李坏死环斑病毒诱导的桃PpPDS沉默体系的优化及验证

doi:10.16420/j.issn.0513-353x.2022-0484

摘要/Abstract

摘要：

病毒诱导的基因沉默（virus-induced gene silencing，VIGS）是在桃（Prunus persica）上进行基因沉默的主要手段，现有VIGS体系效率较低，亟待开发针对桃的高效沉默体系。以桃八氢番茄红素脱氢酶（Phytoene desaturase）基因PpPDS为指示基因，探究不同侵染方式、病毒载体、温度、接种苗龄和菌液浓度对桃叶片VIGS效率的影响。结果表明，在侵染方式上，叶片注射是有效的方式；李坏死环斑病毒（Prunus necrotic ring spot virus，PNRSV）载体（pCaRNA1/2和pCaRNA3）优于烟草脆裂病毒（Tobacco rattle virus，TRV）改造后的TRV载体（pTRV1和pTRV2）；温度为22 ℃左右PpPDS的沉默效率较高；苗龄为5 ~ 6片完全展开叶的桃苗沉默效率较高，达到85%；菌液浓度为OD₆₀₀= 1.0时沉默效率较高。以优化好的VIGS体系参数，选择桃叶绿素合成基因（PpGluTR和PpPOR）为目标基因进行验证，结果显示PpGluTR和PpPOR的表达均被显著抑制，叶片颜色变化明显。这些结果表明优化的VIGS体系稳定性好且沉默效率高，可用于桃基因功能的研究。

关键词: 桃, 病毒诱导的基因沉默, 李坏死环斑病毒, 八氢番茄红素脱氢酶, 叶绿素合成基因

Abstract:

At present，VIGS still works as a main method for gene silencing studies in peach. However，the current VIGS efficiency is low that is urgent to develop a more efficient VIGS system in peach. Using PpPDS（Phytoene desaturase gene）as the indicator gene，the effects of infection mode，viral vector，temperature，age of inoculated seedling and bacterial concentration on the VIGS efficiency in peach leaves，were comprehensively investigated. The results showed that leaf agroinfiltration was an effective way of infection；Prunus necrosis ring spot virus（PNRSV）vectors（pCaRNA1/2 and pCaRNA3）were better than the modified TRV vectors（pTRV1 and pTRV2）of tobacco rattle virus；the infection temperature was better at 22 ℃；peach seedlings with 5-6 fully expanded leaves were higher，reaching 85% of gene silencing efficiency；the bacterial concentration at OD₆₀₀= 1.0 was better. Based on the optimized parameters of VIGS system，two peach chlorophyll biosynthetic genes（PpGluTR and PpPOR）were selected as target to verify the optimized VIGS system. The results showed that the expressions of PpGluTR and PpPOR were significantly repressed，and the change of leaf color was evident. The studies suggest that the optimized VIGS system has good stability and high silencing efficiency，which can be used for gene silencing studies in peach.

Key words: Prunus persica, virus-induced gene silencing（VIGS）, Prunus necrotic ring spot virus （PNRSV）, phytoene desaturase, chlorophyll biosynthetic gene

潘佳佳, 张东梅, 孟建, 高苏南, 朱凯杰, 刘军伟, 李国怀. 李坏死环斑病毒诱导的桃PpPDS沉默体系的优化及验证[J]. 园艺学报, 2023, 50(7): 1587-1600.

PAN Jiajia, ZHANG Dongmei, MENG Jian, GAO Sunan, ZHU Kaijie, LIU Junwei, LI Guohuai. Optimization and Validation of PpPDS Gene Silencing Induced by Prunus Necrotic Ring Spot Virus in Prunus persica[J]. Acta Horticulturae Sinica, 2023, 50(7): 1587-1600.

