Cloning and Expression Characteristics of Pinus massoniana pmCaM Response to Bursaphelenchus xylophilus Infection

XU Liang; LIU Zhen-yu; L&#220; Quan; LIANG Jun; ZHANG Xing-yao

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Cloning and Expression Characteristics of Pinus massoniana pmCaM Response to Bursaphelenchus xylophilus Infection

1.
College of Forestry, Beijing Forestry University, Beijing 100083, China
2.
Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China
3.
College of Plant Protection, Shandong Agricultural University, Tai'an 271018, Shandong, China

Received Date: 2013-08-10

Abstract

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is believed to be the most destructive disease of pine species, causing significant economic and ecological losses around the world. Calmodulin (CaM) is a major calcium-binding messenger protein expressed in all eukaryotic cells. A cDNA sequence of CaM was cloned from Masson pine (P. massoniana) by reverse transcription polymerase chain reaction (RT-PCR) methods, and named as pmCaM. And the pmCaM expression characteristics response to pine wood nematode infection was checked by real-time fluorescent quantitative PCR. The sequence analysis showed that the pmCaM consisted of an opening reading frame of 450 bp, encoded a protein of 149 aa containing four EF-hand domains and having the typical features of calmodulin. The comparison analysis showed that pmCaM shared high homology with CaMs from other plants. Real-time PCR analysis showed that 30 180 min post-PWD inoculation, pmCaM genes in root, stem and leaf of pine seedling were all down-regulated expression, but the times for significantly down-regulated expression of pmCaM were difference among different organs; specific fluctuate characteristics of pmCaM expression amount along with time development were appeared in root, stem and leaf, and the pmCaM expression amount peaked in root and stem 45 min post-PWD inoculation. This study showed that the expression of pmCaM was response to PWD infection, and inferred that the pmCaM in pine may play an important role in the regulation of calcium signal response early interaction between pine wood nematode with Masson pine.
- Pine wilt disease,
- Pinus massoniana,
- Bursaphelenchus xylophilus,
- pmCaM,
- cloning

