Chinese Journal OF Rice Science ›› 2022, Vol. 36 ›› Issue (2): 150-158.DOI: 10.16819/j.1001-7216.2022.210606
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QIU Linlin1, LIU Qiao1, FU Yaping2, LIU Wenzhen2, HU Guocheng2, ZHAI Yufeng1, PANG Bo1, WANG Dekai1,*()
Received:
2021-06-17
Revised:
2021-09-14
Online:
2022-03-10
Published:
2022-03-11
Contact:
WANG Dekai
裘霖琳1, 刘窍1, 付亚萍2, 刘文真2, 胡国成2, 翟玉凤1, 庞礴1, 汪得凯1,*()
通讯作者:
汪得凯
基金资助:
QIU Linlin, LIU Qiao, FU Yaping, LIU Wenzhen, HU Guocheng, ZHAI Yufeng, PANG Bo, WANG Dekai. Identification and Gene Cloning of DSP2 in Rice (Oryza sativa L.)[J]. Chinese Journal OF Rice Science, 2022, 36(2): 150-158.
裘霖琳, 刘窍, 付亚萍, 刘文真, 胡国成, 翟玉凤, 庞礴, 汪得凯. 水稻矮化小穗基因DSP2的鉴定与克隆[J]. 中国水稻科学, 2022, 36(2): 150-158.
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URL: http://www.ricesci.cn/EN/10.16819/j.1001-7216.2022.210606
引物名称 | 正向引物序列 | 反向引物序列 | 实验目的 |
---|---|---|---|
Primer name | Forward primer(5'-3') | Reverse primer(5'-3') | Purpose |
ORF1-qPCR | AGATCAACCTCCAACTACTCTC | AAGCTCAGTAGTACACAGGC | RT-PCR |
ORF2-qPCR | GTGGTTCCAGAAGATCGTGTC | TTGTCGATGGACAGGAGCTC | RT-PCR |
ORF3-qPCR | CACGCCTACGACAACATGAAC | GTACTCGAACGCGTTGACATC | RT-PCR |
ORF4-qPCR | CCGGTGATACAAAGAGGTGC | ATATGCTTCGGCCACCTTG | RT-PCR |
ORF5-qPCR | AGCGGTTGTCTGGTGATATC | AGACAGAAAACCCCTTGACG | RT-PCR |
ORF6-qPCR | CAGCTTCTGATCCTGCAGTAG | ATCTCCCTTACTGATGCTGAC | RT-PCR |
UBQ5-qPCR | ACCACTTCGACCGCCACTACT | ACGCCTAAGCCTGCTGGTT | RT-PCR |
HPTⅡ | CAGAAGAAGATGTTGGCGAC | ATGTCCTGCGGGTAAATAGC | 共分离分析 Co-segregation |
DSP2-ORF | ATGTCGACATGGCTGGTGCTACGGCTGC | ATCTGCAGCTGAGAGTAGTTGGAGGTTGAT | 全长ORF Full length ORF |
NPTⅡ | TATGTCCTGATAGCGGTCCG | GTGCCCTGAATGAACTCCAG | 转化植株检测 Detection of transgenic plants |
Table 1 Primers used for PCR identification and quantitative real-time PCR (qRT-PCR) analysis.
引物名称 | 正向引物序列 | 反向引物序列 | 实验目的 |
---|---|---|---|
Primer name | Forward primer(5'-3') | Reverse primer(5'-3') | Purpose |
ORF1-qPCR | AGATCAACCTCCAACTACTCTC | AAGCTCAGTAGTACACAGGC | RT-PCR |
ORF2-qPCR | GTGGTTCCAGAAGATCGTGTC | TTGTCGATGGACAGGAGCTC | RT-PCR |
ORF3-qPCR | CACGCCTACGACAACATGAAC | GTACTCGAACGCGTTGACATC | RT-PCR |
ORF4-qPCR | CCGGTGATACAAAGAGGTGC | ATATGCTTCGGCCACCTTG | RT-PCR |
ORF5-qPCR | AGCGGTTGTCTGGTGATATC | AGACAGAAAACCCCTTGACG | RT-PCR |
ORF6-qPCR | CAGCTTCTGATCCTGCAGTAG | ATCTCCCTTACTGATGCTGAC | RT-PCR |
UBQ5-qPCR | ACCACTTCGACCGCCACTACT | ACGCCTAAGCCTGCTGGTT | RT-PCR |
HPTⅡ | CAGAAGAAGATGTTGGCGAC | ATGTCCTGCGGGTAAATAGC | 共分离分析 Co-segregation |
DSP2-ORF | ATGTCGACATGGCTGGTGCTACGGCTGC | ATCTGCAGCTGAGAGTAGTTGGAGGTTGAT | 全长ORF Full length ORF |
NPTⅡ | TATGTCCTGATAGCGGTCCG | GTGCCCTGAATGAACTCCAG | 转化植株检测 Detection of transgenic plants |
Fig. 1. Phenotypes of the dsp2-D mutant. AA, Wild-type ‘Nipponbare’ plants; Aa, dsp2-D heterozygous plants; aa, dsp2-D homozygous plants. Values are mean ± SD of ten biological replicates. Samples with different letters are significantly different (P < 0.01; Tukey’s test).
Fig. 2. Location of DSP2 on chromosome 2 and co-segregation analysis of T-DNA. A, Preliminary localization of DSP2 gene; B, Co-segregation analysis of T-DNA and dsp2-D phenotypes. M, 1 kb ladder, P, Plasmid positive control; A1-A3, Wild type; B1-B7, Heterozygote; C1-C4, Homozygous mutant.
Fig. 3. Expression analysis of candidate gene DSP2. A, Schematic diagram of T-DNA insertion sites; B and C, Expression levels of six candidate genes in dsp2-D and wild type analyzed by semi-quantitative RT-PCR and real time PCR; OsUBQ5 was amplified as a control. Bars represent standard deviation (n=3). ** indicates significant difference between Nipponbare and dsp2-D by t-test (P<0.01).
Fig. 4. Overexpression of DSP2 gene and functional identification. A, Expression levels of DSP2 gene in wild-type and transgenic plants. WT, Wild type Nipponbare; CK, Control plasmid transgenic plants; OE-1#-OE-3#, DSP2-OE transgenic plants; Bars represent standard deviation (n=3). **indicates significant difference between Nipponbare and dsp2-D by t-test (P<0.01). B and C, Phenotype of the dsp2-D mutant and transgenic plant. B, Bar=10 cm; C, Bar=5 cm.
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