图/表 13

表1 本研究中所用到的引物序列

Table 1 Primer sequences used in this study

引物	目的	序列（5′-3′）	条带大小/bp
Primer	Purpose	Sequence	Product size
1	PpPDS CDS	F：ATGTCTCAGTGGGCTTGTGTCTCTG；R：TCACCGAACGCTTGCCTCAGCCACC	1 722
2	PpPDS	F：GCTCTAGAAGAAAGCTGAAGAACACAT；R：GCTCTAGAGATTGTAATATTCCTTACAT	100
3	pCaRNA3-PpPDS	F：GCTTCCCTAACGGGGCATCC；R：AGGTCTTGGTTAGGGATTTG	375
4	PpGluTR	F：AAGACCTGCTTGAGATCTAGAGAGAATGTTGGTGATTGGTGCTG； R：GTGTGCTTATCTCACTCTAGATGTTGATATTTCTGTCCCTAGGAATG	320
5	PpPOR	F：AAGACCTGCTTGAGATCTAGATTGATGTGCTTGTGTGCAATGC； R：GTGTGCTTATCTCACTCTAGACAAGGTGTTTGTGTTCCCTGTAAT	209
6	pCaRNA3	F：GCTTCCCTAACGGGGCATCC；R：AGGTCTTGGTTAGGGATTTG	269
7	pTRV1	F：TTACAGGTTATTTGGGCTAG；R：CCGGGTTCAATTCCTTATC	647
8	pTRV2	F：TACGACGAACCAAGGG；R：TGCGAAACTCAAATGCT	427
9	PpPDS qRT-PCR	F：CCTTGCAAAGATCTCCCCTA；R：CGAGCAACAAGCAATTCGTA	145
10	PpGluTR qRT-PCR	F：GGAAGATGGGGAAGCTTGTGA；R：GGTTTCTGATGCCGTGCTTG	200
11	PpPOR qRT-PCR	F：TTGTGTGCAATGCTGCTGTT；R：CGAAAGAAGGAAGTGCCCGA	110
12	PpTEF2 qRT-PCR	F：AGCAAGTCACCCAACAAGCATA；R：CCAACCAAACTCTTCAGCCAAT	147

图1 桃苗取样的模型图 ①表型上叶：表型叶以上没有出现表型的叶片；②表型叶：出现表型的叶片；③表型下叶：表型叶以下没有出现表型的叶片；④注射叶：注射过的叶片；⑤茎：剪掉叶片后剩下的地上部分；⑥根：洗去培养土后的地下部分。

Fig. 1 Schematic illustration of peach seedlings sampling ①Phenotypic upper leaf：The leaves above the phenotypic leaves without phenotypic appearance；②Phenotypic leaf：The leaves with phenotypic appearance；③Phenotypic lower leaf：The leaves below the phenotypic leaves without phenotypic appearance；④Injected leaf：The agroinfiltrated leaves；⑤Stem：the above-ground part left after cutting off the leaves；⑥Root：underground parts after washing culture soil.

图2 PpPDS片段的PCR扩增（A）和pCaRNA3-PpPDS重组载体的电泳检测（B）

Fig. 2 PCR amplification of peach PpPDS gene（A）and positive test of recombinant plasmid pCaRNA3-PpPDS（B）

图3 PCR检测pCaRNA3病毒和pTRV病毒侵入桃苗叶片 M：Marker；1：空白对照（未注射）；2：阴性对照（空载体）；3：PDS沉默。

Fig. 3 PCR analysis of pCaRNA3 virus and pTRV virus infection into peach seedlings M：Marker；1：Blank control（non-infected）；2：Negative control（empty vector）；3：PDS silencing.

图4 桃苗叶片注射pCaRNA3-PpPDS和pTRV2-PpPDS处理后的沉默表型

Fig. 4 Silencing phenotypes of peach seedlings agroinfiltrated with pCaRNA3-PpPDS and pTRV2-PpPDS

图5 不同温度（A）和苗龄大小（B）对桃苗VIGS效率的影响 ‘nd’代表没有检测到数据；不同小写字母代表不同处理间存在显著性差异（P < 0.05）。

Fig. 5 Effect of different temperature（A）and seedlings age（B）on VIGS efficiency in peach ‘nd’ stands for not detected；Different lowercase letters on top of bars indicate statistical significance at P < 0.05 among different treatments.

