水稻抽穗期基因OsDof6功能的初步研究

doi:10.16819/j.1001-7216.2020.0211

摘要/Abstract

摘要：

【目的】以前期通过穗发育芯片筛选到一个水稻Dof家族转录因子OsDof6为研究对象,进一步探究OsDof6的表达模式与生物学功能。【方法】对OsDof6的基因、蛋白及启动子序列进行生物信息学分析,利用CRISPR/Cas9技术对该基因进行定点编辑,通过实时定量PCR和亚细胞定位技术分析该基因的表达模式。【结果】对该基因的启动子序列分析表明,该基因启动子中存在大量与光响应、激素响应及胁迫响应相关的顺式调控元件。利用CRISPR/Cas9技术获得了2种不同突变类型的功能缺失突变体9522^Dof6-3和9522^Dof6-4。对突变体T₁株系进行表型观察发现,相较于对照,两种突变体在营养生长阶段分蘖数明显降低,转入生殖生长阶段后,两种突变体的抽穗时间推迟约3 d。实时定量PCR结果显示OsDof6在水稻根、茎、叶和穗中均有不同程度的表达,在穗发育后期相对表达量明显提高;通过亚细胞定位分析发现OsDof6定位于细胞核。【结论】初步判断OsDof6基因会影响水稻分蘖数与抽穗期。

关键词: 水稻, Dof家族, 抽穗期, CRISPR/Cas9

Abstract:

【Objective】Our goal is to further explore the expression pattern and biological function of OsDof6, a rice Dof family transcription factor screened with the panicle development chip in the previous study. 【Method】Bioinformatics approaches were applied to analyze OsDof6 gene, protein and protein promoter sequences. The gene was edited using CRISPR/Cas9 technology. Real-time quantitative PCR and subcellular localization technology was used to reveal the gene expression pattern. 【Result】The gene promoter contains a large number of photo-, hormone-, and stress-responses and tissue expression elements. Then, two mutants of different mutation types 9522^Dof6-3 and 9522^Dof6-4 were obtained by using CRISPR/Cas9 technology. Through observation of the T₁ generation of the mutant materials, we found that the mutants had significantly less tillers and longer heading dates (delayed by 3 days) compared to the control materials. Real-time quantitative PCR results showed that OsDof6 was expressed in varying degrees in the roots, stems, leaves and panicles, and the relative expression was significantly increased at late stages of panicle development. Subcellular localization analysis showed that OsDof6 belongs to a nuclear localization gene. 【Conclusion】OsDof6 gene affects the tiller number and heading date of rice.

Key words: rice, Dof family, heading date, CRISPR/Cas9

中图分类号:

Q755
S511.01

张立成, 李懿星, 王天抗, 邱牡丹, 宋书锋, 董皓, 李磊, 刘建丰, 李莉. 水稻抽穗期基因OsDof6功能的初步研究[J]. 中国水稻科学, 2020, 34(5): 397-405.

Licheng ZHANG, Yixing LI, Tiankang WANG, Mudan QIU, Shufeng SONG, Hao DONG, Lei LI, Jianfeng LIU, Li LI. A Preliminary Study on the Function of Rice Heading Date Gene OsDof6[J]. Chinese Journal OF Rice Science, 2020, 34(5): 397-405.

图/表 9

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引物名称	序列
Primer name	Sequence (5'-3')
Cas-B1′	TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG
Cas-BL	AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC
SP1	CCCGACATAGATGCAATAACTTC
SP2	GCGCGGTGTCATCTATGTTACT
JC-Dof6-F	AGTAGCTCACCCTGTGATCCAT
JC-Dof6-R	GCCGACGACGAGGACTTC
Dof6-U3-F	GGCACCGGTACAAGCAGGGCGCCG
Dof6-U3-R	AAACCGGCGCCCTGCTTGTACCGG
1300-Dof6-F	GCCCAGATCAACTAGTATGCTGCCGTACGCG
1300-Dof6-R	TGCTCACCATGGATCCTGGCATGTAAAGAGCC
qPCR-Dof6-F	CATGAGCCTCCATGAGTG
qPCR-Dof6-R	ATGTAAAGAGCCTCCAATCC
qPCR-Hd1-F	CTCTTGGCTTCTCCTCTC
qPCR-Hd1-R	CTGTTGTTGTTGTCGTTATTG
qPCR-Ghd7-F	TACAAGGAGAAGAGGAAGAAG
qPCR-Ghd7-R	CATCTCGGCATAGGCTTT
qPCR-Hd3a-F	GTATCTACATTGGTTGGTCACT
qPCR-Hd3a-R	TCGTAGCACATCACCTCTT
qPCR-RFT1-F	TAACCTTAGGGAGTATCTACA
qPCR-RFT1-R	GTAGCACATCACCTCTTG

