马铃薯线粒体靶向表达载体的构建与应用

doi:10.13560/j.cnki.biotech.bull.1985.2023-1178

摘要/Abstract

摘要：

【目的】植物线粒体基因变异是产生细胞质雄性不育（cytoplasmic male sterility，CMS）的主要原因，利用生物技术是创造CMS不育系的重要手段。通过比较启动子、线粒体转运信号肽（mitochondrial targeting signal peptides，MTS）的不同组合对下游eGFP的表达强弱，来探究最佳马铃薯线粒体靶向表达载体。【方法】选择3个启动子AtUBI10、StUBI10、2×35S和两个线粒体转运信号肽ATPγ与Rf1b进行试验，利用烟草叶片瞬时表达系统和PEG介导马铃薯原生质体转化体系进行靶向载体的表达验证。【结果】六个线粒体靶向表达载体中AtUBI10::ATPγ-eGFP在烟草瞬时表达效果最佳，可准确定位到线粒体，其次为StUBI10::ATPγ-eGFP和2×35S::ATPγ-eGFP；在马铃薯原生质体中载体AtUBI10::ATPγ-eGFP有最佳的表达效果，与烟草中表达结果一致，其次是StUBI10::Rf1b-eGFP和AtUBI10::Rf1b-eGFP，而2×35S启动子的两个载体在原生质体均表达较弱。【结论】启动子AtUBI10与线粒体转运信号肽ATPγ所构建的靶向线粒体载体为最佳组合，可用于马铃薯线粒体基因的相关研究中。

关键词: 马铃薯, 线粒体转运信号肽, 靶向表达载体, 亚细胞定位, 细胞质雄性不育

Abstract:

【Objective】The genetic variations of mitochondria genome usually lead to cytoplasmic male sterility（CMS）in plants. Biotechnology is an important measure to produce variations resulting in CMS lines. In order to establish an efficient expression vector system for the research on potato mitochondrial genes, the optimal potato mitochondrial targeting expression vector was explored by comparing the expression level of eGFP, which was promoted by different combinations of promoters and mitochondrial targeting signal peptides（MTS）. 【Method】Three promoters AtUBI10, StUBI10, 2×35S and two mitochondrial transport signal peptides ATPγ and Rf1b were tested. The expression level of the target vector was verified by tobacco leaf transient expression system and PEG-mediated potato protoplast transformation system. 【Result】AtUBI10 :: ATPγ-eGFP among 6 expressing vectors had the best expression effect in tobacco and was accurately located in mitochondria. The second best ones were StUBI10 :: ATPγ-eGFP and 2×35S :: ATPγ-eGFP. The vector AtUBI10 :: ATPγ-eGFP also showed the best performance, followed by StUBI10 :: Rf1b-eGFP and AtUBI10 :: Rf1b-eGFP in the potato protoplast transformation system. The two vectors with 2×35S promoter were weakly expressed in protoplasts. 【Conclusion】The comparative studies showed the mitochondrial vector constructed by the promoter AtUBI10 and the mitochondrial transport signal peptide ATPγ is the best combination, and this vectors will be of great benefit to the relative study on mitochondrial genes.

Key words: potato, mitochondrial transport signal peptides, targeted expression vector, subcellular localization, cytoplasmic male sterility

张震, 李清, 徐菁, 陈凯园, 张春芝, 祝光涛. 马铃薯线粒体靶向表达载体的构建与应用[J]. 生物技术通报, 2024, 40(5): 66-73.

ZHANG Zhen, LI Qing, XU Jing, CHEN Kai-yuan, ZHANG Chun-zhi, ZHU Guang-tao. Construction and Application of Potato Mitochondrial Targeted Expression Vector[J]. Biotechnology Bulletin, 2024, 40(5): 66-73.