图6 pCaRNA3-PpPDS和pTRV2-PpPDS侵染桃苗叶片后不同部位叶片的沉默效果由于pCaRNA3-PpPDS表型叶以上均有表型显现，因此无表型上叶。

Fig. 6 Silencing effect of different peach leaves agroinfiltrated with pCaRNA3-PpPDS and pTRV2-PpPDS The upper leaves of phenotypic leaves of pCaRNA3-PpPDS were all phenotypic leaves, so here no phenotypic upper leaf.

图7 注射pCaRNA3-PpPDS（A）和pTRV2-PpPDS（B）及其空载体的桃苗不同部位PpPDS的相对表达量星号表示处理植株的叶、茎、根与注射空载体对照植株对应组织之间存在显著性差异（*α = 0.05和**α = 0.01）。

Fig. 7 Transcriptional abundance of PpPDS in different parts of peach seedlings agroinfiltrated with pCaRNA3-PpPDS（A），pTRV2-PpPDS（B）and their empty vectors Asterisks indicate statistical significance between treatment and negative control（* α = 0.05 and ** α = 0.01）.

图8 pCaRNA3-PpPDS不同菌液浓度对桃苗基因的沉默效率不同小写字母代表不同处理间存在显著差异（P < 0.05）。下同。

Fig. 8 Silencing efficiency of pCaRNA3-PpPDS at different bacterial concentrations in peach seedlings Different lowercase letters on top of bars indicate statistical significance at P < 0.05 among different treatments. The same below.

图9 桃苗叶片注射pCaRNA3-PpGluTR和pCaRNA3-PpPOR后28 d的表型

Fig. 9 Leaf morphology of peach seedlings agroinfiltrated with pCaRNA3-PpGluTR and pCaRNA3-PpPOR after 28 days

图10 桃苗叶片注射pCaRNA3-PpGluTR和pCaRNA3-PpPOR后的荧光光合参数符号-1、-2和-3：3个生物学重复。

Fig. 10 Chlorophyll fluorescence of peach leaves agroinfiltrated with pCaRNA3-PpGluTR and pCaRNA3-PpPOR Symbol-1，-2 and -3 indicate three biological replicates.

图11 桃苗叶片注射pCaRNA3-PpGluTR和pCaRNA3-PpPOR后的叶绿素含量

Fig. 11 Chlorophyll content of peach leaves agroinfiltrated with pCaRNA3-PpGluTR and pCaRNA3-PpPOR

图12 PNRSV介导的VIGS体系中桃苗叶绿素合成基因PpGluTR和PpPOR的表达水平

Fig. 12 Transcript levels of PpGluTR and PpPOR in PNRSV-mediated VIGS system in peach seedlings

