序列中的第二个CCAAT-box 199 bp contains the second CCAAT-box in the promoter of NRT1.1 |
OsNRT1.1 Q-R2 | CAAGTAATCCATCTAACGCTACGA | ||
OsNRT2.1 | OsNRT2.1 Q-F1 | ACGAATCTTAAGGCAAAT | 194 bp包含NRT2.1基因启动子序列中的一个CCAAT-box 199 bp contains a CCAAT-box in the promoter of NRT2.1 |
OsNRT2.1 Q-R1 | CTTCTTGGAGATGGAATC | ||
OsNRT2.2 | OsNRT2.2 Q-F2 | CACGAGGCAGATATTACAACTTGA | 149 bp 包含NRT2.2基因启动子序列中的第二个CCAAT-box 149 bp contains the second CCAAT-box in the promoter of NRT2.2 |
OsNRT2.2 Q-R2 | CGGTGACGATGATCTTGGC | ||
OsNAR2.1 | OsNAR2.1 Q-F2 | TTCCTCCATTAAGAACGCCTTC | 119 bp 包含NAR2.1基因启动子序列中的第二个CCAAT-box 119 bp contains the second CCAAT-box in the promoter of NAR2.1 |
OsNAR2.1 Q-R2 | TTGAGTGCCTCGGTTGTTG | ||
Negative | OsNAR2.1-NF | GATGGCTGTCCTGCTCTTG | 183 bp 为NAR2.1基因启动子中不含CCAAT-box的序列 183 bp NAR2.1 gene promoter without CCAAT-box |
OsNAR2.1-NR | ATCATTTCGCTCCTCCAAACTATT |
Table 2 Required primer sequences for RT-qPCR validation
基因名称 Gene name | 引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 片段大小及内容 Segment size and content |
---|---|---|---|
OsNRT1.1 | OsNRT1.1 Q-F2 | TGCTACGGTCTCATCTTCTCT | 199 bp 包含NRT1.1基因启动子序列中的第二个CCAAT-box 199 bp contains the second CCAAT-box in the promoter of NRT1.1 |
OsNRT1.1 Q-R2 | CAAGTAATCCATCTAACGCTACGA | ||
OsNRT2.1 | OsNRT2.1 Q-F1 | ACGAATCTTAAGGCAAAT | 194 bp包含NRT2.1基因启动子序列中的一个CCAAT-box 199 bp contains a CCAAT-box in the promoter of NRT2.1 |
OsNRT2.1 Q-R1 | CTTCTTGGAGATGGAATC | ||
OsNRT2.2 | OsNRT2.2 Q-F2 | CACGAGGCAGATATTACAACTTGA | 149 bp 包含NRT2.2基因启动子序列中的第二个CCAAT-box 149 bp contains the second CCAAT-box in the promoter of NRT2.2 |
OsNRT2.2 Q-R2 | CGGTGACGATGATCTTGGC | ||
OsNAR2.1 | OsNAR2.1 Q-F2 | TTCCTCCATTAAGAACGCCTTC | 119 bp 包含NAR2.1基因启动子序列中的第二个CCAAT-box 119 bp contains the second CCAAT-box in the promoter of NAR2.1 |
OsNAR2.1 Q-R2 | TTGAGTGCCTCGGTTGTTG | ||
Negative | OsNAR2.1-NF | GATGGCTGTCCTGCTCTTG | 183 bp 为NAR2.1基因启动子中不含CCAAT-box的序列 183 bp NAR2.1 gene promoter without CCAAT-box |
OsNAR2.1-NR | ATCATTTCGCTCCTCCAAACTATT |
Fig.2 Expressions detection of OsNF-YA4 protein after protoplast transformed After rice protoplast transformed with expression vector and the incubation was performed at different times,the transient expressed proteins were extracted,and the expression of fused protein was detected via anti-Myc antibody 1-4:NF-YA4 protein expressions at 12,18,24 and 36 h. Coomassie brilliant blue staining showed the loaded protein in each lane
甲醛终浓度 Final concentration of formaldehyde/% | 细胞活性百分比 Percentage of cell activity/% |
---|---|
0 | 100+0.01 |
0.1 | 93+2.79 |
0.3 | 80+4.27 |
0.7 | 72+3.98 |
1.0 | 48+2.72 |
Table 3 Effects of different final concentrations of formal-dehyde on protoplasmic cell activity
甲醛终浓度 Final concentration of formaldehyde/% | 细胞活性百分比 Percentage of cell activity/% |
---|---|
0 | 100+0.01 |
0.1 | 93+2.79 |
0.3 | 80+4.27 |
0.7 | 72+3.98 |
1.0 | 48+2.72 |
Fig.3 Optimization of ultrasonic fragmentation time on OsNF-YA4 ChIPs-enriched DNA(A)and validation of the fragmentation(B) A:Effects of ultrasonic crushing time on fragment length(M:DL2000 marker;1-5:ultrasonic duration was 5,10,15,20 and 30 min respectivey)B:The fragmentation of DNA enrichment by ChIPs(1:the fragmentation of input when ultrasonic treated 15 min)
Fig.4 Detection of the effect of chromosome immunopreci-pitation(A)and specificity analysis of enriched DNA(B)A:The level of OsNFYA4 after immunoprecipitation detected by Western blot(1:immunoprecipitated samples(ChIPs-)without anti-Myc antibody;2:immunoprecipitated samples with anti-Myc antibody(ChIPs+)). B:Validation of the specificity of the ChIP-RP enriched DNA by qRT-PCR
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