小麦mRNA剪接因子TaSR与TaHis的互作鉴定及其基因表达分析

doi:10.7668/hbnxb.20194686

摘要/Abstract

摘要：

为研究TaHis基因对小麦赤霉病的抗性机制,首先克隆了TaHis基因全长编码序列,构建了pGBKT7-TaHis诱饵载体,通过酵母双杂交技术对赤霉病诱导的小麦穗部酵母双杂交文库进行筛选,获得互作蛋白后利用酵母双杂交技术和双分子荧光互补技术验证蛋白质之间的互作,并利用RT-qPCR技术对抗感小麦材料中TaHis互作蛋白的赤霉病诱导表达模式进行分析。结果表明,成功构建了pGBKT7-TaHis诱饵载体,经过酵母双杂交文库筛选后,在四缺选择培养基(SD/-Leu/-Trp/-His/-Ade)上获得了18个酵母单克隆。测序后Blast分析发现,共获得5个可能与TaHis互作的蛋白质,其中在6个酵母单克隆中均鉴定到富含丝氨酸/精氨酸的mRNA剪接因子SR45a类蛋白(TaSR)编码序列。以百农4299为材料,克隆了TaSR基因全长编码区,并构建了pGADT7-TaSR载体,酵母双杂交试验表明,pGADT7-TaSR和pGBKT7-TaHis共转化的酵母细胞在四缺培养基(SD/-Leu/-Trp/-His/-Ade/X-α-Gal/AbA)上可以生长且显蓝色,表明TaSR和TaHis在酵母细胞中直接互作;构建了YC-TaHis、YN-TaSR载体,双分子荧光互补试验表明,共转化YC-TaHis和YN-TaSR的烟草细胞中产生强烈荧光信号,进一步验证了TaSR与TaHis的互作。RT-qPCR分析显示,TaSR 在抗性材料百农4299中受赤霉病诱导后上调表达,在感病材料百农607中受赤霉病诱导后下调表达,表明TaSR基因表达量与小麦赤霉病抗性呈正相关。综上,小麦TaSR与TaHis存在相互作用,且TaSR基因表达量与小麦赤霉病抗性呈正相关。

关键词: 小麦赤霉病, TaHis, TaSR, 酵母双杂交, 双分子荧光互补, RT-qPCR

Abstract:

To study the resistance mechanism of TaHis gene to Fusarium head blight(FHB)in wheat,the full-length coding sequence of TaHis was cloned,and the bait vector pGBKT7-TaHis was constructed,which was then used as bait for screening a yeast two-hybrid library of wheat ear induced by FHB.After obtaining the interacting proteins,yeast two-hybridization and bimolecular fluorescence complementation were further used to verify the interaction between these proteins,and RT-qPCR was used to analyze the expression pattern of TaHis interacting protein induced by FHB in resistant and susceptible cultivars respectively.The results showed that the bait vector pGBKT7-TaHis was successfully constructed,and 18 yeast monoclones were obtained on the four deficient selection medium(SD/-Leu/-Trp/-His/-Ade)after yeast two-hybrid library screening.Blast analysis showed that a total of 5 proteins were obtained,and the coding sequence of serine/arginine-rich mRNA splicing factor SR45a-like(TaSR)was identified in 6 colonies.We cloned the full-length coding region of TaSR gene from Bainong 4299 and constructed pGADT7-TaSR vector.The experiment of yeast two-hybrid showed that the yeast cells co-transformed with pGADT7-TaSR and pGBKT7-TaHis grew well and appeared blue on SD/-Leu/-Trp/-His/-Ade/ X-α-Gal/AbA,indicating that TaSR and TaHis directly interacted in yeast cells.The vectors of YC-TaHis and YN-TaSR were constructed,and the bimolecular fluorescence complementary experiments were performed.The results showed that strong fluorescence signals were generated in tobacco cells co-transferred with YC-TaHis and YN-TaSR,which further verified the interaction between TaSR and TaHis.RT-qPCR analysis of TaSR gene expression showed that TaSR expression was up-regulated in resistant cultivar Bainong 4299,while down-regulated in susceptible cultivar Bainong 607 upon FHB infection,indicating a positive correlation between TaSR expression level and FHB resistance in wheat.To sum up,the interaction between wheat TaSR and TaHis was proved,and TaSR expression level was positively correlated with FHB resistance in wheat.

