Cloning and Analysis of a Polygalacturonase Inhibiting Protein Gene and Its Promoter from Lilium regale Wilson

doi:10.7668/hbnxb.20192931

Abstract

Abstract:

Polygalacturonase-inhibiting proteins(PGIPs)play an important role during response to pathogen infection.A PGIP gene and its promoter were isolated from Lilium regale Wilson,which has strong resistance to Fusarium oxysporum.The open reading frame of LrPGIP was 1 008 bp in length,which encoded a protein with a length of 335 amino acid residues and contained 7 leucine-rich repeat domains.LrPGIP had high homology with PGIP of Elaeis guineensis,Cocos nucifera and Nicotiana sylvestris,which was 91%,94% and 86%,respectively.The LrPGIP showed closer relation with Phoenix dactylifera,Ensete ventricosum,E.guineensis and C.nucifera.LrPGIP was expressed in roots,stems,leaves,flowers and scales.It had the highest expression level in roots and the lowest expression level in scales.The expression level of LrPGIP was induced by F.oxysporum,and the highest expression level was at 72 h after F.oxysporum infection.The plant signal molecules including salicylic acid(SA),jasmonic acid(JA),ethephon(ETH)and hydrogen peroxide induced the expression of LrPGIP.Among them,the expression level of LrPGIP induced by ETH was the highest,which was followed by JA.The length of LrPGIP promoter fragment was 706 bp and had some cis-acting elements,such as hormone and stress responding elements.The expression cassette of the β-glucuronidase(GUS)driven by the LrPGIP promoter was transferred into tobacco for expression,and the GUS activity was obviously induced by the infection of F.oxysporum and Alternaria alternata as well as the stress of HgCl₂ and NaCl.It also responded to defense-related plant hormones including JA,SA and ETH.The up-regulation of GUS activity by HgCl₂ treatment was the greatest,followed by A.alternata and ETH.These data showed that the LrPGIP promoter activity was induced by plant hormones,biotic and abiotic stresses.The above results indicated that LrPGIP might be an important disease resistance gene in L.regale against F.oxysporum.

Key words: Lilium regale Wilson, Polygalacturonase-inhibiting proteins, Expression characteristics, Promoter activity

DENG Jie, WANG Hanlin, LI Youyu, WANG Zie, CHEN Xiaohua, LIU Diqiu. Cloning and Analysis of a Polygalacturonase Inhibiting Protein Gene and Its Promoter from Lilium regale Wilson[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(4): 45-52. doi: 10.7668/hbnxb.20192931.