图/表 6

图1 6个线粒体过表达载体的构建示意图

Fig. 1 Construction diagram of 6 mitochondrial overexpressing vectors

表1 本研究所用PCR引物序列

Table 1 PCR primer sequences used in this study

扩增片段Amplified fragment		引物序列Primer sequence（5'-3'）
2×35S启动子组合	2×35S	F	TATGACCATGATTACGAATTCGCTAGAGCAGCTTGCCAACATG
	2×35S	R	CCATTGCCATGGATCCAAGCTTGAAGAGAGAGAC
	2×35S	R*	GGCGTGCCATGGTCGATCGACAGATCTGCG
	ATPγ	F	TCGATCGACCATGGCAATGGCTGTTTTCCG
	ATPγ	R	TGCTCACCATGCCTTGAACTGCTCTAAGCTTGG
	Rf1b	F	TCGATCGACCATGGCACGCCGCGTCGCTG
	Rf1b	R	TGCTCACCATGCGGTTGAAGCGGGACAC
	eGFP	F	AGTTCAAGGCATGGTGAGCAAGGGCGAGG
	eGFP	F*	CTTCAACCGCATGGTGAGCAAGGGCGAGG
	eGFP	R	ACGACGGCCAGTGCCAAGCTTGATCTAGTAACATAGATGACACCGC
StUBI10启动子组合	StUBI10	F	TATGACCATGATTACGAATTCCCAAGACAATTTCAGCTTAAAAAGT
	StUBI10	R	TGCTCACCATGCCTTGAACTGCTCTAAGCTTGG
	StUBI10	R*	CGCGGCGTGCCATGGATCCAAGCTTGAATCTGCAAATTC
	ATPγ	F	GCTTGGATCCATGGCAATGGCTGTTTTCCG
	ATPγ	R	TGCTCACCATGCCTTGAACTGCTCTAAGCTTGG
	Rf1b	F	GCTTGGATCCATGGCACGCCGCGTCGCTG
	Rf1b	R	TGCTCACCATGCGGTTGAAGCGGGACAC
	eGFP	F	AGTTCAAGGCATGGTGAGCAAGGGCGAGG
	eGFP	F*	CTTCAACCGCATGGTGAGCAAGGGCGAGG
	eGFP	R	ACGACGGCCAGTGCCAAGCTTGATCTAGTAACATAGATGACACCGC
AtUBI10启动子组合	AtUBI10	F	TATGACCATGATTACGAATTCATACGCTTCAATGCAGTGG
	AtUBI10	R	CCATTGCCATTGATAACTCTAGAATCTGTTAATCA
	AtUBI10	R*	GGCGTGCCATTGATAACTCTAGAATCTGTTAATCA
	ATPγ	F	AGAGTTATCAATGGCAATGGCTGTTTTCCG
	ATPγ	R	TGCTCACCATGCCTTGAACTGCTCTAAGCTTGG
	Rf1b	F	AGAGTTATCAATGGCACGCCGCGTCGCTG
	Rf1b	R	TGCTCACCATGCGGTTGAAGCGGGACAC
	eGFP	F	AGTTCAAGGCATGGTGAGCAAGGGCGAGG
	eGFP	F*	CTTCAACCGCATGGTGAGCAAGGGCGAGG
	eGFP	R	ACGACGGCCAGTGCCAAGCTTGATCTAGTAACATAGATGACACCGC
	P2300	F	GTTAGCTCACTCATTAGGCAC
	P2300	R	GCTGGCGTAATAGCGAAGAG

图2 烟草瞬时侵染eGFP与线粒体共定位图示 A1为载体AtUBI10::ATPγ-eGFP，A2为载体AtUBI10::Rf1b-eGFP；B1为载体StUBI10::ATPγ-eGFP，B2为载体StUBI10::Rf1b-eGFP；C1为载体2×35S::ATPγ-eGFP，C2为载体2×35S::Rf1b-eGFP；mCherry为线粒体定位Marker。下同

Fig. 2 Colocalization of eGFP and mitochondria in transient tobacco infection A1 is the vector AtUBI10::ATPγ-eGFP, A2 is the vector AtUBI10::Rf1b-eGFP; B1 is the vector StUBI10::ATPγ-eGFP, B2 is the vector StUBI10::Rf1b-eGFP; C1 is the carrier 2×35S::ATPγ-eGFP, C2 is the carrier 2×35S::Rf1b-eGFP. Mitochondrial localization marker by mCherry. The same below

图3 烟草瞬时侵染与马铃薯原生质体转化荧光强度统计 A为烟草瞬时侵染中各载体eGFP与mCherry的平均荧光数值统计，B为马铃薯原生质体转化中各载体eGFP与mCherry的平均荧光数值统计；荧光数值测量重复3次，误差线代表重复之间的标准差；ns代表t检验无差异，*代表t检验所得显著差异（P<0.05）

Fig. 3 Fluorescence intensity statistics of tobacco transient infestation and potato protoplast transformation A is the average fluorescence value statistics of eGFP and mCherry for each vector in tobacco transient infestation, and B is the average fluorescence value statistics of eGFP and mCherry for each vector in potato protoplast transformation. Data are in mean±SD（n=3）. ns indicates no significant differences according to student's t-test, and *（P<0.05）indicates significant differences according to student's t-test

图4 马铃薯原生质体血细胞计数板观察

Fig. 4 Observation of potato protoplasts using a hemocytometer

图5 马铃薯原生质体转化eGFP与线粒体共定位图示

Fig. 5 Diagram of co-location of eGFP and mitochondria in potato protoplast transformation