参考文献 34

[1]	Ali S, Ahmad Nasir I, Rafiq M, Javed Butt S, Ihsan F, Qayyum Rao A, Husnain T. 2017. Sugarcane mosaic virus-based gene silencing in Nicotiana benthamiana. Iranian Journal of Biotechnology, 15 (4):260-267. doi: 10.15171/ijb.1536 URL
[2]	Bai S, Tuan P A, Tatsuki M, Yaegaki H, Ohmiya A, Yamamizo C, Moriguchi T. 2016. Knockdown of carotenoid cleavage dioxygenase 4 (ccd4) via virus-induced gene silencing confers yellow coloration in peach fruit:evaluation of gene function related to fruit traits. Plant Molecular Biology Reporter, 34:257-264. doi: 10.1007/s11105-015-0920-8 URL
[3]	Bernal-Vicente A, Cantabella D, Hernandez J A, Diaz-Vivancos P. 2018. The effect of mandelonitrile,a recently described salicylic acid precursor,on peach plant response against abiotic and biotic stresses. Plant Biology, 20:986-994. doi: 10.1111/plb.12894 pmid: 30098127
[4]	Cai X Z, Xu Q F, Wang C C, Zhong Z. 2006. Development of a virus-induced gene-silencing system for functional analysis of the RPS2-dependent resistance signalling pathways in Arabidopsis. Plant Molecular Biology, 62 (1-2):223-232. doi: 10.1007/s11103-006-9016-z URL
[5]	Clavel D, Diouf O, Khalfaoui J L, Braconnier S. 2005. Genotypes variations in fluorescence parameters among closely related groundnut(Arachis hypogaea L.) lines and their potential for drought screening programs. Field Crops Research, 96:296-306. doi: 10.1016/j.fcr.2005.07.012 URL
[6]	Cui H, Wang A. 2017. An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene. Plant Biotechnology Journal, 15 (3):344-356. doi: 10.1111/pbi.2017.15.issue-3 URL
[7]	Dai Wenshan, Wu Yue, Wang Min. 2022. Preliminary study on mechanisms of resistance to citrus canker of kumquat FcRGA1. Acta Horticulturae Sinica, 49 (11):2325-2335. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0940
	戴文珊, 吴玥, 王敏. 2022. 金柑FcRGA1抗溃疡病机制初探. 园艺学报, 49 (11):2325-2335. doi: 10.16420/j.issn.0513-353x.2021-0940
[8]	Deng C Y, Zhang F, Wang J Y, Li Y F, Huang H, Dai S L. 2021. Tobacco rattle virus-induced phytoene desaturase (PDS) silencing in Centaurea cyanus. Horticultural Plant Journal, 7 (2):159-166. doi: 10.1016/j.hpj.2020.08.002 URL
[9]	Dommes A B, Gross T, Herbert D B, Kivivirta K I, Becker A. 2019. Virus-induced gene silencing:empowering genetics in non-model organisms. Journal of Experimental Botany, 70 (3):757-770. doi: 10.1093/jxb/ery411 pmid: 30452695
[10]	Guo Yan, Liu Zhi-da, Kang Li-ru, Bao Ting-ting, Yang Xu, Zhao Jun, Zhang Zhi-wei. 2023. Optimization of efficient silencing system of tomato VIGS based on PDS gene. Crops,(2):46-50. (in Chinese)
	郭燕, 刘志达, 康立茹, 包婷婷, 杨旭, 赵君, 张之为. 2023. 基于PDS基因的番茄VIGS高效沉默体系的优化. 作物杂志,(2):46-50.
[11]	Hao Meng-yuan, Hang Qi, Shi Gong-yao. 2022. Application and prospect of virus-induced gene silencing in crop gene function research. Journal of Agricultural Science and Technology, 24 (1):1-13. (in Chinese) doi: 10.13304/j.nykjdb.2020.0781
	郝梦媛, 杭琦, 师恭曜. 2022. VIGS基因沉默技术在作物基因功能研究中的应用与展望. 中国农业科技导报, 24 (1):1-13. doi: 10.13304/j.nykjdb.2020.0781
[12]	Jang S, Choi S C, Li H Y, An G, Schmelzer E. 2015. Functional characterization of phalaenopsis aphrodite flowering genes PaFT1 and PaFD. PLoS ONE, 10 (8):e0134987.
