芦笋病毒诱导基因沉默体系的初步构建

吴贺; 李云宾; 董陈文华; 林春; 刘正杰; 毛自朝

doi:10.12101/j.issn.1004-390X(n).202404023

本文排版定稿已在中国知网首发，如需要《中国知网学术期刊网络首发论文出版证书》，可直接在中国知网本稿件的首发页面下载；如果无法下载，请与编辑部联系。

芦笋病毒诱导基因沉默体系的初步构建

吴贺^1,,
李云宾¹,
董陈文华^{1, 2},
林春^{1, 2},
刘正杰^{1, 2},
毛自朝^{1, 2, ,}

1.
云南农业大学农学与生物技术学院，云南昆明 650201
2.
云南农业大学特色小宗作物研究中心，云南昆明 650201

基金项目: 云南省农业联合专项重点项目(202101BD070001-027)；云南省外专引智项目(202305AO350034)；云南省基础研究计划面上项目(202201AT070254)。

详细信息

作者简介:
吴贺(1999—)，男，广东龙川人，在读硕士研究生，主要从事芦笋基因挖掘和表达验证研究。E-mail：wh19990709@163.com

通信作者:
毛自朝(1969—)，男，云南东川人，博士，教授，主要从事作物基因组学研究。E-mail：maozichao@ynau.edu.cn

中图分类号: S435.672
计量
- 文章访问数: 121
- HTML全文浏览量: 26
- PDF下载量: 1
出版历程
- 收稿日期: 2024-04-15
- 修回日期: 2025-01-19
- 录用日期: 2025-02-05
- 网络出版日期: 2025-02-10

摘要:

目的

在多年生雌雄异株植物芦笋(Asparagus officinalis L.)中建立病毒诱导基因沉默(virus-induced gene silencing，VIGS)体系，以快速探究芦笋中关键基因的功能。

方法

以绿芦笋品种‘Guelph Millennium’为试验材料，选用八氢番茄红素脱氢酶编码基因(AoPDS)和镁螯合酶Ⅰ亚基编码基因(AoCH42)为报告基因，基于苹果潜伏球形病毒(apple latent spherical virus，ALSV)沉默载体，采用真空渗透法通过农杆菌对芦笋露白种子进行侵染，经表型和qRT-PCR检测，构建芦笋的VIGS体系，并进一步验证编码芦笋NADH-脱氢酶铁硫蛋白亚基S8基因(AoNDUFS8)的功能。

结果

侵染21 d后，AoPDS和AoCH42沉默植株分别出现白化和黄化表型；AoNDUFS8沉默植株长势瘦弱，发育迟缓。qRT-PCR结果显示：与对照组相比，植株侵染后，目的基因表达均下降，预示成功构建芦笋VIGS体系。

结论

基于ALSV的VIGS体系能有效沉默芦笋中的目的基因，为后续芦笋功能基因组分析提供了实践基础。

关键词:

Preliminary Construction of a Virus-induced Gene Silencing System in Asparagus officinalis

WU He^1,,
LI Yunbin¹,
DONG Chenwenhua^{1, 2},
LIN Chun^{1, 2},
LIU Zhengjie^{1, 2},
MAO Zichao^{1, 2, ,}

1.
College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, China
2.
Institute of Improvement and Utilization of Characteristic Resource Plants, Yunnan Agricultural University, Kunming 650201, China

Abstract:

Purpose

To establish a virus-induced gene silencing (VIGS) system in the perennial dioecious plant of Asparagus officinalis L., exploring the function of key genes.

Methods

The green A. officinalis variety ‘Guelph Millennium’ was used as material, the phytoene dehydrogenase (AoPDS) and the magnesium chelatase subunit Ⅰ (AoCH42) were selected as report genes, and the apple latent spherical virus (ALSV) was used as silencing vector, the geminating A. officinalis seeds were infected by the resulted Agrobacterium tumefaciens with vacuum infiltration method. The VIGS system of A. officinalis was constructed by phenotype and qRT-PCR, and the function of AoNDUFS8 gene encoding A. officinalis NADH-dehydrogenase ferrithione subunit was further verified.

Results

After 21 days of infection, both the AoPDS and AoCH42 silenced plants had an albino and etiolation phenotypes, while the AoNDUFS8 silenced plants grew thin and stunted. qRT-PCR results showed that: compared with the control group, the expression of the target gene decreased after VIGS, indicating that the silencing system was successfully established.

Conclusion

The VIGS system can effectively silence the target gene(s) in A. officinalis, providing a practical basis for subsequent functional genomic analyses of A. officinalis.