Key words: Fusarium head blight, TaHis, TaSR, Yeast two-hybrid, Bimolecular fluorescence complementation, RT-qPCR

宋普文, 邓佳乐, 杜玉新, 陈家美, 敬月婷, 刘俊彤, 李澳, 胡海燕. 小麦mRNA剪接因子TaSR与TaHis的互作鉴定及其基因表达分析[J]. 华北农学报, 2024, 39(3): 166-172. doi: 10.7668/hbnxb.20194686.

SONG Puwen, DENG Jiale, DU Yuxin, CHEN Jiamei, JING Yueting, LIU Juntong, LI Ao, HU Haiyan. Interaction Identification Between mRNA Splicing Factor TaSR and TaHis as well as Expression Analysis of TaSR in Wheat[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 166-172. doi: 10.7668/hbnxb.20194686.

http://www.hbnxb.net/CN/Y2024/V39/I3/166

图/表 8

表1 本研究所用引物

Tab.1 Primer sets used in this study

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
TaSR-F	ATGGCGCCGTCCGCCAG
TaSR-R	TCAATCATAACTGGCATTCAGAAAC
TaHis-F	ATGTGTATATTTGGACAACCATGTA
TaHis-R	TTACACGAGTTGCTTCCCGT
AD-TaSR-F	GGAGGCCAGTGAATTCATGGCGCCGTCCGCCAG
AD-TaSR-R	CGAGCTCGATGGATCCTCAATCATAACTGGCATTCAGAAAC
BD-TaHis-F	CATGGAGGCCGAATTCATGTGTATATTTGGACAACCATGTA
BD-TaHis-R	GCAGGTCGACGGATCCTTACACGAGTTGCTTCCCGT
BiFC-TaSR-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCGCCGTCCGCCAG
BiFC-TaSR-R	GGGGACCACTTTGTACAAGAAAGCTGGGTCATCATAACTGGCATTCAGAAAC
BiFC-TaHis-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTGTATATTTGGACAACCATGTA
BiFC-TaHis-R	GGGGACCACTTTGTACAAGAAAGCTGGGTCCACGAGTTGCTTCCCGTC
qTubulin-F	ATCTCCAACTCCACCAGTGTCG
qTubulin-R	TCATCGCCCTCATCACCGTC
qTaSR-F	AGCTTGAGAAATCCAAGGAAGA
qTaSR-R	CCACTTTCGACAAGTTGTCAAT

图1 TaHis基因的克隆和pGBKT7-TaHis诱饵载体的构建 A.TaHis基因的PCR扩增;B.诱饵载体pGBKT7-TaHis双酶切鉴定。M.DNA分子质量标准 DL2000+。

Fig.1 Cloning of TaHis gene and construction of pGBKT7-TaHis bait vector A.PCR amplification of TaHis gene;B.Identification of bait vector pGBKT7-TaHis by double enzyme digestion.M.DNA Marker DL2000+.

表2 TaHis互作蛋白统计

Tab.2 TaHis interacting protein statistics

蛋白质ID Protein ID	蛋白质名称 Protein name	酵母克隆数量/个 Number of yeast clones
XP_037440604.1	小麦CAX相互作用蛋白	2
XP_037468224.1	丝氨酸苏氨酸蛋白激酶RIO1	3
XP_044367519.1	富含丝氨酸/精氨酸的mRNA剪接因子SR45a类蛋白	6
XP_044437442.1	泛素受体RAD23b	3
XP_044369301.1	二磷酸核酮糖羧化酶/加氧酶活化酶A	4

图2 TaSR基因的克隆和pGADT7-TaSR载体的构建 A.TaSR基因的PCR扩增;B.pGADT7-TaSR载体双酶切鉴定。M.DNA分子质量标准DL2000+。

Fig.2 Cloning of TaSR gene and construction of pGADT7-TaSR vector A.PCR amplification of TaSR gene;B.Identification of pGADT7-TaSR vector by double enzyme digestion.M.DNA Marker DL2000+.

图3 TaHis和TaSR基因双分子荧光互补载体的PCR检测 A.YC-TaHis载体的PCR检测;B.YN-TaSR载体的PCR检测。M.DNA分子质量标准DL2000+。

Fig.3 PCR detection of bimolecular fluorescent complementary vectors for TaHis and TaSR genes A.PCR detection of YC-TaHis vector;B.PCR detection of YN-TaSR vector.M.DNA Marker DL2000+.