[13]	Jia Hai-feng. 2010. The construction of VIGS system in peach leaf and strawberry fruit[M. D. Dissertation]. Beijing: Beijing University of Agriculture. (in Chinese)
	贾海锋. 2010. 桃叶片及草莓果实中VIGS体系的建立[硕士论文]. 北京: 北京农学院.
[14]	Li Ran-ran, Wang Shu-bin, Pan Bao-gui, Diao Wei-ping, Guo Guang-jun, Liu Jin-bing, Ge Wei. 2017. Optimization of gene silencing inoculation method induced by pepper virus. Jiangsu Agricultural Sciences, 45 (14):82-84. (in Chinese)
	李然然, 王述彬, 潘宝贵, 刁卫平, 郭广君, 刘金兵, 戈伟. 2017. 辣椒病毒诱导基因沉默接种方法的优化. 江苏农业科学, 45 (14):82-84.
[15]	Li Rui-xue. 2018. Screening,cloning and functional identification of POR gene in the Albino of Ananas comosus var. Bracteatus leaves using VIGS technique[M. D. Dissertation]. Ya’an: Sichuan Agricultural University. (in Chinese)
	李瑞雪. 2018. 红苞凤梨VIGS基因沉默体系的建立及POR基因的克隆与功能验证[硕士论文]. 雅安: 四川农业大学.
[16]	Liu Jun, Yang Yan, Zhou Xiao-hui, Jiang Xuan-yun, Bao Sheng-you, Zhuang Yong. 2018. Establishment and optimization of virus-induced gene silencing in eggplants. Jiangsu Journal of Agricultural Sciences, 34 (4):880-886. (in Chinese)
	刘军, 杨艳, 周晓慧, 姜悬云, 鲍生有, 庄勇. 2018. 应用于茄子的病毒诱导的基因沉默体系的建立与优化. 江苏农业学报, 34 (4):880-886.
[17]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods, 25 (4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[18]	Ma Meng. 2010. Development of an effective system of virus induced gene silencing by barley stripe mosaic virus(BSMV)in wheat spikes[M. D. Dissertation]. Yangling: Northwest A & F University. (in Chinese)
	马猛. 2010. 大麦条斑花叶病毒(BSMV)诱导小麦穗部基因有效沉默体系的建立[硕士论文]. 杨凌: 西北农林科技大学.
[19]	Qi Dongxia, Zhang Ying, Zhao zhen, Zhang Weiwei, Chen Yuhui, Lian Yong, Tian Shibing, Liu Fuzhong. 2018. Optimization of virus-induced gene silencing system and function identification of SmIAA19 gene in eggplant. Acta Horticulturae Sinica, 45 (4):691-701. (in Chinese)
	齐东霞, 张映, 赵祯, 张伟伟, 陈钰辉, 连勇, 田时炳, 刘富中. 2018. 茄子TRV-VIGS体系的优化及SmIAA19基因功能初步分析. 园艺学报, 45 (4):691-701. doi: 10.16420/j.issn.0513-353x.2017-0643
[20]	Qi Xiliang, Liu Congli, Song Lulu, Li Ming. 2021. Functional analysis of sucrose-phosphate synthase genes(SPS) in sweet cherry. Acta Horticulturae Sinica, 48 (8):1446-1456. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0630
	齐希梁, 刘聪利, 宋露露, 李明. 2021. 甜樱桃磷酸蔗糖合酶基因PavSPS的功能分析. 园艺学报, 48 (8):1446-1456. doi: 10.16420/j.issn.0513-353x.2020-0630
[21]	Sajjad M. 2021. The mechanism of GhWOX4 regulating drought tolerance in arabidopsis and cotton[Ph. D. Dissertation]. Beijing: Chinese Academy of Agricultural Sciences. (in Chinese)
	Sajjad M. 2021. GhWOX4调节拟南芥和棉花抗旱机制的研究[博士论文]. 北京: 中国农业科学院.
[22]	Shafique Khan F, Zeng R F, Gan Z M, Zhang J Z, Hu C G. 2021. Genome-wide identification and expression profiling of the WOX gene family in Citrus sinensis and functional analysis of a CsWUS member. International Journal of Molecular Sciences, 22 (9):4919. doi: 10.3390/ijms22094919 URL
[23]	Shen J S, Si W J, Wu Y T, Xu Y, Wang J, Cheng T R, Zhang Q X, Pan H T. 2021. Establishment and verification of an efficient virus-induced gene silencing system in Forsythia. Horticultural Plant Journal, 7 (1):81-88. doi: 10.1016/j.hpj.2020.09.001 URL
[24]	Shen Z D, Sun J, Yao J, Wang S J, Ding M Q, Zhang H L, Qian Z Y, Zhao N, Sa G, Zhao R, Shen X, Andrea Polle, Chen S L. 2015. High rates of virus-induced gene silencing by tobacco rattle virus in Populus. Tree Physiology, 35 (9):1016-1029. doi: 10.1093/treephys/tpv064 URL
