多个稻曲病菌效应因子的信号肽验证和表达分析

doi:10.16819/j.1001-7216.2024.231012

摘要/Abstract

摘要：

【目的】信号肽对效应因子的分泌至关重要，影响病原微生物的致病进程。前期鉴定到在稻曲病菌侵染水稻花器官过程中显著上调表达的10个效应因子基因，但这些效应因子是否含有功能性信号肽及其表达模式尚不明确。【方法】利用SignaIP-5.0数据库预测、烟草瞬时表达系统和酵母信号肽分泌实验，分析并鉴定这些效应因子的信号肽。利用实时荧光定量PCR技术，检测效应因子基因的表达模式。【结果】UV_44、UV_1548、UV_1567等10个稻曲病菌效应因子均含有信号肽。烟草瞬时表达结果显示，含有信号肽的eYFP融合蛋白在细胞质壁分离后均能分泌到质外体空间。酵母菌信号肽分泌结果显示，分别携带这10个信号肽的酵母菌株可在以棉子糖为唯一碳源的培养基上生长，且与TTC发生显色反应。实时荧光定量结果表明，除了UV_6754，其余9个效应因子基因的表达模式不尽相同，其中UV_44、UV_1567、UV_3667、UV_4213、UV_4989、UV_5215和UV_8184的表达量在接种后显著升高。【结论】这10个稻曲病菌效应因子均含有分泌功能的信号肽，编码效应因子的基因在稻曲病菌侵染过程中具有不同的表达模式。该结果为深入研究以上稻曲病菌效应因子的功能奠定了基础。

关键词: 水稻, 稻曲病菌, 效应因子, 信号肽

Abstract:

【Objective】Signal peptides are crucial for effector secretion and affect the pathogenic process of pathogenic microorganisms. Previously, ten effector genes were identified to be significantly up-regulated during Ustilaginoidea virens infection in rice spikelets. However, it remains unclear whether these effectors contain functional signal peptides and how they express.【Method】The signal peptides of these effectors were predicted and verified by using SignaIP-5.0 database, Agrobacterium-mediated transient expression in Nicotiana benthamiana, and yeast invertase assay. Besides, the expression patterns of these effectors were analyzed by using RT-qPCR.【Results】All the ten effectors, including UV_44, UV_1548, UV_1567, etc., were predicted to contain a signal peptide. The transient expression method showed that eYFP fusion proteins containing signal peptides were successfully secreted into the extracellular space after plasmolysis in N. benthamiana. Yeast invertase assay showed that the yeast strains harboring signal peptides could grow on the medium with raffinose as the only carbon source with the occurrence of a chromogenic reaction using TTC. All the effector genes had distinct expression patterns, except for UV_6754. Seven of them showed significant up-regulation after inoculation, including UV_44, UV_1567, UV_3667, UV_4213, UV_4989, UV_5215, and UV_8184.【Conclusion】The signal peptides of these effectors had secretory functions, and the effector genes have different expression patterns during U. virens infection. These findings lay a foundation for further research on the functions of the above-mentioned effectors from U. virens.

Key words: rice, Ustilaginoidea virens, effector, signal peptide

尹潇潇, 张芷菡, 颜绣莲, 廖蓉, 杨思葭, 郭岱铭, 樊晶, 赵志学, 王文明. 多个稻曲病菌效应因子的信号肽验证和表达分析[J]. 中国水稻科学, 2024, 38(3): 256-265.

YIN Xiaoxiao, ZHANG Zhihan, YAN Xiulian, LIAO Rong, YANG Sijia, Beenish HASSAN, GUO Daiming, FAN Jing, ZHAO Zhixue, WANG Wenming. Signal Peptide Validation and Expression Analysis of Multiple Effectors from Ustilaginoidea virens[J]. Chinese Journal OF Rice Science, 2024, 38(3): 256-265.