参考文献 32

[1]	Stokstad E. The new potato[J]. Science, 2019, 363(6427): 574-577. doi: 10.1126/science.363.6427.574 pmid: 30733400
[2]	屈冬玉, 谢开云, 金黎平, 等. 中国马铃薯产业发展与食物安全[J]. 中国农业科学, 2005, 38(2): 358-362.
	Qu DY, Xie KY, Jin LP, et al. Development of potato industry and food safety in China[J]. Sci Agric Sin, 2005, 38(2): 358-362.
[3]	Fan L, Wu DF, Goremykin V, et al. Phylogenetic analyses with systematic taxon sampling show that mitochondria branch within Alphaproteobacteria[J]. Nat Ecol Evol, 2020, 4(9): 1213-1219. doi: 10.1038/s41559-020-1239-x pmid: 32661403
[4]	Bohra A, Jha UC, Adhimoolam P, et al. Cytoplasmic male sterility(CMS)in hybrid breeding in field crops[J]. Plant Cell Rep, 2016, 35(5): 967-993.
[5]	Chase CD. Cytoplasmic male sterility: a window to the world of plant mitochondrial-nuclear interactions[J]. Trends Genet, 2007, 23(2): 81-90. doi: 10.1016/j.tig.2006.12.004 pmid: 17188396
[6]	Bentolila S, Stefanov S. A reevaluation of rice mitochondrial evolution based on the complete sequence of male-fertile and male-sterile mitochondrial genomes[J]. Plant Physiol, 2012, 158(2): 996-1017. doi: 10.1104/pp.111.190231 pmid: 22128137
[7]	Cheng SH, Zhuang JY, Fan YY, et al. Progress in research and development on hybrid rice: a super-domesticate in China[J]. Ann Bot, 2007, 100(5): 959-966.
[8]	Xie F, Yuan JL, Li YX, et al. Transcriptome analysis reveals candidate genes associated with leaf etiolation of a cytoplasmic male sterility line in Chinese cabbage(Brassica rapa L. ssp. pekinensis)[J]. Int J Mol Sci, 2018, 19(4): 922.
[9]	燕厚兴, 林春晶, 范亚军, 等. 植物细胞质雄性不育基因克隆及分子机制研究进展[J]. 植物生理学报, 2022, 58(1): 61-76.
	Yan HX, Lin CJ, Fan YJ, et al. Research progress on cloning and molecular mechanism of cytoplasmic male sterility genes in plants[J]. Plant Physiol J, 2022, 58(1): 61-76.
[10]	Yang JH, Liu XY, Yang XD, et al. Mitochondrially-targeted expression of a cytoplasmic male sterility-associated orf220 gene causes male sterility in Brassica juncea[J]. BMC Plant Biol, 2010, 10: 231.
[11]	Jiang HC, Lu Q, Qiu SQ, et al. Fujian cytoplasmic male sterility and the fertility restorer gene OsRf19 provide a promising breeding system for hybrid rice[J]. Proc Natl Acad Sci USA, 2022, 119(34): e2208759119.
[12]	Luo DP, Xu H, Liu ZL, et al. A detrimental mitochondrial-nuclear interaction causes cytoplasmic male sterility in rice[J]. Nat Genet, 2013, 45(5): 573-577. doi: 10.1038/ng.2570 pmid: 23502780
[13]	He S, Abad AR, Gelvin SB, et al. A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco[J]. Proc Natl Acad Sci USA, 1996, 93(21): 11763-11768. doi: 10.1073/pnas.93.21.11763 pmid: 8876211
[14]	Wang ZH, Zou YJ, Li XY, et al. Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing[J]. Plant Cell, 2006, 18(3): 676-687. doi: 10.1105/tpc.105.038240 pmid: 16489123
[15]	Yoo SD, Cho YH, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[J]. Nat Protoc, 2007, 2(7): 1565-1572.
[16]	叶明旺. 二倍体马铃薯遗传转化体系的建立及S-RNase基因编辑的研究[D]. 昆明: 云南师范大学, 2018.
	Ye MW. Establishment of transformation system for diploid potato and the study of transgenic with S-RNase gene editing[D]. Kunming: Yunnan Normal University, 2018.
[17]	Zhang CZ, Yang ZM, Tang D, et al. Genome design of hybrid potato[J]. Cell, 2021, 184(15): 3873-3883.e12. doi: 10.1016/j.cell.2021.06.006 pmid: 34171306
