Effects of Exogenous Ethephon on Fertility of Thermo-sensitive Genic Male Sterile Rice Lines Under Low Temperature Inducement

doi:10.16819/j.1001-7216.2018.7045

Abstract

Abstract:

【Objective】Thisresearch aims toelucidate the mechanism behind sterility conversion(infertile convert into fertile)in low temperatureofthermo-sensitive genic male sterile ricein seed production in South China by applying exogenous ethephon.【Method】The thermo-sensitive genic male sterile rice cultivars Zhu 1S and Zhun S weretreated with artificial cooling water tank (low temperature, 22.5℃) induction for 7 days and sprayed withethephon (which promote synthesis of ethylene) and the chlorine dioxide solution (inhibitory substances)during fertility-sensitive period.Then the pollendyeing rate and the baggedseed-setting rate were calculatedand the quantitative real-time PCR was conducted.In addition, the promoters of OsACO5 and OsACS5, which are key enzymes of ethylene synthesis pathway, were isolated and analyzed.【Result】Under22.5℃for 7 days, the pollen rate of Zhu 1S and Zhun Swas 4.3% and 8.9%, and bagged seed-setting rate was 1.6% and 2.5%, respectively. Compared with the water spray control, the pollen rate of lines Zhu 1S and Zhun S was reduced by 3.3and 7.2 percent, respectively when spraying with 1600mg/L ethephon. When spraying with 1500 mg/L of chlorine dioxide solution, the pollen rate was increased by 3.5and 3.3 percent, and the bagged seed setting rate increased by 0.78and 0.52 percent, respectively. The expression of OsACO5 and OsACS5in young panicles was significantly increased at 1600 mg/L of ethephon under 22.5℃ inductionfor 7 days, which were 1.19, 1.24 (Zhu 1S) and 1.24, 1.1 (Zhun S) times of that of the control (water spray) respectively. When sprayed with 1500mg/L of chlorine dioxide solution for 7 days, the expression of OsACS5 gene in the Zhu 1S and Zhun S was significantly reduced, which were 0.93 and 0.89 times of that of the control (water spray), respectively, but the expression of OsACO5 was not significantly changed. Compared with normal field temperature (control) for 7 days, water spraying, 1600 mg/L of ethephon and 1500 mg/L of chlorine dioxide solution at22.5℃down-regulated the expression of OsACO5 and OsACS5 in young panicles of Zhu 1S and Zhun S.The promoters of OsACO5 and OsACS5contained multiple tissue-specific expression elements and ethylene-regulated cis-acting elements in addition to the basic structures such as TATA-box and CAAT-box. 【Conclusion】Under low temperature condition, spraying 1600mg/L ethephon in the sensitive period of thermo-sensitive genic male sterile rice had inhibitory effect on fertility conversion from sterility to fertility.

Key words: thermo-sensitive genic male sterile rice, low-temperature inducement, ethephon, fertility

摘要：

【目的】为解决我国南方温敏核不育水稻制种过程中遇低温易发生育性转换问题,对处于育性转换敏感期的温敏核不育水稻进行低温诱导并喷施不同浓度乙烯利,探讨其对温敏核不育水稻育性的影响。【方法】利用温敏核不育水稻株1S和准S为材料,在育性敏感时期对其进行连续7d冷灌池低温(22.5℃)诱导并分别喷施乙烯合成促进物质乙烯利和抑制物质二氧化氯溶液,统计花粉可染率及套袋自交结实率,对乙烯合成关键酶进行实时荧光定量PCR分析。分离并分析了乙烯合成途径关键酶OsACO5和OsACS5的启动子。【结果】在22.5℃低温诱导条件下,株1S和准S连续7 d喷水处理后的可染花粉率分别是4.3%和8.9%,套袋结实率分别是1.6%和2.5%;与喷水对照相比,喷施1600mg/L乙烯利,株1S和准S可染花粉率分别下降3.3和7.2个百分点,且都无结实;喷施1500mg/L二氧化氯溶液的株1S和准S可染花粉率分别上升3.5和3.3个百分点,套袋结实率分别上升0.78和0.52个百分点。在低温诱导条件下(以喷水为对照),连续7 d喷施1600mg/L乙烯利时,株1S和准S幼穗中OsACO5和OsACS5基因表达量显著升高,分别是对照(喷水)的1.19、1.24(株1S)和1.24、1.1(准S)倍;连续7 d喷施1500mg/L二氧化氯溶液时,株1S和准S幼穗中OsACO5基因表达量变化不显著,而株1S和准S中OsACS5基因表达量显著降低,分别是对照(喷水)的93%和89%。与正常大田温度相比,在22.5℃低温诱导7 d时,喷水、喷施1600mg/L乙烯利及1500mg/L二氧化氯溶液处理分别会导致株1S和准S幼穗中OsACO5和OsACS5基因下调表达。此外,发现OsACO5和OsACS5的启动子除含TATA盒和CAAT盒等基本结构以外,还含有多个组织特异表达及乙烯调控顺式作用元件。【结论】低温诱导条件下,在温敏核不育水稻育性敏感期,喷施1600mg/L乙烯利可抑制育性转换。

关键词: 温敏核不育水稻, 低温诱导, 乙烯利, 育性分析

CLC Number:

Linzhi LI, Jiaping WEI, Yanfeng JIA, Xiaolin LIU, Haiqing ZHANG, Ming WANG, Hao MA. Effects of Exogenous Ethephon on Fertility of Thermo-sensitive Genic Male Sterile Rice Lines Under Low Temperature Inducement[J]. Chinese Journal OF Rice Science, 2018, 1(1): 128-136.