图/表 6

参考文献 30

[1]	Brooks S A, Anders M M, Yeater K M. Effect of cultural management practices on the severity of false smut and kernel smut of rice[J]. Plant Disease, 2009, 93(11): 1202-1208.
[2]	Tang Y X, Jin J, Hu D W, Yong M L, Xu Y, He L P. Elucidation of the infection process of Ustilaginoidea virens (teleomorph: Villosiclava virens) in rice spikelets[J]. Plant Pathology, 2013, 62(1): 1-8.
[3]	Cooke M C. Some extra-european fungi[J]. Grevillea, 1878, 7: 13-15.
[4]	Sun W, Fan J, Fang A, Li Y, Tariqjaveed M, Li D, Hu D, Wang W M. Ustilaginoidea virens: Insights into an emerging rice pathogen[J]. Annual Review of Phytopathology, 2020, 58(1): 363-385.
[5]	Fan J, Liu J, Gong Z Y, Xu P Z, Hu X H, Wu J L, Li G B, Yang J, Wang Y Q, Zhou Y F, Li S C, Wang L, Chen X Q. The false smut pathogen Ustilaginoidea virens requires rice stamens for false smut ball formation[J]. Environmental Microbiology, 2020, 22(2): 646-659.
[6]	Fan J, Guo X Y, Li L, Huang F, Sun W X, Li Y, Huang Y Y, Xu Y J, Shi J, Zheng A P, Wang W M. Infection of Ustilaginoidea virens intercepts rice seed formation but activates grain-filling-related genes[J]. Journal of Integrative Plant Biology, 2015, 57(6): 577-590.
[7]	Fan J, Yang J, Wang Y Q, Li G B, Li Y, Wang W M. Current understanding on Villosiclava virens, a unique flower-infecting fungus causing rice false smut disease[J]. Molecular Plant Pathology, 2016, 17(9): 1321-1330.
[8]	Wang B, Liu L, Li Y, Zou J, Li D, Zhao D, Li W, Sun W. Ustilaginoidin D induces hepatotoxicity and behaviour aberrations in zebrafish larvae[J]. Toxicology, 2021, 456: 152786.
[9]	Lin X, Bian Y, Mou R, Cao Z, Cao Z, Zhu Z, Chen M. Isolation, identification, and characterization of Ustilaginoidea virens from rice false smut balls with high ustilotoxin production potential[J]. Journal of Basic Microbiology, 2018, 58(8): 670-678.
[10]	Rafiqi M, Ellis J G, Ludowici V A, Hardham A R, Dodds P N. Challenges and progress towards understanding the role of effectors in plant-fungal interactions[J]. Current Opinion in Plant Biology, 2012, 15(4): 477-482.
[11]	Zhang K, Zhao Z, Zhang Z, Li Y, Li S, Yao N, Hsiang T. Insights into genomic evolution from chromosomal and mitochondrial genomes of Ustilaginoidea virens[J]. Phytopathology Research, 2021, 3(1): 9.
[12]	Zhang N, Yang J, Fang A, Wang J, Li D, Li Y, Wang S, Cui F, Yu J, Liu Y, Peng Y L, Sun W. The essential effector SCRE1 in Ustilaginoidea virens suppresses rice immunity via a small peptide region[J]. Molecular Plant Pathology, 2020, 21(4): 445-459.
[13]	Zheng X, Fang A, Qiu S, Zhao G, Wang J, Wang S, Wei J, Gao H, Yang J, Mou B, Cui F, Zhang J, Liu J, Sun W. Ustilaginoidea virens secretes a family of phosphatases that stabilize the negative immune regulator OsMPK6 and suppress plant immunity[J]. The Plant Cell, 2022, 34(8): 3088-3109.
[14]	Chen X, Duan Y, Qiao F, Liu H, Huang J, Luo C, Chen X, Li G, Xie K, Hsiang T, Zheng L. A secreted fungal effector suppresses rice immunity through host histone hypoacetylation[J]. New Phytologist, 2022, 235(5): 1977.
[15]	Li G B, He J X, Wu J L, Wang H, Zhang X, Liu J, Hu X H, Zhu Y, Shen S, Bai Y F, Wang W M, Fan J. Overproduction of OsRACK1A, an effector-targeted scaffold protein promoting OsRBOHB-mediated ROS production, confers rice floral resistance to false smut disease without yield penalty[J]. Molecular Plant, 2022, 15(11): 1790-1806.