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References 28

[1]	di Matteo A, Bonivento D, Tsernoglou D, Federici L, Cervone F. Polygalacturonase-inhibiting protein(PGIP)in plant defence:A structural view[J]. Phytochemistry, 2006, 67(6):528-533. doi: 10.1016/j.phytochem.2005.12.025. doi: 10.1016/j.phytochem.2005.12.025 pmid: 16458942
[2]	Bacete L, Mélida H, Miedes E, Molina A. Plant cell wall-mediated immunity:Cell wall changes trigger disease resistance responses[J]. The Plant Journal:for Cell and Molecular Biology, 2018, 93(4):614-636. doi: 10.1111/tpj.13807. doi: 10.1111/tpj.13807 pmid: 29266460
[3]	Kalunke R M, Tundo S, Benedetti M, Cervone F, de Lorenzo G, D'Ovidio R. An update on polygalacturonase-inhibiting protein(PGIP),a leucine-rich repeat protein that protects crop plants against pathogens[J]. Frontiers in Plant Science, 2015, 6:146. doi: 10.3389/fpls.2015.00146. doi: 10.3389/fpls.2015.00146
[4]	Mattei B, Bernalda M S, Federici L, Roepstorff P, Cervone F, Boffi A. Secondary structure and post-translational modifications of the leucine-rich repeat protein PGIP(polygalacturonase-inhibiting protein)from Phaseolus vulgaris[J]. Biochemistry, 2001, 40(2):569-576. doi: 10.1021/bi0017632. doi: 10.1021/bi0017632 pmid: 11148052
[5]	Hou W N, Singh R K, Zhao P, Martins V, Aguilar E, Canto T, Tenllado F, Franklin G, Dias A C P. Overexpression of polygalacturonase-inhibiting protein(PGIP)gene from Hypericum perforatum alters expression of multiple defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens in tobacco[J]. Journal of Plant Physiology, 2020, 253:153268. doi: 10.1016/j.jplph.2020.153268. doi: 10.1016/j.jplph.2020.153268 URL
[6]	Li H Y, Smigocki A C. Wound induced Beta vulgaris polygalacturonase-inhibiting protein genes encode a longer leucine-rich repeat domain and inhibit fungal polygalacturonases[J]. Physiological and Molecular Plant Pathology, 2016, 96:8-18. doi: 10.1016/j.pmpp.2016.06.004. doi: 10.1016/j.pmpp.2016.06.004 URL
[7]	Feng C S, Zhang X, Wu T, Yuan B, Ding X H, Yao F Y, Chu Z H. The polygalacturonase-inhibiting protein 4(OsPGIP4),a potential component of the qBlsr5a locus,confers resistance to bacterial leaf streak in rice[J]. Planta, 2016, 243(5):1297-1308. doi: 10.1007/s00425-016-2480-z. doi: 10.1007/s00425-016-2480-z URL
[8]	关瑞攀, 白智伟, 王倩, 唐笔锋, 崔秀明, 刘迪秋. 三七多聚半乳糖醛酸酶抑制蛋白基因的功能分析[J]. 华北农学报, 2018, 33(2):65-71. doi: 10.7668/hbnxb.2018.02.010. doi: 10.7668/hbnxb.2018.02.010
	Guan R P, Bai Z W, Wang Q, Tang B F, Cui X M, Liu D Q. Functional analysis of polygalacturonase-inhibiting protein gene from Panax notoginseng[J]. Acta Agriculturae Boreali-Sinica, 2018, 33(2):65-71.
[9]	Zhu G, Liang E X, Lan X, Li Q, Qian J J, Tao H X, Zhang M J, Xiao N, Zuo S M, Chen J M, Gao Y. ZmPGIP3 gene encodes a polygalacturonase-inhibiting protein that enhances resistance to sheath blight in rice[J]. Phytopathology, 2019, 109(10):1732-1740. doi: 10.1094/PHYTO-01-19-0008-R. doi: 10.1094/PHYTO-01-19-0008-R URL