References

[1]	Forestry Agency. Annual report on trends of forest and forestry-fiscal year 2003[R]; The ministry of agriculture, forestry and fisheries of Japan: Tokyo, Japan, 2004
[2]	Shin H, Lee H, Woo K S, et al. Identification of genes upregulated by pine wood nematode inoculation in Japanese red pine [J]. Tree Physiol, 2009, 29: 411-421
[3]	Yang B J, Pan H Y, Tang J, et al. Pine wood nematode disease [M]. Beijing: Chinese Forestry Press, 2003: 6-143
[4]	张星耀,骆有庆.中国森林重大生物灾害[M].北京:中国林业出版社,2003
[5]	王艳辉,贾慧,司贺龙,等。不同基因型玉米受HT-毒素胁迫后细胞内CaM的动态变化 [J]. 河北农业大学学报, 2007, 30: 4-7
[6]	Yang T, Poovaiah B W. Hydrogen peroxide homeostasis: Activation of plant catalase by calcium/calmodulin [J]. Proc Natl Acad Sci USA, 2002, 99: 4097-4102
[7]	Harding S A,Oh S,Roberts D M. Transgenic tobacco expressing a foreign calmodulin gene shows an enhanced production of active oxygen species [J].EMBO J, 1997, 16: 1137-1144
[8]	Kim M C, Lee S H, Kim J K, et al. Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein [J]. J Biol Chem, 2002, 277: 19304-19314
[9]	Kim M C, Panstruga R, Ellio C, et al. Calmodulin interacts with MLo protein to regulate defense against mildew in barley [J]. Nature, 2002, 416: 447-451
[10]	Kang C H, Jung W Y, Kang Y H, et al. AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants[J]. Cell Death Differ. 2006, 13: 84-95
[11]	金钢. 黑松与松材线虫互作过程中细胞程序性死亡的研究[D]. 南京:南京林业大学,2007
[12]	Tavernier E, Wendehenne D, Blein J-P, et al. Involvement of free calcium in action of cryptogein, a proteinaceous elicitor of hypersensitive reaction in tobacco cells [J]. Plant Physiol, 1995, 109: 1025-1031
[13]	Zielinski R E. Calmodulin and calmodulin-binding proteins in plants [J]. Annu Rev Plant Physiol Plant Mol Biol, 1998, 49: 697-725
[14]	Xu L, Liu Z Y, Zhang K, et al. Characterization of the Pinus massoniana transcriptional response to Bursaphelenchus xylophilus infection using suppression subtractive hybridization [J]. Int J Mol Sci, 2013, 14: 11356-11375
[15]	Heo W D, Lee S H, Kim M C, et al. Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses [J]. Proc Natl Acad Sci USA, 1999, 96: 766-771
[16]	霍建飞,宋水山,李星,等. CaM及各亚型基因参与小麦抗叶锈病反应的研究 [J].华北农学报, 2010, 25(4): 175-179
[17]	Yamakawa H, Mitsuhara I, Ito N, et al. Transcriptionally and post-transcriptionally regulated response of 13 calmodulin genes to tobacco mosaic virus-induced cell death and wounding in tobacco plant [J]. Eur J Biochem, 2001, 268: 3916-3929
[18]	Takabatake R, Karita E, Seo S, et al. Pathogen-induced calmodulin isoforms in basal resistance against bacterial and fungal pathogens in tobacco [J]. Plant Cell Physiol, 2007, 48(3): 414-423
[19]	Lee S H, Johnson J D, Walsh M P, et al. Differential regulation of Ca²⁺/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca²⁺ concentration [J]. Biochem J, 2000, 350: 299-306
[20]	Nath M, Goel A, Taj G, et al. Molecular cloning and comparative in silico analysis of calmodulin genes from cereals and millets for understanding the mechanism of differential calcium accumulation [J]. J Proteomics Bioinform, 2010, 3: 294-301
[21]	霍建飞, 宋水山, 李星,等. Ca²⁺·CaM信使系统参与小麦抗叶锈病反应的初步研究 [J]. 华北农学报, 2008, 23: 234-239

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Cloning and Expression Characteristics of Pinus massoniana pmCaM Response to Bursaphelenchus xylophilus Infection

1. College of Forestry, Beijing Forestry University, Beijing 100083, China

2. Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China

3. College of Plant Protection, Shandong Agricultural University, Tai'an 271018, Shandong, China

Received Date: 2013-08-10

Abstract: Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is believed to be the most destructive disease of pine species, causing significant economic and ecological losses around the world. Calmodulin (CaM) is a major calcium-binding messenger protein expressed in all eukaryotic cells. A cDNA sequence of CaM was cloned from Masson pine (P. massoniana) by reverse transcription polymerase chain reaction (RT-PCR) methods, and named as pmCaM. And the pmCaM expression characteristics response to pine wood nematode infection was checked by real-time fluorescent quantitative PCR. The sequence analysis showed that the pmCaM consisted of an opening reading frame of 450 bp, encoded a protein of 149 aa containing four EF-hand domains and having the typical features of calmodulin. The comparison analysis showed that pmCaM shared high homology with CaMs from other plants. Real-time PCR analysis showed that 30 180 min post-PWD inoculation, pmCaM genes in root, stem and leaf of pine seedling were all down-regulated expression, but the times for significantly down-regulated expression of pmCaM were difference among different organs; specific fluctuate characteristics of pmCaM expression amount along with time development were appeared in root, stem and leaf, and the pmCaM expression amount peaked in root and stem 45 min post-PWD inoculation. This study showed that the expression of pmCaM was response to PWD infection, and inferred that the pmCaM in pine may play an important role in the regulation of calcium signal response early interaction between pine wood nematode with Masson pine.