[16]	Chen X, Li X, Duan Y, Pei Z, Liu H, Yin W, Huang J, Luo C, Chen X, Li G, Xie K, Hsiang T, Zheng L. A secreted fungal subtilase interferes with rice immunity via degradation of SUPPRESSOR OF G2 ALLELE OF skp1[J]. Plant Physiology, 2022, 190(2): 1474-1489.
[17]	Owji H, Nezafat N, Negahdaripour M, Hajiebrahimi A, Ghasemi Y. A comprehensive review of signal peptides: Structure, roles, and applications[J]. European Journal of Cell Biology, 2018, 97(6): 422-441.
[18]	von Heijne G. Signal sequences: Limits of variation[J]. Journal of Molecular Biology, 1985, 184(1): 99-105.
[19]	Rafiqi M, Gan P H P, Ravensdale M, Lawrence G J, Ellis J G, Jones D A, Hardham A R, Dodds P N. Internalization of flax rust avirulence proteins into flax and tobacco cells can occur in the absence of the pathogen[J]. The Plant Cell, 2010, 22(6): 2017-2032.
[20]	Yin W, Wang Y, Chen T, Lin Y, Luo C. Functional evaluation of the signal peptides of secreted proteins[J]. Bio-protocol, 2018, 8(9): e2839.
[21]	Gu B, Kale S D, Wang Q, Wang D, Pan Q, Cao H, Meng Y, Kang Z, Tyler B M, Shan W. Rust secreted protein Ps87 is conserved in diverse fungal pathogens and contains a RXLR-like motif sufficient for translocation into plant cells[J]. PLOS ONE, 2011, 6(11): e27217.
[22]	Bao J, Wang R, Gao S, Wang Z, Fang Y, Wu L, Wang M. High-Quality genome sequence resource of a rice false smut fungus Ustilaginoidea virens isolate, UV-FJ-1[J]. Phytopathology, 2021, 111(10): 1889-1892.
[23]	Zhang Y, Zhang K, Fang A, Han Y, Yang J, Xue M, Bao J, Hu D, Zhou B, Sun X, Li S, Wen M, Yao N, Ma L J, Liu Y, Zhang M, Huang F, Luo C, Zhou L, Li J, Chen Z. Specific adaptation of Ustilaginoidea virens in occupying host florets revealed by comparative and functional genomics[J]. Nature Communications, 2014, 5(1): 3849.
[24]	Wang H, Yang X, Wei S, Wang Y. Proteomic analysis of mycelial exudates of Ustilaginoidea virens[J]. Pathogens, 2021, 10(3): 364.
[25]	Fang A, Han Y, Zhang N, Zhang M, Liu L, Li S, Lu F, Sun W. Identification and characterization of plant cell death-inducing secreted proteins from Ustilaginoidea virens[J]. Molecular Plant-Microbe Interactions, 2016, 29(5): 405-416.
[26]	Wei S, Wang Y, Zhou J, Xiang S, Sun W, Peng X, Li J, Hai Y, Wang Y, Li S. The conserved effector UvHrip1 interacts with OsHGW and infection of Ustilaginoidea virens regulates defense- and heading date-related signaling pathway[J]. International Journal of Molecular Sciences, 2020, 21(9): 3376.
[27]	Qiu S, Fang A, Zheng X, Wang S, Wang J, Fan J, Sun Z, Gao H, Yang J, Zeng Q, Cui F, Wang W M, Chen J, Sun W. Ustilaginoidea virens nuclear effector SCRE4 suppresses rice immunity via inhibiting expression of a positive immune regulator OsARF17[J]. International Journal of Molecular Sciences, 2022, 23(18): 10527.
[28]	Fang A, Gao H, Zhang N, Zheng X, Qiu S, Li Y, Zhou S, Cui F, Sun W. A novel effector gene SCRE2 contributes to full virulence of Ustilaginoidea virens to rice[J]. Frontiers in Microbiology, 2019, 10.
[29]	Presti L L, Lanver D, Schweizer G, Tanaka S, Liang L, Tollot M, Zuccaro A, Reissmann S, Kahmann R. Fungal Effectors and plant susceptibility[J]. Annual Review of Plant Biology, 2015, 66(1): 513-545.
[30]	Fan J, Du N, Li L, Li G B, Wang Y Q, Zhou Y F, Hu X H, Liu J, Zhao J Q, Li Y, Huang F, Wang W M. A core effector UV_1261 promotes Ustilaginoidea virens infection via spatiotemporally suppressing plant defense[J]. Phytopathology Research, 2019, 1(1): 11.