图4 酵母双杂交检测TaSR与TaHis的相互作用

Fig.4 Interaction between TaSR and TaHis detected by yeast two-hybrid system

图5 双分子荧光互补试验检测TaHis和TaSR的相互作用

Fig.5 Interaction between TaHis and TaSR detected by bimolecular fluorescent complementation

图6 赤霉病感染后TaSR表达的定量PCR分析不同大写字母表示同一时间点不同品种之间的差异显著(P<0.05),不同小写字母表示不同时间点同一品种间差异显著(P<0.05)。

Fig.6 Quantitative PCR analysis of TaSR expression after F.graminearum infection Different capital letters indicate significant differences(P<0.05)between different cultivars at the same time point,and different lowercase letters indicate significant difference(P<0.05)between different time points for the same cultivar.

参考文献 20

[1]	苏培森. 小麦赤霉病抗病机制研究进展[J]. 生物技术进展, 2021, 11(5):599-609.doi:10.19586/j.2095-2341.2021.0111.
	Su P S. Research advances in wheat FHB resistance mechanism[J]. Current Biotechnology, 2021, 11(5):599-609. doi: 10.19586/j.2095-2341.2021.0111
[2]	张立勇, 夏明聪, 徐文, 王琦, 张洁, 孙润红, 潘娅梅, 陈瑞雪, 吴坤, 杨丽荣. 拮抗小麦赤霉病内生细菌YB-144的鉴定及其对DON的影响[J]. 华北农学报, 2020, 35(6):172-179.doi:10.7668/hbnxb.20191619.
	Zhang L Y, Xia M C, Xu W, Wang Q, Zhang J, Sun R H, Pan Y M, Chen R X, Wu K, Yang L R. Identification of antagonistic endophytic bacterium YB-144 strain from Fusarium head blight against and its effect on DON[J]. Acta Agriculturae Boreali-Sinica, 2020, 35(6):172-179.
[3]	张颖君, 高慧敏, 李子千, 胡梦芸, 孙丽静, 刘茜, 吕亮杰, 李辉. 黄淮北片冬麦区抗赤霉病基因Fhb1种质挖掘及溯源[J]. 华北农学报, 2020, 35(2):196-202.doi:10.7668/hbnxb.20190265.
	Zhang Y J, Gao H M, Li Z Q, Hu M Y, Sun L J, Liu Q, Lü L J, Li H. Screening of Fhb1 germplasms resistant to Fusarium head blight and its putative ancestor in north of Huang and Huai winter wheat region[J]. Acta Agriculturae Boreali-Sinica, 2020, 35(2):196-202.doi:10.7668/hbnxb.20190265.
[4]	Li G Q, Zhou J Y, Jia H Y, Gao Z X, Fan M, Luo Y J, Zhao P T, Xue S L, Li N, Yuan Y, Ma S W, Kong Z X, Jia L, An X, Jiang G, Liu W X, Cao W J, Zhang R R, Fan J C, Xu X W, Liu Y F, Kong Q Q, Zheng S H, Wang Y, Qin B, Cao S Y, Ding Y X, Shi J X, Yan H S, Wang X, Ran C F, Ma Z Q. Mutation of a histidine-rich calcium-binding-protein gene in wheat confers resistance to Fusarium head blight[J]. Nature Genetics, 2019, 51(7):1106-1112.doi:10.1038/s41588-019-0426-7.
[5]	Su Z Q, Bernardo A, Tian B, Chen H, Wang S, Ma H X, Cai S B, Liu D T, Zhang D D, Li T, Trick H, St Amand P, Yu J M, Zhang Z Y, Bai G H. A deletion mutation in TaHRC confers Fhb1 resistance to Fusarium head blight in wheat[J]. Nature Genetics, 2019, 51(7):1099-1105.doi:10.1038/s41588-019-0425-8.
[6]	Chen H, Su Z Q, Tian B, Liu Y, Pang Y H, Kavetskyi V, Trick H N, Bai G H. Development and optimization of a Barley stripe mosaic virus-mediated gene editing system to improve Fusarium head blight resistance in wheat[J]. Plant Biotechnology Journal, 2022, 20(6):1018-1020.doi:10.1111/pbi.13819.
[7]	瓮巧云, 黄聪聪, 王娜, 梁晨曦, 刘高然, 郝丛丛, 邢继红, 董金皋. 拟南芥抗灰霉病基因T1N6_22互作蛋白的筛选与分析[J]. 华北农学报, 2017, 32(2):45-49.doi:10.7668/hbnxb.2017.02.007.
	Wang Q Y, Huang C C, Wang N, Liang C X, Liu G R, Hao C C, Xing J H, Dong J A. Screening and analysis of interacting proteins of T1N6_22 gene from Arabidopsis against Botrytis cinerea[J]. Acta Agriculturae Boreali-Sinica, 2017, 32(2):45-49.
[8]	顾佳, 李浩, 安瑞朋, 刘刚, 王冬梅. 小麦富含半胱氨酸的类受体激酶TaCRK2与翻译控制肿瘤蛋白TaTCTP的相互作用[J]. 河北农业大学学报, 2020, 43(4):16-22.doi:10.13320/j.cnki.jauh.2020.0067.