李林芝, 魏家萍, 贾彦凤, 刘小林, 张海清, 王明, 麻浩. 低温条件下喷施乙烯利对温敏核不育水稻育性的影响[J]. 中国水稻科学, 2018, 1(1): 128-136.

Figures/Tables 6

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引物名称 Primer name	引物序列 Primersequence (5′-3′)	产物大小 Amplified productsize / bp	引物用途 Application of primer
Promoter-OsACO5-F	AAATGTCGCCATCACCTTTACC	2056	启动子扩增
Promoter-OsACO5-R	GAAGAATCCCCACTCCTCGC		Promoter amplification
Promoter-OsACS5-F	CCCATTTTTATGTAATTGAAGTAG	1838	启动子扩增
Promoter-OsACS5-R	GTAGGGGTCCTCGTCGTAG		Promoter amplification
ORF-OsACO5-F	AAAAACTTGAGAAATTAACTAAACA	927	基因扩增
ORF-OsACO5-R	TCCTACTGACAAACCGTGACA		Gene amplification
ORF-OsACS5-F	GAGAACGAGAGAGAACATCAT	1386	基因扩增
ORF-OsACS5-R	AACTAACTAATTAACCAAATTAACT		Gene amplification
qRT-OsACO5-F	GACAATGGCTGCGAGGAGTG	87	荧光定量RT-PCR
qRT-OsACO5-R	AGCACACCTTCTTCACCCGAT		Fluorescent quantitative RT-PCR
qRT-OsACS5-F	CAACGACATCCACCTCATCT	148	荧光定量RT-PCR
qRT-OsACS5-R	GGTCCTTGGAGAGGCTGTAG		Fluorescent quantitative RT-PCR
18S-F	ATGGTGGTGACGGGTGAC	150	cDNA质量检测
18S-R	CAGACACTAAAGCGCCCGGTA		cDNAquality assay

引物名称 Primer name	引物序列 Primersequence (5′-3′)	产物大小 Amplified productsize / bp	引物用途 Application of primer
Promoter-OsACO5-F	AAATGTCGCCATCACCTTTACC	2056	启动子扩增
Promoter-OsACO5-R	GAAGAATCCCCACTCCTCGC		Promoter amplification
Promoter-OsACS5-F	CCCATTTTTATGTAATTGAAGTAG	1838	启动子扩增
Promoter-OsACS5-R	GTAGGGGTCCTCGTCGTAG		Promoter amplification
ORF-OsACO5-F	AAAAACTTGAGAAATTAACTAAACA	927	基因扩增
ORF-OsACO5-R	TCCTACTGACAAACCGTGACA		Gene amplification
ORF-OsACS5-F	GAGAACGAGAGAGAACATCAT	1386	基因扩增
ORF-OsACS5-R	AACTAACTAATTAACCAAATTAACT		Gene amplification
qRT-OsACO5-F	GACAATGGCTGCGAGGAGTG	87	荧光定量RT-PCR
qRT-OsACO5-R	AGCACACCTTCTTCACCCGAT		Fluorescent quantitative RT-PCR
qRT-OsACS5-F	CAACGACATCCACCTCATCT	148	荧光定量RT-PCR
qRT-OsACS5-R	GGTCCTTGGAGAGGCTGTAG		Fluorescent quantitative RT-PCR
18S-F	ATGGTGGTGACGGGTGAC	150	cDNA质量检测
18S-R	CAGACACTAAAGCGCCCGGTA		cDNAquality assay

不育系 Sterile line	处理时间Treatment time/d		喷水处理 Water spraying	ClO₂					乙烯利Ethephon
不育系 Sterile line	处理时间Treatment time/d		喷水处理 Water spraying	750 mg/L			1500 mg/L		1600 mg/L		8000 mg/L
株1S Zhu 1S		1	0.0		0.0	0.0		0.0		0.0
		4	2.1		1.0	5.6		0.0		0.0
		7	4.3		5.3	7.8		1.0		0.0
准SZhun S		1	0.0		0.0	0.0		0.0		0.0
		4	6.5		1.8	10.1		0.0		0.0
		7	8.9		9.0	12.2		1.7		0.0

不育系 Sterile line	处理时间Treatment time/d		喷水处理 Water spraying	ClO₂					乙烯利Ethephon
不育系 Sterile line	处理时间Treatment time/d		喷水处理 Water spraying	750 mg/L			1500 mg/L		1600 mg/L		8000 mg/L
株1S Zhu 1S		1	0.0		0.0	0.0		0.0		0.0
		4	2.1		1.0	5.6		0.0		0.0
		7	4.3		5.3	7.8		1.0		0.0
准SZhun S		1	0.0		0.0	0.0		0.0		0.0
		4	6.5		1.8	10.1		0.0		0.0
		7	8.9		9.0	12.2		1.7		0.0

不育系 Sterile line	处理时间Treatment time/d		喷水处理 Water spraying	ClO₂					乙烯利Ethephon
不育系 Sterile line	处理时间Treatment time/d		喷水处理 Water spraying	750 mg/L			1500 mg/L		1600 mg/L		8000 mg/L
株1S Zhu 1S		1	0.0		0.0	0.0		0.0		0.0
		4	0.7		0.0	1.8		0.0		0.0
		7	1.6		1.8	2.4		0.0		0.0
准SZhun S		1	0.0		0.0	0.0		0.0		0.0
		4	1.2		0.0	2.6		0.0		0.0
		7	2.5		2.5	3.0		0.0		0.0