引物名称 Primer name	引物序列（5′-3′） Primer sequence（5′-3′）		用途 Usage
Psuc2-44-F	AATTCATGAAGGCCTCGACGCTGCTGGCTCTCCTGCCCTTTGCCGCGGCCC		酵母系统鉴定信号肽功能 Functional validation of the signal peptides using yeast
Psuc2-44-R	TCGAGGGCCGCGGCAAAGGGCAGGAGAGCCAGCAGCGTCGAGGCCTTCATG
Psuc2-1548-F	AATTCATGAACCCAATCCTGCTTCTCCTCGCCGCATCAGTCCCCGCCCGCAGCC
Psuc2-1548-R	TCGAGGCTGCGGGCGGGGACTGATGCGGCGAGGAGAAGCAGGATTGGGTTCATG
Psuc2-1567-F	AATTCATGCAGTTGTCCTACCTTCTCTCGCTCGTCTCCCTTGTGGCCGCTC
Psuc2-1567-R	TCGAGAGCGGCCACAAGGGAGACGAGCGAGAGAAGGTAGGACAACTGCATG
Psuc2-1990-F	AATTCATGCGCCCAGCAACCGTCATCTTCGCACTGGCCGTCTCCGTCAAGGCTC
Psuc2-1990-R	TCGAGAGCCTTGACGGAGACGGCCAGTGCGAAGATGACGGTTGCTGGGCGCATG
Psuc2-3667-F	AATTCATGAAGTACACTGCTGCTCTCGTCGCCCTGGCTGCTGCCGTCATGGCCC
Psuc2-3667-R	TCGAGGGCCATGACGGCAGCAGCCAGGGCGACGAGAGCAGCAGTGTACTTCATG
Psuc2-4213-F	AATTCATGATTCTGCTGCTCCTCTTGCTATGCACGACAACATGTTTAGGAC
Psuc2-4213-R	TCGAGTCCTAAACATGTTGTCGTGCATAGCAAGAGGAGCAGCAGAATCATG
Psuc2-4989-F	AATTCATGCACCAGCTCCTTGCACTACTCCTCGCCAGCCTCACCGCTGCCC
Psuc2-4989-R	TCGAGGGCAGCGGTGAGGCTGGCGAGGAGTAGTGCAAGGAGCTGGTGCATG
Psuc2-5215-F	AATTCATGAAGTTTATTTCCCTCGTAGCCGCGGCCGGTGTGGCGTATGCCC
Psuc2-5215-R	TCGAGGGCATACGCCACACCGGCCGCGGCTACGAGGGAAATAAACTTCATG
Psuc2-6754-F	AATTCATGGCTGCAGCATCAGCCCTTTTCTTCCTCACGGCGGCTGTCTCCTGGACAC
Psuc2-6754-R	TCGAGTGTCCAGGAGACAGCCGCCGTGAGGAAGAAAAGGGCTGATGCTGCAGCCATG
Psuc2-8184-F	AATTCATGCAAATCGTCCTCCCTCTCCTGGCCTGCGCAGCCGTGGCTGCCGCCC
Psuc2-8184-R	TCGAGGGCGGCAGCCACGGCTGCGCAGGCCAGGAGAGGGAGGACGATTTGCATG
P1300-44-F	CATGAAGGCCTCGACGCTGCTGGCTCTCCTGCCCTTTGCCGCGGCCGGTAC		烟草瞬时表达鉴定信号肽功能 Functional validation of the signal peptides using N. benthamiana
P1300-44-R	CGGCCGCGGCAAAGGGCAGGAGAGCCAGCAGCGTCGAGGCCTTCATGGTAC
P1300-1548-F	CATGAACCCAATCCTGCTTCTCCTCGCCGCATCAGTCCCCGCCCGCAGCGGTAC
