Expression of OsBC88, a Rice Cellulose Synthase Catalytic Subunit Gene

doi:10.3969/j.issn.1001-7216.2015.02.003

Abstract

Abstract:

Brittle culm mutant in gramineae plants is an ideal material for secondary cell wall research. Previous studies about bc88 mutant in rice show that BC88 encodes a cellulose synthase catalytic subunit, OsCesA9. The genetic transformation of rice by BC88 promoter fused with GUS vector showed that BC88 was expressed ubiquitously in root, stem, leaf, sheath, flower, with high expression in culm and root. The mutation in BC88 may affect normal development and function of root, thereby affect the shoot growth and lead to semi-dwarf phenotype. Transfer BC88 fused with GFP protein into rice and tobacco epidermal cells show that, OsCesA9 is located at plasma membrane where cellulose synthesized, coinciding with that OsCesA9 is one of the essential functional CESA units for cellulose synthesis of secondary cell wall. These results provided new information for analysis of BC88 biological function and it’s of great significance for the further understanding of BC88 in regulating the secondary cell wall synthesis or other related process.

Key words: rice, BC88, OsCesA, expression, localization

摘要：

禾本科植物的脆秆突变体是进行次生细胞壁研究的理想材料。对水稻 bc88突变体的前期研究表明, BC88编码一个纤维素合酶催化亚基OsCesA9。通过水稻遗传转化 BC88启动子融合GUS的载体, 检测到BC88在根、茎、叶、鞘、花中均有表达,并且在茎和根中表达量较高,这可能是由于 BC88突变后影响了根系的正常发育及功能,进而影响地上部的生长,最终导致 bc88半矮表型。将 BC88与GFP的融合表达载体转入水稻和烟草表皮细胞中,共聚焦显微观察显示,OsCesA9定位在质膜上。质膜是纤维素合成的主要场所,这充分说明了OsCesA9是水稻次生细胞壁中纤维素合成必不可少的纤维素合酶催化亚基之一。本研究为 BC88执行其生物学功能提供了新依据,对进一步认识该基因在调节次生细胞壁合成等方面的作用具有重要的意义。

关键词: 水稻, BC88, OsCesA, 表达, 定位

CLC Number:

Q786
S511.01

Xiao-jing LI, Duo-duo XU, Yi-min XU, Kai-en ZHAI, Yao-long YANG, Jian-wei PAN, Yu-chun RAO. Expression of OsBC88, a Rice Cellulose Synthase Catalytic Subunit Gene[J]. Chinese Journal OF Rice Science, 2015, 29(2): 126-134.

李晓静, 徐多多, 徐益敏, 翟开恩, 杨窑龙, 潘建伟, 饶玉春. 水稻纤维素合酶催化亚基的编码基因BC88的表达分析[J]. 中国水稻科学, 2015, 29(2): 126-134.

Figures/Tables 6

Table 1 Primer sequences for OsBC88 cloning and Real-time PCR.

引物名称 Primer	引物序列(5'-3') Primer sequence(5'-3')
pBC88-2035-HindⅢ-F	CCGAAGCTTCTTTTCCATTTTCATATTTCCCTGCCATCA
pBC88-3896-BamHⅠ-R	GCCGGATCCGTGTGGGAGACGGTGGCGGGTTGGTTG
BC88-no ATG-XbaⅠ-F	GGCTCTAGAGAGGCGAGCGCCGGGCTGGTGGCCGG
BC88-stop codon-XbaⅠ-R	AGCTCTAGATCAGCAGTTGATGCCGCACTGCCTGACGTC
BC88-600-R	CTCAGACACCGGGTAGGGGTGGATGC
OsActin-Real-F	TGGCATCTCTCAGCACATTCC
OsActin-Real-R	TGCACAATGGATGGGTCAGA
OsBC-104-F	AGGTGTGCGAGATATGCGGCGACGAG
OsBC-213-R	CTCGTACTCGTAGCAGGGGCGGCACAC

Fig.1. Construction of PBI121 BC88 pro :: GUS vector. A, Structure of PBI121 BC88 pro :: GUS vector; B, PCR products of BC88 promoter; C, PCR identification of positive bacterial suspension; D, PCR identification of positive bacterial colony; M, DNA marker; CK, Positive control.

