sua-CMS烟草不育系COXⅡ的功能分析

doi:10.3864/j.issn.0578-1752.2023.16.003

摘要/Abstract

摘要：

【目的】细胞质雄性不育由细胞质基因组（主要是线粒体基因组）控制，在杂交种的育种和生产中起重要作用。长期以来，我国烟草种植以不育系和杂交种为主，其面积占烟草总种植面积的80%以上，不育类型均为sua-CMS，但该不育类型的控制基因、不育机制尚不清楚。COXⅡ是细胞色素氧化酶（COX）的第二亚基，由线粒体基因组编码。在sua-CMS烟草不育系中，COXⅡ末端发生多个碱基变异，导致C端编码蛋白Thr²⁵⁷被Ser取代。研究sua-CMS烟草COXⅡ功能，为阐明sua-CMS烟草不育分子机制奠定基础。【方法】以4组sua-CMS不育系和可育对照为材料，RNA印迹杂交和qRT-PCR方法分析COXⅡ在sua-CMS不育系和可育对照之间的转录本、基因表达差异，Western杂交、COX酶活性试验分析COX蛋白表达和COX酶活性，并测定ATP含量；将可育烟草的COXⅡ在sua-CMS不育系中过表达，研究COXⅡ过表达对COXⅡ表达量、COX酶活性、ATP含量以及烟草雄蕊发育的影响。【结果】通过对COXⅡ多序列比对，发现C端Thr²⁵⁷在物种间高度保守，表达分析显示不育系中COXⅡ转录本显著低于可育对照，COXⅡ在不育系烟草中的表达量只有对照的0.3%—0.4%，COX蛋白表达量和COX酶活性均显著低于可育对照，ATP含量为可育对照的52%。将可育烟草COXⅡ在不育系烟草中过表达，转基因植株COXⅡ在花芽中的表达量最高提高171倍，COX酶活性提高70%，ATP含量提高90%，但转基因植株在花药中的COXⅡ表达量、COX酶活性和ATP含量等各项指标均低于可育对照。转基因植株的花药形态发生了明显的改变，并出现了花药壁的分化，但花药内无胞母细胞分化，最终也无花粉粒产生。【结论】sua-CMS烟草不育系COXⅡ变异影响COX功能，是其花药能量缺陷的重要原因，也是影响花药发育的一个重要因素。

关键词: 烟草, sua-CMS, COXⅡ, 功能鉴定

Abstract:

【Objective】Cytoplasmic male sterility (CMS) is controlled by the cytoplasmic genome, mainly the mitochondrial genome, which plays an important role in breeding and production of hybrids. Sterile lines and hybrids are the main types of tobacco cultivation in China, accounting for more than 80% of the total tobacco cultivation area. The sterile type of tobacco in China is sua-CMS, which is also the only sterile type of tobacco production, but the control gene and sterility mechanism of sua-CMS are not clear. COXⅡ is the second subunit of cytochrome oxidase (COX), encoded by the mitochondrial genome. Nucleotide base variants occur at the terminal of the COXⅡ in sua-CMS lines, which resulted in the replacement of the C-terminal of COXⅡ Thr²⁵⁷ by Ser. The function analysis of COXⅡ in sua-CMS tobacco provides a foundation for the elucidation of the molecular mechanism of sterility in tobacco.【Method】In this study, four sets of sua-CMS lines and fertile controls were used as materials, and RNA blot hybridization and qRT-PCR methods were used to analyze the transcript and gene expression of COXⅡ between sua-CMS lines and fertile controls, and Western blot and COX enzyme activity experiments analyzed COX protein expression and COX enzyme activity, and ATP content were measured. The COXⅡ of fertile tobacco was overexpressed in sua-CMS line, and the effects of COXⅡ overexpression on COXⅡ expression, COX enzyme activity, ATP content and stamens development of tobacco were studied.【Result】COXⅡ multi-sequence alignment showed that Thr²⁵⁷at C-terminal was highly conserved between species. Expression analysis showed that the COXⅡ transcript in sua-CMS line was significantly lower than that of the fertile control, and quantitative expression of four sets of sua-CMS lines and fertile controls showed that the expression amount of COXⅡ in sua-CMS lines was only 0.3%-0.4% of the fertile controls, the COX protein abundances and enzyme activity were significantly lower than that of the fertile control, and the ATP content of sua-CMS line was 52% of that of the fertile control. When COXⅡ was overexpressed in sua-CMS line, the expression levels of COXⅡ in transgenic plants increased by up to 171 times, the COX enzyme activity increased by 70%, the ATP content increased by 90%. However, all indicators including COXⅡ expression levels, COX enzyme activity and ATP content in transgenic plants were lower than those of fertile tobacco. We also observed significant changes in anther morphology in transgenic plants, and the differentiation of the anther wall appeared, but there was no pollen grains in the anthers.【Conclusion】This study shows that the mutant COXⅡ of sua-CMS line affects COX function, which is a main reason of anther energy deficiency, and it is also an important factor affecting the development of anthers.

Key words: tobacco, cytoplasmic male sterility, COXⅡ, functional identification

望锦, 刘艳芳, 刘文文, 王雅琦, 宋时洋, 张幸子, 李凤霞. sua-CMS烟草不育系COXⅡ的功能分析[J]. 中国农业科学, 2023, 56(16): 3077-3087.

WANG Jin, LIU YanFang, LIU WenWen, WANG YaQi, SONG ShiYang, ZHANG XingZi, LI FengXia. COXⅡ Functional Analysis in Tobacco sua-CMS Line[J]. Scientia Agricultura Sinica, 2023, 56(16): 3077-3087.

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材料分组 Material set	材料名称 Material name	类型 Type	育性 Fertility
Ⅰ	MS中烟100 MS ZY100	sua-CMS不育系sua-CMS line	不育Sterile
Ⅰ	中烟100 ZY100	保持系Maintainer line	可育Fertile
Ⅱ	MS云烟87 MS YY87	sua-CMS不育系sua-CMS line	不育Sterile
Ⅱ	云烟87 YY87	保持系Maintainer line	可育Fertile
Ⅲ	MS中烟300 MS ZY300	sua-CMS不育系sua-CMS line	不育Sterile
Ⅲ	中烟300 ZY300	保持系Maintainer line	可育Fertile
Ⅳ	中烟202 ZY202	sua-CMS杂交种sua-CMS hybrid	不育Sterile
Ⅳ	中烟98 ZY98	可育亲本Fertile parent	可育Fertile

引物 Primer	正向引物 F-Primer (5′-3′)	反向引物 R-Primer (5′-3′)
N-COXⅡ	ATGATTGTTCTAGAATGGCTATTC	TTAAGCTTCCCCGGTTTGTG
N-Actin	CTATTCTCCGCTTTGGACTTGGCA	AGGACCTCAGGACAACGGAAACG
Actin	CAAGGAAATCACCGCTTTGG	AAGGGATGCGAGGATGGA
qCOXⅡ	TGTGATGCTGTACCTGGTCG	TAAGCTTCCCCGGTTTGTGG
AP3	TATGACCATGATTACGAATTCCAGTAACTGTGGCCAACTTAGTTTTG	GACGTAGTGAAAGCATATTCTTCTCTCTTTG
MTS	CAAAGAGAGAAGAATATGCTTTCACTACGT	CCGAATACTCATAAGACCCTCTTTAGCACC
COXⅡ	GGTGCTAAAGAGGGTCTTATGAGTATTCGG	ATTCTGGTGTGTGCGCAATG
P		ATTCTGGTGTGTGCGCAATG