[18]	陈彦儒. 粘果山羊草(Aegilops kotschyi)细胞质雄性不育小麦KTP116A育性恢复基因位点Rfk1的精细定位及分子克隆[D]. 杨凌: 西北农林科技大学, 2021.
	Chen YR. Fine mapping and molecular clone of fertility restorer locus Rfk1 for cytoplasmic male sterile wheat KTP116A with Aegilops kotschyi cytoplasm[D]. Yangling: Northwest A & F University, 2021.
[19]	Xue ZY, Xu X, Zhou Y, et al. Deficiency of a triterpene pathway results in humidity-sensitive genic male sterility in rice[J]. Nat Commun, 2018, 9(1): 604. doi: 10.1038/s41467-018-03048-8 pmid: 29426861
[20]	易冬莲, 陈卫江. 甘蓝型油菜杂种优势利用研究与应用[J]. 湖南农业科学, 2002(S1): 21-22, 26.
	Yi DL, Chen WJ. Study and application of heterosis utilization in Brassica napus L[J]. Hunan Agric Sci, 2002(S1): 21-22, 26.
[21]	Ding XL, Guo QL, Li Q, et al. Comparative transcriptomics analysis and functional study reveal important role of high-temperature stress response gene GmHSFA2 during flower bud development of CMS-based F1 in soybean[J]. Front Plant Sci, 2020, 11: 600217.
[22]	周瑞阳, 张新, 张加强, 等. 红麻细胞质雄性不育系的选育及杂种优势利用取得突破[J]. 中国农业科学, 2008, 41(1): 314.
	Zhou RY, Zhang X, Zhang JQ, et al. A breakthrough in kenaf cytoplasmic male sterile lines breeding and heterosis utilization[J]. Sci Agric Sin, 2008, 41(1): 314.
[23]	段琉颖, 吴婷, 李霞, 等. 水稻细胞质雄性不育及其育性恢复基因的研究进展[J]. 作物杂志, 2022(1): 20-30.
	Duan LY, Wu T, Li X, et al. Progress on cytoplasmic male sterility and fertility restoration genes in rice[J]. Crops, 2022(1): 20-30.
[24]	Yang HL, Xue YD, Li B, et al. The chimeric gene atp6c confers cytoplasmic male sterility in maize by impairing the assembly of the mitochondrial ATP synthase complex[J]. Mol Plant, 2022, 15(5): 872-886.
[25]	Toriyama K. Molecular basis of cytoplasmic male sterility and fertility restoration in rice[J]. Plant Biotechnol, 2021, 38(3): 285-295. doi: 10.5511/plantbiotechnology.21.0607a pmid: 34782814
[26]	Sanetomo R, Akai K, Nashiki A. Discovery of a novel mitochondrial DNA molecule associated with tetrad pollen sterility in potato[J]. BMC Plant Biol, 2022, 22(1): 302. doi: 10.1186/s12870-022-03669-8 pmid: 35725378
[27]	Peng XJ, Wang K, Hu CF, et al. The mitochondrial gene orfH79 plays a critical role in impairing both male gametophyte development and root growth in CMS-Honglian rice[J]. BMC Plant Biol, 2010, 10: 125. doi: 10.1186/1471-2229-10-125 pmid: 20576097
[28]	Kim DH, Kang JG, Kim BD. Isolation and characterization of the cytoplasmic male sterility-associated orf456 gene of chili pepper(Capsicum annuum L.)[J]. Plant Mol Biol, 2007, 63(4): 519-532.
[29]	Ji JJ, Huang W, Li Z, et al. Tapetum-specific expression of a cytoplasmic orf507 gene causes semi-male sterility in transgenic peppers[J]. Front Plant Sci, 2015, 6: 272.
[30]	Xie XR, Zhang ZX, Zhao Z, et al. The mitochondrial aldehyde dehydrogenase OsALDH2b negatively regulates tapetum degeneration in rice[J]. J Exp Bot, 2020, 71(9): 2551-2560. doi: 10.1093/jxb/eraa045 pmid: 31989154
[31]	景兵. 芥菜型油菜胞质不育Hau CMS不育相关基因的鉴定及其功能分析[D]. 武汉: 华中农业大学, 2012.
	Jing B. Identification and function analysis of hau CMS-associated gene in Brassica juncea[D]. Wuhan: Huazhong Agricultural University, 2012.
[32]	张垲, 余冰菲, 陈瑞川, 等. 荧光定量检测细胞绿色荧光蛋白技术的建立与应用[J]. 厦门大学学报: 自然科学版, 2008, 47(S2): 264-267.
	Zhang K, Yu BF, Chen RC, et al. Establishment and application of fluorescence quantitative detection technology for cell green fluorescent protein[J]. J Xiamen Univ Nat Sci, 2008, 47(S2): 264-267.