Fig. 2. Construction of pcambia 1300-2×35s:: eGFP: BC88 vector. A, Structure of pcambia 1300-2×35s:: eGFP: BC88 vector; B, PCR products of BC88; C, PCR identification of positive bacterial suspension; D, PCR identification of positive bacterial colony, M, DNA marker; CK, Positive control.

Fig. 3. Tissue expression analysis of OsBC88. A, GUS staining of root, culm, leaf, sheath of Nipponbare at seedling stage; B, GUS staining of flower of Nipponbare at flowering stage, serving as negative control; C, GUS staining of root, culm, sheath, leaf of pBI121: BC88 pro :: GUS transgenic lines at seedling stage; D, GUS staining of flower of pBI121:BC88 pro:: GUS transgenic lines at flowering stage.

Fig.4. Expression pattern of OsBC88.

Fig. 5. Subcellular localization of BC88. A, Localization of 35S:: eGFP and 35S:: eGFP: BC88 in tobacco epidermal cells; B, Localization of 35S:: eGFP in rice root tip cells, the bottom is the amplification of the upside. GFP indicates the green fluorescence of proteins, DIC indicates the differential interference contrast phase, and Merged indicates the merging of GFP and DIC.

References 41

[1]	Cosgrove D J.Relaxation in a high-stress environment: The molecular bases of extensible cell walls and cell enlargement.Plant Cell, 1997, 9(7): 1031-1041.
[2]	Kotilainen M, Helariutta Y, Mehto M, et al.GEG participates in the regulation of cell and organ shape during corolla and carpel development in Gerbera hybrida.Plant Cell, 1999, 11(6): 1093-1104.
[3]	Martin C, Bhatt K, Baumann K.Shaping in plant cells.Curr Opin Plant Biol, 2001, 4(6): 540-549.
[4]	Campbell M M, Sederoff R R.Variation in lignin content and composition: Mechanism of control and implications for the genetic improvement of plants.Plant Physiol, 1996, 110(1): 3-13.
[5]	Keegstra K. Plant Cell Walls.Plant Physiol, 2010, 154(2): 483-486.
[6]	Cosgrove D J.Growth of the plant cell wall.Nat Rev Mol Cell Biol, 2005, 6(11): 850-861.
[7]	Crowell E F, Gonneau M, Vernhettes S, et al.Regulation of anisotropic cell expansion in higher plants.C R Biol, 2010, 333(4): 320-324.
[8]	汪海峰, 朱军莉, 刘建新, 等.饲喂脆茎全株水稻对生长肥育猪生长性能、养分消化和胴体品质的影响.畜牧兽医学报, 2005, 36(11): 1139-1144.
[9]	Carpita N, McCann M. The cell wall//Buchanan R B, Gruissem W, Jones R L. Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, 2000: 52-108.
[10]	Somerville C.Cellulose synthesis in higher plants.Annu Rev Cell Dev Biol, 2006, 22:53-78.
[11]	Taylor N G, Scheible W R, Cutler S, et al.The irregular xylern3 locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis.Plant Cell, 1999, 11(5): 769-779.
[12]	Lerouxel O, Cavalier D M, Liepman A H, et al.Biosynthesis of plant cell wall polysaccharides — a complex process.Curr Opin Plant Biol, 2006, 9(6): 621-630.
[13]	Baskin T I.Anisotropic expansion of the plant cell wall.Annu Rev Cell Dev Biol, 2005, 21: 203-222.
[14]	Pear J R, Kawagoe Y, Schreckengost W E, et al.Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase.Proc Natl Acad Sci USA, 1996, 93(22): 12637-12642.
[15]	Arioli T, Peng L, Betzner A S, et al.Molecular analysis of cellulose biosynthesis in Arabidopsis.Science, 1998, 279(5351): 717-720.
[16]	Fagard M, Desnoes T, Desprez T, et al.PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis.Plant Cell, 2001, 12(12): 2409-2423.
[17]	Taylor NG, Laurie S, Turner S R.Mutiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis.Plant Cell, 2000, 12(12): 2529-2539.
[18]	Tanaka K, Murata K, Yamazaki M, et al.Three distinct rice cellulose synthase catalytic subunit genes required for cellulose synthesis in the secondary wall.Plant Physiol, 2003, 133(1): 73-83.
