甘蔗NAD(P)H脱氢酶复合体O亚基基因克隆及其与甘蔗花叶病毒VPg互作

doi:10.3724/SP.J.1006.2019.94002

Abstract

Abstract:

NAD(P)H dehydrogenase (NDH) complex mediates cyclic electron transports, playing key role in efficient photosynthesis in chloroplast. The involvement of NDH complex in Sugarcane mosaic virus (SCMV) infection of sugarcane (Saccharum spp. hybrid) has not been reported. In this study, we isolated the coding sequence of the subunit of the NAD(P)H dehydrogenase complex from sugarcane and designated it as ScNdhO. The open reading frame (ORF) of ScNdhO is 471 bp and encodes a 156 aa length protein. Bioinformatics analysis showed that ScNdhO is a stable hydrophilic protein with no signal peptide and transmembrane domain. The secondary structure of ScNdhO is composed of mostly random coilα-helices, with a typical domain of NDH complex O subunit. Phylogenetic tree analysis showed that ScNdhO belongs to the NDHO supperfamily. Real-time quantitative PCR analysis showed that ScNdhO gene was tissue specific in sugarcane, with the lowest expression level in roots or stem, and the highest in leaf. The expression of ScNdhO was upregulated in the early stage of SCMV infection, but downregulated with time going. Subcellular location assays showed that ScNdhO was located in chloroplast. ScNdhO interacted with the VPg from SCMV as demonstrated by yeast two hybrid and bimolecular fluorescence complementation assays. We proposed that ScNdhO should be selectively employed by SCMV and involved in the mosaic symptom.

Key words: sugarcane, SCMV, chloroplast, NAD(P)H dehydrogenase complex, O subunit

ZHAI Yu-Shan,ZHAO He,ZHANG Hai,DENG Yu-Qing,CHENG Guang-Yuan,YANG Zong-Tao,WANG Tong,PENG Lei,XU Qian,DONG Meng,XU Jing-Sheng. Cloning of NAD(P)H complex O subunit gene and its interaction with VPg of Sugarcane mosaic virus[J].Acta Agronomica Sinica, 2019, 45(10): 1478-1487.

Figures/Tables 7

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	用途 Strategy
ScNdhO-F	ACCAAGCCGAGCAATCCCTC	基因克隆
ScNdhO-R	CACCGTGTCCTGACCCCAAG	Gene cloning
ScNdhO-AD-F	GGAATTCCATATGATGGAAGCCCTCGCCTCG	酵母双杂交捕获载体构建
ScNdhO-AD-R	CGGAATTCTATCCTCTCGTAGTCGAGCTTG	Vector generation for Y2H
ScNdhO-YC-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGAAGCCCTCGCCTCG	双分子荧光互补载体构建
ScNdhO-YC-R	GGGGACCACTTTGTACAAGAAAGCTGGGTCTATCCTCTCGTAGTCGAGCTTG	Vector generation for BiFC
ScNdhO-qF	CGATGAAGAAAGGGCAGA	定量PCR
ScNdhO-qR	TGTAGAATGGCGGGTGTC	Real-time-qPCR
GAPDH-F	CACGGCCACTGGAAGCA	内参基因
GAPDH-R	TCCTCAG GGTTCCTGATGCC	Reference gene
eEF-1α-F	TTTCACACTTGGAGTGAAGCAGAT	内参基因
eEF-1α-R	GACTTCCTTCACAATCTCATCATAA	Reference gene
SCMV-CP-F	TACAGAGAGACACACAGCTG	SCMV检测
SCMV-CP-R	ACGCTACACCAGAAGACACT	Detection of SCMV

Cloning of NAD(P)H complex O subunit gene and its interaction with VPg of Sugarcane mosaic virus