P1300-1548-R	CGCTGCGGGCGGGGACTGATGCGGCGAGGAGAAGCAGGATTGGGTTCATGGTAC
P1300-1567-F	CATGCAGTTGTCCTACCTTCTCTCGCTCGTCTCCCTTGTGGCCGCTGGTAC
P1300-1567-R	CAGCGGCCACAAGGGAGACGAGCGAGAGAAGGTAGGACAACTGCATGGTAC
P1300-1990-F	CATGCGCCCAGCAACCGTCATCTTCGCACTGGCCGTCTCCGTCAAGGCTGGTAC
P1300-1990-R	CAGCCTTGACGGAGACGGCCAGTGCGAAGATGACGGTTGCTGGGCGCATGGTAC
P1300-3667-F	CATGAAGTACACTGCTGCTCTCGTCGCCCTGGCTGCTGCCGTCATGGCCGGTAC
P1300-3667-R	CGGCCATGACGGCAGCAGCCAGGGCGACGAGAGCAGCAGTGTACTTCATGGTAC
P1300-4213-F	CATGATTCTGCTGCTCCTCTTGCTATGCACGACAACATGTTTAGGAGGTAC
P1300-4213-R	CTCCTAAACATGTTGTCGTGCATAGCAAGAGGAGCAGCAGAATCATGGTAC
P1300-4989-F	CATGCACCAGCTCCTTGCACTACTCCTCGCCAGCCTCACCGCTGCCGGTAC
P1300-4989-R	CGGCAGCGGTGAGGCTGGCGAGGAGTAGTGCAAGGAGCTGGTGCATGGTAC
P1300-5215-F	CATGAAGTTTATTTCCCTCGTAGCCGCGGCCGGTGTGGCGTATGCCGGTAC
P1300-5215-R	CGGCATACGCCACACCGGCCGCGGCTACGAGGGAAATAAACTTCATGGTAC
P1300-6754-F	CATGGCTGCAGCATCAGCCCTTTTCTTCCTCACGGCGGCTGTCTCCTGGACAGGTAC
P1300-6754-R	CTGTCCAGGAGACAGCCGCCGTGAGGAAGAAAAGGGCTGATGCTGCAGCCATGGTAC
P1300-8184-F	CATGCAAATCGTCCTCCCTCTCCTGGCCTGCGCAGCCGTGGCTGCCGCCGGTAC
P1300-8184-R	CGGCGGCAGCCACGGCTGCGCAGGCCAGGAGAGGGAGGACGATTTGCATGGTAC
Uv_Tublin2α-2F/R	ACCAGCTCGTTGAGAACTCG / AATCAGAGTTGAGCTGGCCG	实时荧光定量PCR RT-qPCR
UV_44-RT-F/R	GCGTTGCCAAGAAGACCAAG / ATTCCAGCAATCACGGCAGA
UV_1548-RT- F/R	GACAACGGGCAGACAAACTG / CTAGCAATCCCAGCAGTCGT
UV_1567-RT-F/R	TGCTAGCGCTATCGTTCCTG / GTCGGCATGGTTTTGCAACT
UV_1990-RT-F/R	ACTCACTTCCAAACACGGCA / GCCTTGTCTTCTCAACACGC
UV_3667-RT-F/R	GCCTCCTCTGCAATGTGGAA / CTTGAGGCAGTTGAGTCCGA
UV_4213-RT-F/R	ATGCGAGAACTTGGTCGTGT / AACCAACGCCTCCAACTTGA
UV_4989-RT-F/R	CCTGCCTCCTTCGTCCAAAT / GGTCGATCTGGCAAAGTGGA
UV_5215-RT-F/R	TAGCGTACTGCGACGGTAAC / GAAACAGCGCCCAATTCCTG
UV_6754-RT-F/R	AAATGCGAGGACTAGGGCAC / CTTTTTGCCGGAGGAGGTCT
UV_8184-RT-F/R	CATCGCTGCGAAGACAAAGG / GCGCCTGTGAACTTTTCCTG

