Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (1): 224-231.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0461
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YU Xiao-ling1,2(), LI Wen-bin1,2, LI Zhi-bo1, RUAN Meng-bin1,2()
Received:
2022-04-15
Online:
2023-01-26
Published:
2023-02-02
Contact:
RUAN Meng-bin
E-mail:yuxiaoling@itbb.org.cn;ruanmengbin@itbb.org.cn
YU Xiao-ling, LI Wen-bin, LI Zhi-bo, RUAN Meng-bin. Cold Resistance Function Analysis of Cassava MeMYC2.2[J]. Biotechnology Bulletin, 2023, 39(1): 224-231.
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 引物用途 Primer usage |
---|---|---|
MeMYC 2.2-F | GCTCTAGAATGAATCTGT- GGACGGACGA | 基因克隆 Gene cloning |
MeMYC 2.2-R | GTGGATCCGGAGTCACCA- ACTTTGGTTG | |
MeActin-qF | TGATGAGTCTGGTCCATCCA | 内参基因 |
MeActin-qR | CCTCCTACGACCCAATCTCA | Reference gene |
MeMYC 2.2-qF | CCCTGACCAGGGTGAGAATGAT | 实时荧光定量 RT-qPCR |
MeMYC 2.2-qR | CCCTTTCGACACATGCTGAT | |
AtActin1-qF | GGGCAAGTGATCACCATTGG | 转基因拟南芥基因表达分析 Gene expression analysis of transgenic Arabidopsis |
AtActin1-qR | TGGAGCCAAAGCAGTGATCTC | |
AtCBF1-qF | ATTATTGTCCGACGTTGGCCA | |
AtCBF1-qR | GCGAAGTTGAGACATGCTGAT | |
AtCBF2-qF | TGCCTGTCTCAATTTCGCTGA | |
AtCBF2-qR | CGGCCATGTTATCCAACAAAC | |
AtCBF3-qF | CAAGGATTTGGCTCGGAACAT | |
AtCBF3-qR | TCCACCAACGTCTCCTCCATG | |
AtCBF4-qF | AAGCTGCAATGGCGTTTCAGA | |
AtCBF4-qR | CGTCACCCACTCCGTCAAAGT |
Table 1 Primers used in this study
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 引物用途 Primer usage |
---|---|---|
MeMYC 2.2-F | GCTCTAGAATGAATCTGT- GGACGGACGA | 基因克隆 Gene cloning |
MeMYC 2.2-R | GTGGATCCGGAGTCACCA- ACTTTGGTTG | |
MeActin-qF | TGATGAGTCTGGTCCATCCA | 内参基因 |
MeActin-qR | CCTCCTACGACCCAATCTCA | Reference gene |
MeMYC 2.2-qF | CCCTGACCAGGGTGAGAATGAT | 实时荧光定量 RT-qPCR |
MeMYC 2.2-qR | CCCTTTCGACACATGCTGAT | |
AtActin1-qF | GGGCAAGTGATCACCATTGG | 转基因拟南芥基因表达分析 Gene expression analysis of transgenic Arabidopsis |
AtActin1-qR | TGGAGCCAAAGCAGTGATCTC | |
AtCBF1-qF | ATTATTGTCCGACGTTGGCCA | |
AtCBF1-qR | GCGAAGTTGAGACATGCTGAT | |
AtCBF2-qF | TGCCTGTCTCAATTTCGCTGA | |
AtCBF2-qR | CGGCCATGTTATCCAACAAAC | |
AtCBF3-qF | CAAGGATTTGGCTCGGAACAT | |
AtCBF3-qR | TCCACCAACGTCTCCTCCATG | |
AtCBF4-qF | AAGCTGCAATGGCGTTTCAGA | |
AtCBF4-qR | CGTCACCCACTCCGTCAAAGT |
分析内容 Item | MeMYC2.1 | MeMYC2.2 |
---|---|---|
开放阅读框Open read frame(ORF) | 2 055 bp | 2 019 bp |
内含子 Intron | - | - |
蛋白质的分子量Molecular weight(Mw) | 75 043.89 | 74 015.17 |
等电点 Isoelectric point(pI) | 5.49 | 5.14 |
Plant-mPLoc软件预测 Plant-mPLoc software prediction | 定位于细胞核 | 定位于细胞核 |
SignalP | 非分泌蛋白 | 非分泌蛋白 |
保守功能结构域Conservative functional domains | bHLH-MYC保守结构域(N69-20),及HLH DNA结合域(N505-556) | bHLH-MYC保守结构域(N5-243),及HLH DNA结合域(N218-239) |
Table 2 Bioinformatics analysis of MeMYC2
分析内容 Item | MeMYC2.1 | MeMYC2.2 |
---|---|---|
开放阅读框Open read frame(ORF) | 2 055 bp | 2 019 bp |
内含子 Intron | - | - |
蛋白质的分子量Molecular weight(Mw) | 75 043.89 | 74 015.17 |
等电点 Isoelectric point(pI) | 5.49 | 5.14 |
Plant-mPLoc软件预测 Plant-mPLoc software prediction | 定位于细胞核 | 定位于细胞核 |
SignalP | 非分泌蛋白 | 非分泌蛋白 |
保守功能结构域Conservative functional domains | bHLH-MYC保守结构域(N69-20),及HLH DNA结合域(N505-556) | bHLH-MYC保守结构域(N5-243),及HLH DNA结合域(N218-239) |
Fig. 1 Expression patterns of MeMYC2 under different cold stress(CS)time In vitro cassava seedling leaves were treated by low temperature at 10℃. New expanding leaves of stressed or unstressed seedlings were used in qPCR analysis. The MeActin1 was used as housekeep gene. Error bars are ± SD(n=3), different letters indicate significant differences with P≤0.05(ANOVA test), the same below
Fig. 4 Wild-type and transgenic plants demonstrating different freezing tolerance abilities Two-week-old seedlings were treated by cold at -20℃ for 4-6 h, and then these treated seedlings were recovered for 5 d under normal conditions
基因型 Genotype | 冷冻处理 Freezing treatment/h | 存活率 Survival rate/% |
---|---|---|
Col-0 | 4 | 100 |
5 | 74.2±1.5 | |
6 | 29.4±7.1 | |
MYC2.2 OE | 4 | 100 |
5 | 92.2±2.3 | |
6 | 83.6±3.6 |
Table 3 Survival rates of wild-type and transgenic plants with cold treatment (n>30)
基因型 Genotype | 冷冻处理 Freezing treatment/h | 存活率 Survival rate/% |
---|---|---|
Col-0 | 4 | 100 |
5 | 74.2±1.5 | |
6 | 29.4±7.1 | |
MYC2.2 OE | 4 | 100 |
5 | 92.2±2.3 | |
6 | 83.6±3.6 |
Fig. 5 Expression analysis of CBF genes in transgenic Arabidopsis under cold stress The relative expression of indicated CBF gene in Col-0 Arabidopsis under normal conditions was set to 1. Error bars are ± SD(n=3), and different letters indicate differences with P≤0.05(ANOVA test)
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