[25]	Song Zhen, Li Zhong-an, Zhou Chang-yong. 2014. Research advances of virus-induced gene silencing(VIGS). Acta Horticulturae Sinica, 41 (9):1885-1894. (in Chinese)
	宋震, 李中安, 周常勇. 2014. 病毒诱导的基因沉默(VIGS)研究进展. 园艺学报, 41 (9):1885-1894.
[26]	Tuttle J R, Idris A M, Brown J K, Haigler C H, Robertson D. 2008. Geminivirus-mediated gene silencing from cotton leaf crumple virus is enhanced by low temperature in cotton. Plant Physiology, 148 (1):41-50. doi: 10.1104/pp.108.123869 pmid: 18621976
[27]	Verde I, Abbott A G, Scalabrin S, Jung S, Shu S Q, Marroni F, Zhebentyayeva T, Dettori M T, Grimwood J, Cattonaro F, Zuccolo A, Rossini L, Jenkins J, Vendramin E, Meisel L A, Decroocq V, Sosinski B, Prochnik S, Mitros T, Policriti A, Cipriani G, Dondini L, Ficklin S, Goodstein D M, Xuan P F, Fabbro C D, Aramini V, Copetti D, Gonzalez S, Horner D S, Falchi R, Lucas S, Mica E, Maldonado J, Lazzari B, Bielenberg D, Pirona R, Miculan M, Barakat A, Testolin R, Stella A, Tartarini S, Tonutti P, Arús P, Orellana A, Wells C, Main D, Vizzotto G, Silva H, Salamini F, Schmutz J, Morgante M, Rokhsar D S. 2013. The high-quality draft genome of peach(Prunus persica)identifies unique patterns of genetic diversity,domestication and genome evolution. Nature Genetics, 45 (5):487-494. doi: 10.1038/ng.2586
[28]	Wu Jie, Liu Shuai, Ma Jing-jing, Yang Zhao-guang, Qiao Yan-yan, Zhao Pei, Wu Zhen-ping, Cao Yu-lin, Zhang Chao-jun. 2021. Silencing efficiency of different VIGS systems in cotton. Southwest China Journal of Agricultural Sciences, 34 (5):950-955. (in Chinese)
	吴洁, 刘帅, 马晶晶, 杨兆光, 乔艳艳, 赵沛, 吴珍平, 操宇琳, 张朝军. 2021. 不同VIGS体系在棉花中沉默效率的研究. 西南农业学报, 34 (5):950-955.
[29]	Yan H X, Fu D Q, Zhu B Z, Liu H P, Shen X Y. 2012. Sprout vacuum-infiltration: a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species. Plant Cell Reports, 31:1713-1722. doi: 10.1007/s00299-012-1285-1 URL
[30]	Yaronskaya E, Vershilovskaya I, Poers Y, Alawady A E, Averina N, Grimm B. 2006. Cytokinin effects on tetrapyrrole biosynthesis and photosynthetic activity in barley seedlings. Planta, 224 (3):700-709. doi: 10.1007/s00425-006-0249-5 pmid: 16506064
[31]	Zhang H, Zhang D M, Wang F, Hsiang T, Liu J W, Li G H. 2020. Lasiodiplodia theobromae-induced alteration in ROS metabolism and its relation to gummosis development in Prunus persica. Plant Physiology and Biochemistry, 154:43-53. doi: S0981-9428(20)30242-4 pmid: 32526610
[32]	Zhang Xin-hua, Ji Na-na, Min De-dong, Shao Shu-jun, Li Fu-jun. 2017. Progress of research on application of VIGS vectors in fruit trees. Journal of Fruit Science, 34 (4):507-514. (in Chinese)
	张新华, 季娜娜, 闵德栋, 邵淑君, 李富军. 2017. VIGS载体在果树中的应用研究进展. 果树学报, 34 (4):507-514.
[33]	Zhang Z, Li D W, Jin J H, Yin Y X, Zhang H X, Chai W G, Gong Z H. 2015. VIGS approach reveals the modulation of anthocyanin biosynthetic genes by CaMYB in chili pepper leaves. Frontiers in Plant Science, 6:500. doi: 10.3389/fpls.2015.00500 pmid: 26217354
[34]	Zhou P, Peng J Y, Zeng M J, Wu L X, Fan Y X, Zeng L H. 2021. Virus-induced gene silencing (VIGS) in Chinese narcissus and its use in functional analysis of NtMYB3. Horticultural Plant Journal, 7 (6):565-572. doi: 10.1016/j.hpj.2021.04.009 URL