[10]	张燕梅, 王瑞芳, 杨子平, 李俊峰, 鹿志伟, 赵艳龙, 陆军迎, 周文钊. 剑麻PGIP基因克隆和表达研究[J]. 热带作物学报, 2019, 40(12):2397-2404. doi: 10.3969/j.issn.1000-2561.2019.12.012. doi: 10.3969/j.issn.1000-2561.2019.12.012
	Zhang Y M, Wang R F, Yang Z P, Li J F, Lu Z W, Zhao Y L, Lu J Y, Zhou W Z. Cloning and expression analysis of polygalacturonase-inhibiting protein in sisal[J]. Chinese Journal of Tropical Crops, 2019, 40(12):2397-2404.
[11]	Lu L X, Zhou F, Zhou Y, Fan X L, Ye S F, Wang L, Chen H, Lin Y J. Expression profile analysis of the polygalacturonase-inhibiting protein genes in rice and their responses to phytohormones and fungal infection[J]. Plant Cell Reports, 2012, 31(7):1173-1187. doi: 10.1007/s00299-012-1239-7. doi: 10.1007/s00299-012-1239-7 URL
[12]	Zhang N N, Guan R P, Yang Y, Bai Z W, Ge F, Liu D Q. Isolation and characterization of a Fusarium oxysporum-resistant gene LrGLP1 from Lilium regale Wilson[J]. In Vitro Cellular & Developmental Biology-Plant, 2017, 53(5):461-468. doi: 10.1007/s11627-017-9829-2. doi: 10.1007/s11627-017-9829-2
[13]	Han Q, Chen R, Yang Y, Cui X M, Ge F, Chen C Y, Liu D Q. A glutathione S-transferase gene from Lilium regale Wilson confers transgenic tobacco resistance to Fusarium oxysporum[J]. Scientia Horticulturae, 2016, 198:370-378. doi: 10.1016/j.scienta.2015.11.047. doi: 10.1016/j.scienta.2015.11.047 URL
[14]	Li S, Liu G Z, Pu L M, Liu X Y, Wang Z E, Zhao Q, Chen H J, Ge F, Liu D Q. WRKY transcription factors actively respond to Fusarium oxysporum in Lilium regale[J]. Phytopathology, 2021, 111(9):1625-1637. doi: 10.1094/PHYTO-10-20-0480-R. doi: 10.1094/PHYTO-10-20-0480-R URL
[15]	Zhao Q, Qiu B L, Li S, Zhang Y P, Cui X M, Liu D Q. Osmotin-like protein gene from Panax notoginseng is regulated by jasmonic acid and involved in defense responses to Fusarium solani[J]. Phytopathology, 2020, 110(8):1419-1427. doi: 10.1094/PHYTO-11-19-0410-R. doi: 10.1094/PHYTO-11-19-0410-R pmid: 32301678
[16]	Márton L, Hrouda M, Pécsváradi A, Czakó M. T-DNA-insert-independent mutations induced in transformed plant cells during Agrobacterium co-cultivation[J]. Transgenic Research, 1994, 3(5):317-325. doi: 10.1007/BF01973592. doi: 10.1007/BF01973592 pmid: 7951334
[17]	Chen R, He H, Yang Y, Qu Y, Ge F, Liu D Q. Functional characterization of a pathogenesis-related protein family 10 gene,LrPR10-5,from Lilium regale Wilson[J]. Australasian Plant Pathology, 2017, 46(3):251-259. doi: 10.1007/s13313-017-0485-0. doi: 10.1007/s13313-017-0485-0 URL
[18]	陈夕军, 唐滔, 李丽丽, 陈宸, 陈煜文, 张亚芳, 左示敏. 水稻多聚半乳糖醛酸酶抑制蛋白家族OsPGIP结构及基因表达特征分析[J]. 作物学报, 2020, 46(12):1884-1893. doi: 10.3724/SP.J.1006.2020.02011. doi: 10.3724/SP.J.1006.2020.02011
	Chen X J, Tang T, Li L L, Chen C, Chen Y W, Zhang Y F, Zuo S M. Analysis on the structures of polygalacturonase-inhibiting proteins and the expression profile of its encoding genes in rice[J]. Acta Agronomica Sinica, 2020, 46(12):1884-1893.
