甘蔗类钙调素ScCML13与SCMV运动蛋白P3N-PIPO的互作研究

doi:10.3724/SP.J.1006.2024.34191

Abstract

Abstract:

Calmodulin-like (CML), one of the calcium signal receptor proteins unique to plants, is involved in plant growth and development and in response to environmental signal transduction. However, the response of CML to Sugarcane mosaic virus (SCMV) infection in sugarcane (Saccharum spp. hybrid) has not been reported. In the present study, a CML gene was cloned from a Badila (S. officinarum) and designated as ScCML13. The open reading frame (ORF) of ScCML13 gene is 519 bp in length and encodes a protein with 172 aa in length. Bioinformatics analysis showed that ScCML13 was a stable hydrophilic lipoprotein in with no transmembrane domain and contains 4 Ca²⁺-binding EF-hand domains. Phylogenetic tree analysis showed that the ScCML13 protein differentiated in monocotyledon and dicotyledon plants and in monocotyledonous C₃ and C₄ plants. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments indicated that ScCML13 interacted with P3N-PIPO, the movement protein of SCMV. Subcellular localization assays demonstrated that ScCML13 was localized to the endoplasmic reticulum and nucleus, whereas the co-localization assays showed that ScCML13 interfered with the localization of SCMV-P3N-PIPO to plasma membrane or plasmodesmata. The RT-qPCR showed that ScCML13 gene was mainly expressed in sugarcane leaves, but relatively low in internodes and roots. The ScCML13 gene was significantly up-regulated at 2 h upon SCMV infection, and then down-regulated to the levels compared with the control group, while up-regulated at the later stage of infection.

Key words: calmodulin-like protein, Sugarcane mosaic virus, P3N-PIPO, protein interaction

YU Quan-Xin, YANG Zong-Tao, ZHANG Hai, CHENG Guang-Yuan, JIAO Wen-Di, ZENG Kang, LUO Ting-Xu, HUANG Guo-Qiang, WANG Lu, XU Jing-Sheng. Interaction between calmodulin-like ScCML13 of sugarcane and SCMV movement protein P3N-PIPO[J].Acta Agronomica Sinica, 2024, 50(7): 1855-1866.

Figures/Tables 7

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	用途 Strategy
ScCML13-F	ATGAGTTTCAACCAGTCTACTGTCAAG	基因克隆
ScCML13-R	CTAGTAGCCATAGCTAGTCCTCCT	Gene cloning
221-ScCML13-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAGTTTCAACCAGTCTAC	亚细胞定位及双分子荧光互补载体构建 Vectors generation for subcellular localization and BiFC
221-ScCML13-R	GGGGACCACTTTGTACAAGAAAGCTGGGTCGTAGCCATAGCTAGTCCTCC
pPR3-ScCML13-F	GCAGAGTGGCCATTACGATGAGTTTCAACCAGTCTACT	酵母双杂交载体构建
pPR3-ScCML13-R	ATTCTCGAGAGGCCGCTAGTAGCCATAGCTAGTCCT	Vector generation for Y2H
ScCML13-qF	TCGAGGAGTTTGAGCACATGATGAC	定量PCR
ScCML13-qR	TTACACCCAGCTCTTTGGCAATCC	Real-time-qPCR
Actin-qF	CTGGAATGGTCAAGGCTGGT	内参基因^[64]
Actin-qR	TCCTTCTGTCCCATCCCTACC	Reference gene^[64]
eEF-1α-qF	TTCACACTTGGAGTGAAGCAGAT	内参基因^[65]
eEF-1α-qR	GACTTCCTTCACAATCTCATCATAA	Reference gene^[65]
SCMV-CP-F	TACAGAGAGACACACAGCTG	SCMV检测^[66]
SCMV-CP-R	ACGCTACACCAGAAGACACT	Detection of SCMV^[66]

Interaction between calmodulin-like ScCML13 of sugarcane and SCMV movement protein P3N-PIPO

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