引物名称 Primer name	引物序列（5′-3′） Primer sequence（5′-3′）		用途 Usage
Psuc2-44-F	AATTCATGAAGGCCTCGACGCTGCTGGCTCTCCTGCCCTTTGCCGCGGCCC		酵母系统鉴定信号肽功能 Functional validation of the signal peptides using yeast
Psuc2-44-R	TCGAGGGCCGCGGCAAAGGGCAGGAGAGCCAGCAGCGTCGAGGCCTTCATG
Psuc2-1548-F	AATTCATGAACCCAATCCTGCTTCTCCTCGCCGCATCAGTCCCCGCCCGCAGCC
Psuc2-1548-R	TCGAGGCTGCGGGCGGGGACTGATGCGGCGAGGAGAAGCAGGATTGGGTTCATG
Psuc2-1567-F	AATTCATGCAGTTGTCCTACCTTCTCTCGCTCGTCTCCCTTGTGGCCGCTC
Psuc2-1567-R	TCGAGAGCGGCCACAAGGGAGACGAGCGAGAGAAGGTAGGACAACTGCATG
Psuc2-1990-F	AATTCATGCGCCCAGCAACCGTCATCTTCGCACTGGCCGTCTCCGTCAAGGCTC
Psuc2-1990-R	TCGAGAGCCTTGACGGAGACGGCCAGTGCGAAGATGACGGTTGCTGGGCGCATG
Psuc2-3667-F	AATTCATGAAGTACACTGCTGCTCTCGTCGCCCTGGCTGCTGCCGTCATGGCCC
Psuc2-3667-R	TCGAGGGCCATGACGGCAGCAGCCAGGGCGACGAGAGCAGCAGTGTACTTCATG
Psuc2-4213-F	AATTCATGATTCTGCTGCTCCTCTTGCTATGCACGACAACATGTTTAGGAC
Psuc2-4213-R	TCGAGTCCTAAACATGTTGTCGTGCATAGCAAGAGGAGCAGCAGAATCATG
Psuc2-4989-F	AATTCATGCACCAGCTCCTTGCACTACTCCTCGCCAGCCTCACCGCTGCCC
Psuc2-4989-R	TCGAGGGCAGCGGTGAGGCTGGCGAGGAGTAGTGCAAGGAGCTGGTGCATG
Psuc2-5215-F	AATTCATGAAGTTTATTTCCCTCGTAGCCGCGGCCGGTGTGGCGTATGCCC
Psuc2-5215-R	TCGAGGGCATACGCCACACCGGCCGCGGCTACGAGGGAAATAAACTTCATG
Psuc2-6754-F	AATTCATGGCTGCAGCATCAGCCCTTTTCTTCCTCACGGCGGCTGTCTCCTGGACAC
Psuc2-6754-R	TCGAGTGTCCAGGAGACAGCCGCCGTGAGGAAGAAAAGGGCTGATGCTGCAGCCATG
Psuc2-8184-F	AATTCATGCAAATCGTCCTCCCTCTCCTGGCCTGCGCAGCCGTGGCTGCCGCCC
Psuc2-8184-R	TCGAGGGCGGCAGCCACGGCTGCGCAGGCCAGGAGAGGGAGGACGATTTGCATG
P1300-44-F	CATGAAGGCCTCGACGCTGCTGGCTCTCCTGCCCTTTGCCGCGGCCGGTAC		烟草瞬时表达鉴定信号肽功能 Functional validation of the signal peptides using N. benthamiana
P1300-44-R	CGGCCGCGGCAAAGGGCAGGAGAGCCAGCAGCGTCGAGGCCTTCATGGTAC
P1300-1548-F	CATGAACCCAATCCTGCTTCTCCTCGCCGCATCAGTCCCCGCCCGCAGCGGTAC