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[5]	YANG Zong-Tao, LIU Shu-Xian, CHENG Guang-Yuan, ZHANG Hai, ZHOU Ying-Shuan, SHANG He-Yang, HUANG Guo-Qiang, XU Jing-Sheng. Sugarcane ubiquitin-like protein UBL5 responses to SCMV infection and interacts with SCMV-6K2 [J]. Acta Agronomica Sinica, 2022, 48(2): 332-341.
[6]	ZHANG Hai, CHENG Guang-Yuan, YANG Zong-Tao, LIU Shu-Xian, SHANG He-Yang, HUANG Guo-Qiang, XU Jing-Sheng. Sugarcane PsbR subunit response to SCMV infection and its interaction with SCMV-6K2 [J]. Acta Agronomica Sinica, 2021, 47(8): 1522-1530.
[7]	SU Ya-Chun, LI Cong-Na, SU Wei-Hua, YOU Chui-Huai, CEN Guang-Li, ZHANG Chang, REN Yong-Juan, QUE You-Xiong. Identification of thaumatin-like protein family in Saccharum spontaneum and functional analysis of its homologous gene in sugarcane cultivar [J]. Acta Agronomica Sinica, 2021, 47(7): 1275-1296.
[8]	WANG Heng-Bo, CHEN Shu-Qi, GUO Jin-Long, QUE You-Xiong. Molecular detection of G1 marker for orange rust resistance and analysis of candidate resistance WAK gene in sugarcane [J]. Acta Agronomica Sinica, 2021, 47(4): 577-586.
[9]	ZHANG Rong-Yue, WANG Xiao-Yan, YANG Kun, SHAN Hong-Li, CANG Xiao-Yan, LI Jie, WANG Chang-Mi, YIN Jiong, LUO Zhi-Ming, LI Wen-Feng, HUANG Ying-Kun. Identification of brown rust resistance and molecular detection of Bru1 gene in new and main cultivated sugarcane varieties [J]. Acta Agronomica Sinica, 2021, 47(2): 376-382.
[10]	CANG Xiao-Yan, XIA Hong-Ming, LI Wen-Feng, WANG Xiao-Yan, SHAN Hong-Li, WANG Chang-Mi, LI Jie, ZHANG Rong-Yue, HUANG Ying-Kun. Evaluation of natural resistance to smut in elite sugarcane varieties (lines) [J]. Acta Agronomica Sinica, 2021, 47(11): 2290-2296.
[11]	ZHANG Hai, CHENG Guang-Yuan, YANG Zong-Tao, WANG Tong, LIU Shu-Xian, SHANG He-Yang, ZHAO He, XU Jing-Sheng. Cloning of sugarcane ScCRT1 gene and its response to SCMV infection [J]. Acta Agronomica Sinica, 2021, 47(1): 94-103.
[12]	ZHENG Qing-Lei,YU Chen-Jing,YAO Kun-Cun,HUANG Ning,QUE You-Xiong,LING Hui,XU Li-Ping. Cloning and expression analysis of sugarcane Fe/S precursor protein gene ScPetC [J]. Acta Agronomica Sinica, 2020, 46(6): 844-857.
[13]	LUO Jun,LIN Zhao-Li,LI Shi-Yan,QUE You-Xiong,ZHANG Cai-Fang,YANG Zai-Qi,YAO Kun-Cun,FENG Jing-Fang,CHEN Jian-Feng,ZHANG Hua. Effects of different soil improvement measures on soil physicochemical properties and microbial community structures in mechanically compacted acidified sugarcane field [J]. Acta Agronomica Sinica, 2020, 46(4): 596-613.
[14]	WANG Heng-Bo,QI Shu-Ting,CHEN Shu-Qi,GUO Jin-Long,QUE You-Xiong. Development and application of SSR loci in monoploid reference genome of sugarcane cultivar [J]. Acta Agronomica Sinica, 2020, 46(4): 631-642.
[15]	ZHANG Hai, LIU Shu-Xian, YANG Zong-Tao, WANG Tong, CHENG Guang-Yuan, SHANG He-Yang, XU Jing-Sheng. Sugarcane PsbS subunit response to Sugarcane mosaic virus infection and its interaction with 6K2 protein [J]. Acta Agronomica Sinica, 2020, 46(11): 1722-1733.