引物名称	序列
Primer name	Sequence (5'-3')
Cas-B1′	TTCAGAGGTCTCTCTCGCACTGGAATCGGCAGCAAAGG
Cas-BL	AGCGTGGGTCTCGACCGGGTCCATCCACTCCAAGCTC
SP1	CCCGACATAGATGCAATAACTTC
SP2	GCGCGGTGTCATCTATGTTACT
JC-Dof6-F	AGTAGCTCACCCTGTGATCCAT
JC-Dof6-R	GCCGACGACGAGGACTTC
Dof6-U3-F	GGCACCGGTACAAGCAGGGCGCCG
Dof6-U3-R	AAACCGGCGCCCTGCTTGTACCGG
1300-Dof6-F	GCCCAGATCAACTAGTATGCTGCCGTACGCG
1300-Dof6-R	TGCTCACCATGGATCCTGGCATGTAAAGAGCC
qPCR-Dof6-F	CATGAGCCTCCATGAGTG
qPCR-Dof6-R	ATGTAAAGAGCCTCCAATCC
qPCR-Hd1-F	CTCTTGGCTTCTCCTCTC
qPCR-Hd1-R	CTGTTGTTGTTGTCGTTATTG
qPCR-Ghd7-F	TACAAGGAGAAGAGGAAGAAG
qPCR-Ghd7-R	CATCTCGGCATAGGCTTT
qPCR-Hd3a-F	GTATCTACATTGGTTGGTCACT
qPCR-Hd3a-R	TCGTAGCACATCACCTCTT
qPCR-RFT1-F	TAACCTTAGGGAGTATCTACA
qPCR-RFT1-R	GTAGCACATCACCTCTTG

元件名称	代表序列	功能	数量
Element name	Representative sequence	Function	Number
3-AF1 binding site	AAGAGATATTT	光响应元件	1
Box 4	ATTAAT	参与光响应的保守DNA模块的一部分	2
GT1-motif	GGTTAA	光响应元件	3
Sp1	CC(G/A)CCC	光响应元件	2
TCCC-motif	TCTCCCT	光响应元件的一部分	3
TGGCA-motif	GATGGAAGTGGCA	光响应元件的一部分	1
Circadian	CAANNNNATC	参与昼夜节律控制的顺式作用调节元件	4
CGTCA-motif	CGTCA	参与茉莉酸甲酯反应的顺式作用调节元件	1
TGACG-motif	TGACG	参与茉莉酸甲酯反应的顺式作用调节元件	1
GARE-motif	AAACAGA	赤霉素应答元件	1
CAT-box	GCCACT	与分生组织表达相关的顺式作用调控元件	1
Skn-1_motif	GTCAT	胚乳表达所需的顺式作用调节元件	2
NON-box	AGATCGACG	与分生组织特异性激活相关的顺式作用调控元件	1
TC-rich repeats	ATTTTCTTCA	参与防御和应激反应的顺式作用元件	1
HSE	AAAAAATTTC	热应激反应中的顺式作用元件	1
MBS	CAACTG	MYB结合位点参与干旱诱导	1
MBSⅠ	TTTTTACGGTTA	MYB结合位点参与类黄酮生物合成基因调控	1
Box III	CATTTACACT	蛋白结合位点	1
CAAT-box	CCAAT	启动子和增强子区常见顺式作用元件	36
TATA-box	TTTTA/TATA	核心启动子元件	51

元件名称	代表序列	功能	数量
Element name	Representative sequence	Function	Number
3-AF1 binding site	AAGAGATATTT	光响应元件	1
Box 4	ATTAAT	参与光响应的保守DNA模块的一部分	2
GT1-motif	GGTTAA	光响应元件	3
Sp1	CC(G/A)CCC	光响应元件	2
TCCC-motif	TCTCCCT	光响应元件的一部分	3
TGGCA-motif	GATGGAAGTGGCA	光响应元件的一部分	1
Circadian	CAANNNNATC	参与昼夜节律控制的顺式作用调节元件	4
CGTCA-motif	CGTCA	参与茉莉酸甲酯反应的顺式作用调节元件	1
TGACG-motif	TGACG	参与茉莉酸甲酯反应的顺式作用调节元件	1
GARE-motif	AAACAGA	赤霉素应答元件	1
CAT-box	GCCACT	与分生组织表达相关的顺式作用调控元件	1
Skn-1_motif	GTCAT	胚乳表达所需的顺式作用调节元件	2
NON-box	AGATCGACG	与分生组织特异性激活相关的顺式作用调控元件	1
TC-rich repeats	ATTTTCTTCA	参与防御和应激反应的顺式作用元件	1
HSE	AAAAAATTTC	热应激反应中的顺式作用元件	1
MBS	CAACTG	MYB结合位点参与干旱诱导	1
MBSⅠ	TTTTTACGGTTA	MYB结合位点参与类黄酮生物合成基因调控	1
Box III	CATTTACACT	蛋白结合位点	1
CAAT-box	CCAAT	启动子和增强子区常见顺式作用元件	36
TATA-box	TTTTA/TATA	核心启动子元件	51

编号 Mutant No.	株高	分蘖个数 Tiller number	穗长	每穗粒数	结实率	千粒重	单株产量
编号 Mutant No.	Plant height/cm	分蘖个数 Tiller number	Panicle length/cm	Grain number per panicle	Seed setting rate/%	1000-grain weight/g	Yield per plant/g
9522	87.3±2.1	19.0±1.3	17.7±0.2	167.0±6.6	96.4±1.0	23.82±0.21	73.51±4.21
9522^Dof6-1	86.9±0.9	18.7±1.0	17.3±0.5	172.0±5.4	96.8±1.2	23.76±0.11	73.62±3.36
9522^Dof6-3	86.6±0.8	15.7±1.2^*	17.7±0.4	164.8±4.1	96.9±1.8	23.79±0.15	60.32±5.24^*
9522^Dof6-4	87.0±0.8	15.7±1.2^*	17.6±0.5	162.0±4.8	96.5±1.1	23.74±0.23	59.45±5.72^*