[19]	Kalunke R M, Janni M, Sella L, David P, Geffroy V, Favaron F, D'Ovidio R. Transcript analysis of the bean polygalacturonase inhibiting protein gene family reveals that PVPGIP2 is expressed in the whole plant and is strongly induced by pathogen infection[J]. Journal of Plant Pathology, 2011, 93(1):141-148. doi: 10.4454/jpp.v93i1.284. doi: 10.4454/jpp.v93i1.284
[20]	D'Ovidio R, Raiola A, Capodicasa C, Devoto A, Pontiggia D, Roberti S, Galletti R, Conti E, O'Sullivan D, Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects[J]. Plant Physiology, 2004, 135(4):2424-2435. doi: 10.1104/pp.104.044644. doi: 10.1104/pp.104.044644 pmid: 15299124
[21]	De-Jesús-García R, Rosas U, Dubrovsky J G. The barrier function of plant roots:Biological bases for selective uptake and avoidance of soil compounds[J]. Functional Plant Biology, 2020, 47(5):383-397. doi: 10.1071/FP19144. doi: 10.1071/FP19144 pmid: 32213271
[22]	潘其云, 朱明德, 邓建玲, 唐祥宁. 百合镰刀菌枯萎病的发生与防治研究[J]. 上海农业科技, 2004(3):103-104. doi: 10.3969/j.issn.1001-0106.2004.03.099. doi: 10.3969/j.issn.1001-0106.2004.03.099
	Pan Q Y, Zhu M D, Deng J L, Tang X N. Research on the occurrence and control of lily Fusarium Wilt[J]. Shanghai Agricultural Science and Technology, 2004(3):103-104.
[23]	Liu N N, Zhang X Y, Sun Y, Wang P, Li X C, Pei Y K, Li F G, Hou Y X. Molecular evidence for the involvement of a polygalacturonase-inhibiting protein,GhPGIP1,in enhanced resistance to Verticillium and Fusarium wilts in cotton[J]. Scientific Reports, 2017, 7:39840. doi: 10.1038/srep39840. doi: 10.1038/srep39840 URL
[24]	甘红豪, 赵帅, 高明远, 阴文华, 郭光智, 褚建民. 外源水杨酸对NaCl胁迫下白榆幼苗光合作用及离子分配的影响[J]. 西北植物学报, 2020, 40(3):478-489. doi: 10.7606/j.issn.1000-4025.2020.03.0478. doi: 10.7606/j.issn.1000-4025.2020.03.0478
	Gan H H, Zhao S, Gao M Y, Yin W H, Guo G Z, Chu J M. Effect of salicylic acid on photosynthesis and ion distribution of Ulmus pumila seedlings under NaCl stress[J]. Acta Botanica Boreali-Occidentalia Sinica, 2020, 40(3):478-489.
[25]	Ding P T, Ding Y L. Stories of salicylic acid:A plant defense hormone[J]. Trends in Plant Science, 2020, 25(6):549-565. doi: 10.1016/j.tplants.2020.01.004. doi: 10.1016/j.tplants.2020.01.004 URL
[26]	Ali S, Ganai B A, Kamili A N, Bhat A A, Mir Z A, Bhat J A, Tyagi A, Islam S T, Mushtaq M, Yadav P, Rawat S, Grover A. Pathogenesis-related proteins and peptides as promising tools for engineering plants with multiple stress tolerance[J]. Microbiological Research, 2018, 212/213:29-37. doi: 10.1016/j.micres.2018.04.008. doi: 10.1016/j.micres.2018.04.008 URL
[27]	Howe G A, Major I T, Koo A J. Modularity in jasmonate signaling for multistress resilience[J]. Annual Review of Plant Biology, 2018, 69:387-415. doi: 10.1146/annurev-arplant-042817-040047. doi: 10.1146/annurev-arplant-042817-040047 URL
[28]	Li S, Hai J, Wang Z E, Deng J, Liang T T, Su L L, Liu D Q. Lilium regale Wilson WRKY2 regulates chitinase gene expression during the response to the root rot pathogen Fusarium oxysporum[J]. Frontiers in Plant Science, 2021, 12:741463. doi: 10.3389/fpls.2021.741463. doi: 10.3389/fpls.2021.741463 URL