[19]	童川, 童杰鹏, 孙出,等.水稻脆性基因的功能研究进展.分子植物育种, 2013, 11(2): 286-292.
[20]	Song X Q, Liu L F, Jiang Y J, et al.Disruption of secondary wall cellulose biosynthesis alters cadmium translocation and tolerance in rice plants.Mol Plant, 2013, 6(3): 768-780.
[21]	Rao Y C, Yang Y L, Xin D D, et al.Characterization and cloning of a brittle culm mutant ( bc88) in rice (Oryza sativa L.).Chin Sci Bull, 2013, 58(24): 3000-3006.
[22]	Kotake T, Aohara T, Hirano K, et al.Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls.J Exp Bot, 2011, 62(6): 2053-2062.
[23]	王超. 拟南芥网格蛋白轻链的功能分析.金华:浙江师范大学, 2012.
[24]	沈金秋. 青藏高原野生大麦HsCIPKs基因克隆及其在水稻中的功能验证.金华:浙江师范大学, 2014.
[25]	Livak K J, Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method.Methods, 2001, 25(4): 402-408.
[26]	Li Y H, Qian Q, Zhou Y H, et al.BRITTLE CULM1, which encodes a COBRA-like protein, affects the mechanical properties of rice plants.Plant Cell, 2003, 15(9): 2020-2031.
[27]	Dai X X, You C J, Chen G X, et al.OsBC1L4 encodes a COBRA-like protein that affects cellulose synthesis in rice.Plant Mol Biol, 2011, 75(4-5): 333-345.
[28]	Hirano K, Kotake T, Kamihara K, et al.Rice BRITTLE CULM 3 (BC3) encodes a classical dynamin OsDRP2B essential for proper secondary cell wall synthesis.Planta, 2010, 232(1): 95-108.
[29]	Aohara T, Kotake T, Kaneko Y, et al.Rice BRITTLE CULM 5 (BRITTLE NODE) is involved in secondary cell wall formation in the sclerenchyma tissue of nodes.Plant Cell Physiol, 2009, 50(11): 1886-1897.
[30]	Zhou Y H, Li S B, Qian Q, et al.BC10, a DUF266-containing and Golgi-located typeⅡ membrane protein, is required for cell-wall biosynthesis in rice (Oryza sativa L.).Plant J, 2009, 57(3): 446-462.
[31]	Zhang M, Zhang B C, Qian Q, et al. Brittle Culm 12, a dual-targeting kinesin-4 protein, controls cell-cycle progression and wall properties in rice.Plant J, 2010, 63(2): 312-328.
[32]	Zhang B C, Liu X L, Qian Q, et al.Golgi nucleotide sugar transporter modulates cell wall biosynthesis and plant growth in rice.PNAS, 2011, 108(12): 5110-5115.
[33]	Wu B, Zhang B C, Dai Y, et al.Brittle Culm 15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice.Plant Physiol, 2012, 159(4): 1440-1452.
[34]	Yan C, Yan S, Zeng X H, et al.Fine mapping and isolation of Bc7(t), allelic to OsCesA4.J Genet Genom, 2007, 34(11): 1019-1027.
[35]	Zhang B C, Deng L W, Qian Q, et al.A missense mutation in the transmembrane domain of CESA4 affects protein abundance in the plasma membrane and results in abnormal cell wall biosynthesis in rice.Plant Mol Biol, 2009, 71(4-5): 509-524.
[36]	Kimura S, Laosinchai W, Itoh T, et al.Immunogold labeling of rosette terminal cellulose-synthesizing complexes in the vascular plant Vigna angularis.Plant Cell, 1999, 11(11): 2075-2086.
[37]	Peng L, Kawagoe Y, Hogan P, et al.Sitosterol-β-glucoside as primer for cellulose synthesis in plants.Science, 2002, 295(5552): 147-150.
[38]	Taylor N G, Howells R M, Huttly A K, et al.Interactions among three distinct CesA proteins essential for cellulose synthesis.P Natl Acad Sci USA, 2003, 100(3): 1450-1455.
[39]	Wang D F, Yuan S J, Yin L, et al.A missense mutation in the transmembrane domain of CESA9 affects cell wall biosynthesis and plant growth in rice.Plant Sci, 2012, 196: 117-124.
[40]	Ellis C, Karafyllidis I, Wasternack C, et al.The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses.Plant Cell, 2002, 14(17): 1557-1566.
[41]	Xiong G Y, Li R, Qian Q, et al.The rice dynamin-related protein DRP2B mediates membrane trafficking and thereby plays a critical role in secondary cell wall cellulose biosynthesis.Plant J, 2010, 64(1): 56-70.