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[1]	Forestry Agency. Annual report on trends of forest and forestry-fiscal year 2003[R]; The ministry of agriculture, forestry and fisheries of Japan: Tokyo, Japan, 2004
[2]	Shin H, Lee H, Woo K S, et al. Identification of genes upregulated by pine wood nematode inoculation in Japanese red pine [J]. Tree Physiol, 2009, 29: 411-421
[3]	Yang B J, Pan H Y, Tang J, et al. Pine wood nematode disease [M]. Beijing: Chinese Forestry Press, 2003: 6-143
[4]	张星耀,骆有庆.中国森林重大生物灾害[M].北京:中国林业出版社,2003
[5]	王艳辉,贾慧,司贺龙,等。不同基因型玉米受HT-毒素胁迫后细胞内CaM的动态变化 [J]. 河北农业大学学报, 2007, 30: 4-7
[6]	Yang T, Poovaiah B W. Hydrogen peroxide homeostasis: Activation of plant catalase by calcium/calmodulin [J]. Proc Natl Acad Sci USA, 2002, 99: 4097-4102
[7]	Harding S A,Oh S,Roberts D M. Transgenic tobacco expressing a foreign calmodulin gene shows an enhanced production of active oxygen species [J].EMBO J, 1997, 16: 1137-1144
[8]	Kim M C, Lee S H, Kim J K, et al. Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein [J]. J Biol Chem, 2002, 277: 19304-19314
[9]	Kim M C, Panstruga R, Ellio C, et al. Calmodulin interacts with MLo protein to regulate defense against mildew in barley [J]. Nature, 2002, 416: 447-451
[10]	Kang C H, Jung W Y, Kang Y H, et al. AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants[J]. Cell Death Differ. 2006, 13: 84-95
[11]	金钢. 黑松与松材线虫互作过程中细胞程序性死亡的研究[D]. 南京:南京林业大学,2007
[12]	Tavernier E, Wendehenne D, Blein J-P, et al. Involvement of free calcium in action of cryptogein, a proteinaceous elicitor of hypersensitive reaction in tobacco cells [J]. Plant Physiol, 1995, 109: 1025-1031
[13]	Zielinski R E. Calmodulin and calmodulin-binding proteins in plants [J]. Annu Rev Plant Physiol Plant Mol Biol, 1998, 49: 697-725
[14]	Xu L, Liu Z Y, Zhang K, et al. Characterization of the Pinus massoniana transcriptional response to Bursaphelenchus xylophilus infection using suppression subtractive hybridization [J]. Int J Mol Sci, 2013, 14: 11356-11375
[15]	Heo W D, Lee S H, Kim M C, et al. Involvement of specific calmodulin isoforms in salicylic acid-independent activation of plant disease resistance responses [J]. Proc Natl Acad Sci USA, 1999, 96: 766-771
[16]	霍建飞,宋水山,李星,等. CaM及各亚型基因参与小麦抗叶锈病反应的研究 [J].华北农学报, 2010, 25(4): 175-179
[17]	Yamakawa H, Mitsuhara I, Ito N, et al. Transcriptionally and post-transcriptionally regulated response of 13 calmodulin genes to tobacco mosaic virus-induced cell death and wounding in tobacco plant [J]. Eur J Biochem, 2001, 268: 3916-3929
[18]	Takabatake R, Karita E, Seo S, et al. Pathogen-induced calmodulin isoforms in basal resistance against bacterial and fungal pathogens in tobacco [J]. Plant Cell Physiol, 2007, 48(3): 414-423
[19]	Lee S H, Johnson J D, Walsh M P, et al. Differential regulation of Ca²⁺/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca²⁺ concentration [J]. Biochem J, 2000, 350: 299-306
[20]	Nath M, Goel A, Taj G, et al. Molecular cloning and comparative in silico analysis of calmodulin genes from cereals and millets for understanding the mechanism of differential calcium accumulation [J]. J Proteomics Bioinform, 2010, 3: 294-301
[21]	霍建飞, 宋水山, 李星,等. Ca²⁺·CaM信使系统参与小麦抗叶锈病反应的初步研究 [J]. 华北农学报, 2008, 23: 234-239

Cloning and Expression Characteristics of Pinus massoniana pmCaM Response to Bursaphelenchus xylophilus Infection

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Cloning and Expression Characteristics of Pinus massoniana pmCaM Response to Bursaphelenchus xylophilus Infection

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