	Gu J, Li H, An R P, Liu G, Wang D M. Interaction between wheat cysteine-rich receptor-like kinases TaCRK2 and translationally controlled tumor protein TaTCTP[J]. Journal of Hebei Agricultural University, 2020, 43(4):16-22.
[9]	Chaudhary S, Jabre I, Reddy A S N, Staiger D, Syed N H. Perspective on alternative splicing and proteome complexity in plants[J]. Trends in Plant Science, 2019, 24(6):496-506.doi:10.1016/j.tplants.2019.02.006. pmid: 30852095
[10]	Pan Q, Shai O, Lee L J, Frey B J, Blencowe B J. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing[J]. Nature Genetics, 2008, 40(12):1413-1415.doi:10.1038/ng.259. pmid: 18978789
[11]	Marquez Y, Brown J W S, Simpson C, Barta A, Kalyna M. Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis[J]. Genome Research, 2012, 22(6):1184-1195.doi:10.1101/gr.134106.111. pmid: 22391557
[12]	喻锦莉, 刘长爱, 符霖, 王鑫, 欧阳解秀. 水稻OsHSP40基因可变剪接体鉴定及表达分析[J]. 华北农学报, 2021, 36(3):1-6.doi:10.7668/hbnxb.20191962.
	Yu J L, Liu C A, Fu L, Wang X, Ouyang J X. Analyzing splice variants and expression of variable the OsHSP40 gene in rice[J]. Acta Agriculturae Boreali-Sinica, 2021, 36(3):1-6.
[13]	Erkelenz S, Mueller W F, Evans M S, Busch A, Schöneweis K, Hertel K J, Schaal H. Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms[J]. RNA, 2013, 19(1):96-102.doi:10.1261/rna.037044.112. pmid: 23175589
[14]	Li Y, Guo Q H, Liu P, Huang J G, Zhang S Z, Yang G D, Wu C G, Zheng C C, Yan K. Dual roles of the serine/arginine-rich splicing factor SR45a in promoting and interacting with nuclear cap-binding complex to modulate the salt-stress response in Arabidopsis[J]. The New Phytologist, 2021, 230(2):641-655.doi:10.1111/nph.17175.
[15]	Ling Z H, Zhou W W, Baldwin I T, Xu S Q. Insect herbivory elicits genome-wide alternative splicing responses in Nicotiana attenuata[J]. The Plant Journal, 2015, 84(1):228-243.doi:10.1111/tpj.12997.
[16]	Lorkovic Z J, Hilscher J, Barta A. Co-localisation studies of Arabidopsis SR splicing factors reveal different types of speckles in plant cell nuclei[J]. Experimental Cell Research, 2008, 314(17):3175-3186. doi:10.1016/j.yexcr.2008.06.020.
[17]	Jia Z C, Das D, Zhang Y J, Fernie A R, Liu Y G, Chen M X, Zhang J H. Plant serine/arginine-rich proteins: versatile players in RNA processing[J]. Planta, 2023, 257(6):109. doi:10.1007/s00425-023-04132-0.
[18]	Park H J, You Y N, Lee A, Jung H, Jo S H, Oh N, Kim H S, Lee H J, Kim J K, Kim Y S, Jung C, Cho H S. OsFKBP20-1b interacts with the splicing factor OsSR45 and participates in the environmental stress response at the post-transcriptional level in rice[J]. The Plant Journal, 2020, 102(5):992-1007.doi:10.1111/tpj.14682. pmid: 31925835
[19]	Yan Q Q, Xia X, Sun Z F, Fang Y D. Depletion of Arabidopsis SC35 and SC35-like serine/arginine-rich proteins affects the transcription and splicing of a subset of genes[J]. PLoS Genetics, 2017, 13(3):e1006663.doi:10.1371/journal.pgen.1006663.
[20]	Huang J, Lu X Y, Wu H W, Xie Y C, Peng Q, Gu L F, Wu J Y, Wang Y C, Reddy A S N, Dong S M. Phytophthora effectors modulate genome-wide alternative splicing of host mRNAs to reprogram plant immunity[J]. Molecular Plant, 2020, 13(10):1470-1484.doi:10.1016/j.molp.2020.07.007. pmid: 32693165

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