用途 Use	名称 Name	序列(5'-3') Sequence(5'-3')
ORF克隆	LrPGIP-F	GAGGAGGATTATAGGTTGTGA
ORF cloning	LrPGIP-R	ACTTCGCTTTTTCGTATTG
qRT-PCR	LrPGIP-QF	TGAGGCTGTTGTGGTCGAGC
	LrPGIP-QR	GGTCTGGCACTATCCCCTCC
qRT-PCR	LrGAPDH-QF	ACTTGGTTTCCACTGATTTCCTCG
	LrGAPDH-QR	CTTGCTAATGTGGCGGATGAGAT
启动子克隆	PLrPGIP-Pa-1	CTTCGACGGTCCGACATCGTTCAAC
Promoter cloning	PLrPGIP-Pa-2	TCGTCTACATACAAAACAAGTGCCTCTGTG
启动子扩增	PLrPGIP-F	AAGCTTTTGAATGGGTAAGTTTTCATG
Promoter amplification	PLrPGIP-R	GGATCCCTCTACAGTTACTTGATAATTACCG

用途 Use	名称 Name	序列(5'-3') Sequence(5'-3')
ORF克隆	LrPGIP-F	GAGGAGGATTATAGGTTGTGA
ORF cloning	LrPGIP-R	ACTTCGCTTTTTCGTATTG
qRT-PCR	LrPGIP-QF	TGAGGCTGTTGTGGTCGAGC
	LrPGIP-QR	GGTCTGGCACTATCCCCTCC
qRT-PCR	LrGAPDH-QF	ACTTGGTTTCCACTGATTTCCTCG
	LrGAPDH-QR	CTTGCTAATGTGGCGGATGAGAT
启动子克隆	PLrPGIP-Pa-1	CTTCGACGGTCCGACATCGTTCAAC
Promoter cloning	PLrPGIP-Pa-2	TCGTCTACATACAAAACAAGTGCCTCTGTG
启动子扩增	PLrPGIP-F	AAGCTTTTGAATGGGTAAGTTTTCATG
Promoter amplification	PLrPGIP-R	GGATCCCTCTACAGTTACTTGATAATTACCG

顺式元件名称 The name of cis-element	特征序列 Sequence	元件功能 Element's function	位置/bp Position
ARE	AAACCA	厌氧响应元件	-308~-313
ATC-motif	TGCTATCCA	光调控元件	-136~-144
ATCT-motif	AATCTAATCC	光调控元件	-333~-341
CGTCA-motif	CGTCA	茉莉酸甲酯响应元件	-678~-682
GT1-motif	GGTTAA	光调控元件	-211~-216
LTR	CCGAAA	低温响应元件	-229~-234
MRE	AACCTAA	光调控元件	-186~-192
MYB binding site	CAACCA	MYB结合位点	-46~-51
		胁迫响应元件
		脱落酸响应元件
MYC binding site	CATTTG	MYC结合位点	-424~-429
		冷胁迫响应元件
		脱水响应元件
Myb	TAACTG	水胁迫响应元件	-695~-700
TCA-element	CCATCTTTTT	水杨酸响应元件	-559~-567
TGACG-motif	TGACG	茉莉酸甲酯响应元件	-678~-682
Circadian	CAAAGATATC	昼夜节律调控元件	-128~-136

顺式元件名称 The name of cis-element	特征序列 Sequence	元件功能 Element's function	位置/bp Position
ARE	AAACCA	厌氧响应元件	-308~-313
ATC-motif	TGCTATCCA	光调控元件	-136~-144
ATCT-motif	AATCTAATCC	光调控元件	-333~-341
CGTCA-motif	CGTCA	茉莉酸甲酯响应元件	-678~-682
GT1-motif	GGTTAA	光调控元件	-211~-216
LTR	CCGAAA	低温响应元件	-229~-234
MRE	AACCTAA	光调控元件	-186~-192
MYB binding site	CAACCA	MYB结合位点	-46~-51
		胁迫响应元件
		脱落酸响应元件
MYC binding site	CATTTG	MYC结合位点	-424~-429
		冷胁迫响应元件
		脱水响应元件
Myb	TAACTG	水胁迫响应元件	-695~-700
TCA-element	CCATCTTTTT	水杨酸响应元件	-559~-567
TGACG-motif	TGACG	茉莉酸甲酯响应元件	-678~-682
Circadian	CAAAGATATC	昼夜节律调控元件	-128~-136

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