Keywords:

Asparagus officinalis /
virus-induced gene silencing (VIGS) system /
PDS /
CH42 /
gene function

参考文献

[1]	VAN KAMMEN A. Virus-induced gene silencing in infected and transgenic plants[J]. Trends in Plant Science, 1997, 2(11): 409. DOI: 10.1016/S1360-1385(97)01128-X.
[2]	BURCH-SMITH T M, ANDERSON J C, MARTIN G B, et al. Applications and advantages of virus-induced gene silencing for gene function studies in plants[J]. Plant Journal, 2004, 39(5): 734. DOI: 10.1111/j.1365-313X.2004.02158.x.
[3]	KUMAR G, KUMARI K, DASGUPTA I. RTBV-based VIGS vector for functional genomics in rice: methodology, advances, challenges, and future implications[J]. Methods in Molecular Biology, 2022, 2408: 117. DOI: 10.1007/978-1-0716-1875-2_8.
[4]	张鑫, 桂敏, 刘弟, 等. 辣椒CaDXS和CaPDS基因沉默的表型比较[J]. 云南农业大学学报(自然科学), 2024, 39(1): 125. DOI: 10.12101/j.issn.1004-390X(n).202304032.
[5]	张林. 烟草八氢番茄红素合成酶基因家族鉴定与功能分析[D]. 郑州: 河南农业大学, 2024.
[6]	VELÁSQUEZ A C, CHAKRAVARTHY S, MARTIN G B. Virus-induced gene silencing (VIGS) in Nicotiana benthamiana and tomato[J]. Journal of Visualized Experiments, 2009, 10: 28. DOI: 10.3791/1292.
[7]	BURCH-SMITH T M, SCHIFF M, LIU Y L, et al. Efficient virus-induced gene silencing in Arabidopsis[J]. Plant Physiology, 2006, 142(1): 21. DOI: 10.1104/pp.106.084624.
[8]	HOLZBERG S, BROSIO P, GROSS C, et al. Barley stripe mosaic virus-induced gene silencing in a monocot plant[J]. Plant Journal, 2002, 30(3): 315. DOI: 10.1046/j.1365-313x.2002.01291.x.
[9]	ZHANG J, YU D S, ZHANG Y, et al. Vacuum and co-cultivation agroinfiltration of (germinated) seeds results in tobacco rattle virus (TRV) mediated whole-plant virus-induced gene silencing (VIGS) in wheat and maize[J]. Frontiers in Plant Science, 2017, 8: 393. DOI: 10.3389/fpls.2017.00393.
[10]	张萍萍, 桂敏, 李慧芳, 等. 利用病毒诱导的基因沉默技术沉默TSWV的NSm基因[J]. 云南农业大学学报(自然科学), 2021, 36(3): 409. DOI: 10.12101/j.issn.1004-390X(n).202004035.
[11]	黄家莉, 唐红梅, 凡彩凤, 等. 芦笋的营养保健作用及其加工研究进展[J]. 食品安全导刊, 2022(23): 122. DOI: 10.16043/j.cnki.cfs.2022.23.026.
[12]	鹿志伟, 侯晓婉, 高建明, 等. 农杆菌介导的芦笋遗传转化体系的建立[J]. 热带作物学报, 2016, 37(8): 1494. DOI: 10.3969/j.issn.1000-2561.2016.08.009.
[13]	ZHOU J S, HE R Q, LIU X J, et al. Manipulation of plant height in garden asparagus (Asparagus officinalis L.) through CRISPR/Cas9-mediated aspSPL14 allele editing[J]. Horticulture Research, 2023, 10(7): uhad096. DOI: 10.1093/hr/uhad096.
[14]	FOFANA I B, SANGARE A, COLLIER R, et al. A geminivirus-induced gene silencing system for gene function validation in cassava[J]. Plant Molecular Biology, 2004, 56(4): 613. DOI: 10.1007/s11103-004-0161-y.
[15]	CZARNECKI O, GRIMM B. Post-translational control of tetrapyrrole biosynthesis in plants, algae, and cyanobacteria[J]. Journal of Experimental Botany, 2012, 63(4): 1675. DOI: 10.1093/jxb/err437.
[16]	IGARASHI A, YAMAGATA K, SUGAI T, et al. Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes[J]. Virology, 2009, 386(2): 407. DOI: 10.1016/j.virol.2009.01.039.