P1300-1548-R	CGCTGCGGGCGGGGACTGATGCGGCGAGGAGAAGCAGGATTGGGTTCATGGTAC
P1300-1567-F	CATGCAGTTGTCCTACCTTCTCTCGCTCGTCTCCCTTGTGGCCGCTGGTAC
P1300-1567-R	CAGCGGCCACAAGGGAGACGAGCGAGAGAAGGTAGGACAACTGCATGGTAC
P1300-1990-F	CATGCGCCCAGCAACCGTCATCTTCGCACTGGCCGTCTCCGTCAAGGCTGGTAC
P1300-1990-R	CAGCCTTGACGGAGACGGCCAGTGCGAAGATGACGGTTGCTGGGCGCATGGTAC
P1300-3667-F	CATGAAGTACACTGCTGCTCTCGTCGCCCTGGCTGCTGCCGTCATGGCCGGTAC
P1300-3667-R	CGGCCATGACGGCAGCAGCCAGGGCGACGAGAGCAGCAGTGTACTTCATGGTAC
P1300-4213-F	CATGATTCTGCTGCTCCTCTTGCTATGCACGACAACATGTTTAGGAGGTAC
P1300-4213-R	CTCCTAAACATGTTGTCGTGCATAGCAAGAGGAGCAGCAGAATCATGGTAC
P1300-4989-F	CATGCACCAGCTCCTTGCACTACTCCTCGCCAGCCTCACCGCTGCCGGTAC
P1300-4989-R	CGGCAGCGGTGAGGCTGGCGAGGAGTAGTGCAAGGAGCTGGTGCATGGTAC
P1300-5215-F	CATGAAGTTTATTTCCCTCGTAGCCGCGGCCGGTGTGGCGTATGCCGGTAC
P1300-5215-R	CGGCATACGCCACACCGGCCGCGGCTACGAGGGAAATAAACTTCATGGTAC
P1300-6754-F	CATGGCTGCAGCATCAGCCCTTTTCTTCCTCACGGCGGCTGTCTCCTGGACAGGTAC
P1300-6754-R	CTGTCCAGGAGACAGCCGCCGTGAGGAAGAAAAGGGCTGATGCTGCAGCCATGGTAC
P1300-8184-F	CATGCAAATCGTCCTCCCTCTCCTGGCCTGCGCAGCCGTGGCTGCCGCCGGTAC
P1300-8184-R	CGGCGGCAGCCACGGCTGCGCAGGCCAGGAGAGGGAGGACGATTTGCATGGTAC
Uv_Tublin2α-2F/R	ACCAGCTCGTTGAGAACTCG / AATCAGAGTTGAGCTGGCCG	实时荧光定量PCR RT-qPCR
UV_44-RT-F/R	GCGTTGCCAAGAAGACCAAG / ATTCCAGCAATCACGGCAGA
UV_1548-RT- F/R	GACAACGGGCAGACAAACTG / CTAGCAATCCCAGCAGTCGT
UV_1567-RT-F/R	TGCTAGCGCTATCGTTCCTG / GTCGGCATGGTTTTGCAACT
UV_1990-RT-F/R	ACTCACTTCCAAACACGGCA / GCCTTGTCTTCTCAACACGC
UV_3667-RT-F/R	GCCTCCTCTGCAATGTGGAA / CTTGAGGCAGTTGAGTCCGA
UV_4213-RT-F/R	ATGCGAGAACTTGGTCGTGT / AACCAACGCCTCCAACTTGA
UV_4989-RT-F/R	CCTGCCTCCTTCGTCCAAAT / GGTCGATCTGGCAAAGTGGA
UV_5215-RT-F/R	TAGCGTACTGCGACGGTAAC / GAAACAGCGCCCAATTCCTG
UV_6754-RT-F/R	AAATGCGAGGACTAGGGCAC / CTTTTTGCCGGAGGAGGTCT
UV_8184-RT-F/R	CATCGCTGCGAAGACAAAGG / GCGCCTGTGAACTTTTCCTG