[17]	GUERRERO-CASTILLO S, BAERTLING F, KOWNATZKI D, et al. The assembly pathway of mitochondrial respiratory chain complex Ⅰ[J]. Cell Metabolism, 2017, 25(1): 128. DOI: 10.1016/j.cmet.2016.09.002.
[18]	CAI X R, ZHANG S, LIN J, et al. Role of the gene ndufs8 located in respiratory complex Ⅰ from Monascus purpureus in the cell growth and secondary metabolites biosynthesis[J]. Journal of Fungi, 2022, 8(7): 655. DOI: 10.3390/jof8070655.
[19]	BILICHAK A, KOVALCHUK I. Increasing a stable transformation efficiency of Arabidopsis by manipulating the endogenous gene expression using virus-induced gene silencing[J]. Methods in Molecular Biology, 2017, 1456: 225. DOI: 10.1007/978-1-4899-7708-3_17.
[20]	王利飞, 邢涪喆, 高潇潇. 玉米ZmPDS基因VIGS转化体系的构建与鉴定[EB/OL]. 分子植物育种: 1-9[2024-04-13]. http://kns.cnki.net/kcms/detail/46.1068.s.20220516.1511.016.html.
[21]	南富波, 李淼淼, 王昊龙, 等. 小麦PDS基因VIGS载体构建体系的优化及验证[J]. 麦类作物学报, 2014, 34(3): 285. DOI: 10.7606/j.issn.1009-1041.2014.03.01.
[22]	石源睿, 董海涛, 常璐, 等. 马铃薯PDS和ZDS基因的克隆与表达特性分析[J]. 山西农业科学, 2023, 51(4): 347. DOI: 10.3969/j.issn.1002-2481.2023.04.01.
[23]	陈段芬, 彭镇华, 高志民. 中国水仙八氢番茄红素脱氢酶基因(PDS)的克隆及表达分析[J]. 分子植物育种, 2008, 6(3): 574. DOI: 10.3969/j.issn.1672-416X.2008.03.027.
[24]	许静. 拟南芥叶色突变体ch42-4及v1的基因定位与功能分析[D]. 上海: 上海师范大学, 2009.
[25]	CHEN S F, ZHOU Y Q, CHEN Y R, et al. Fastp: an ultra-fast all-in-one FASTQ preprocessor[J]. Bioinformatics, 2018, 34(17): i884. DOI: 10.1093/bioinformatics/bty560.
[26]	KIM D, LANGMEAD B, SALZBERG S L. HISAT: a fast spliced aligner with low memory requirements[J]. Nature Methods, 2015, 12(4): 357. DOI: 10.1038/nmeth.3317.
[27]	席海秀, 李洪艳, 佟少明. 植物八氢番茄红素脱氢酶(PDS)基因保守结构域模式与系统进化分析[J]. 植物生理学报, 2013, 49(12): 1407. DOI: 10.13592/j.cnki.ppj.2013.12.025.
[28]	BAERTLING F, RODENBURG R J, SCHAPER J, et al. A guide to diagnosis and treatment of leigh syndrome[J]. Journal of Neurology Neurosurgery and Psychlatry, 2014, 85(3): 257. DOI: 10.1136/jnnp-2012-304426.
[29]	姚丹青, 张微微, 原丽华, 等. VIGS: 植物功能基因组学研究的革命[J]. 分子植物育种, 2009, 7(1): 155. DOI: 10.3969/j.issn.1672-416X.2009.01.026.
[30]	叶艳英, 尹玉玲, 张冰冰, 等. 芦笋主要病毒病的研究进展[J]. 山西农业科学, 2022, 50(7): 1069. DOI: 10.3969/j.issn.1002-2481.2022.07.21.
[31]	CAI X Z, WANG C C, XU Y P, et al. Efficient gene silencing induction in tomato by a viral satellite DNA vector[J]. Virus Research, 2007, 125(2): 169. DOI: 10.1016/j.virusres.2006.12.016.
[32]	GOSSELÉ V, FACHÉ I, MEULEWAETER F, et al. SVISS: a novel transient gene silencing system for gene function discovery and validation in tobacco plants[J]. Plant Journal, 2002, 32(5): 859. DOI: 10.1046/j.1365-313x.2002.01471.x.

图(7) / 表(2)

计量

文章访问数: 121
PDF下载量: 1
被引次数: 0

芦笋病毒诱导基因沉默体系的初步构建

作者简介: 吴贺(1999—)，男，广东龙川人，在读硕士研究生，主要从事芦笋基因挖掘和表达验证研究。E-mail：wh19990709@163.com

通信作者: 毛自朝(1969—)，男，云南东川人，博士，教授，主要从事作物基因组学研究。E-mail：maozichao@ynau.edu.cn

计量

出版历程

Preliminary Construction of a Virus-induced Gene Silencing System in Asparagus officinalis

参考文献

计量

出版历程

目录

作者简介:
吴贺(1999—)，男，广东龙川人，在读硕士研究生，主要从事芦笋基因挖掘和表达验证研究。E-mail：wh19990709@163.com

通信作者:
毛自朝(1969—)，男，云南东川人，博士，教授，主要从事作物基因组学研究。E-mail：maozichao@ynau.edu.cn