纖維素是最具經(jīng)濟(jì)價(jià)值的細(xì)胞壁成分,，其高級(jí)結(jié)構(gòu)與纖維素的理化性質(zhì)和經(jīng)濟(jì)價(jià)值密切相關(guān),。然而纖維素的組裝過(guò)程極其復(fù)雜，除了纖維素鏈自組裝假說(shuō)外,，一直沒(méi)有證據(jù)證明纖維素的組裝需要一些關(guān)鍵蛋白的參與,。另一方面，早在2001年已有報(bào)道表明COBRA蛋白參與了纖維素的生物合成過(guò)程,，但其作用機(jī)理一直未知,，使其成為本領(lǐng)域的“未解之謎”。
BC1是一個(gè)類(lèi)COBRA蛋白,，李家洋院士課題組在2003年首次報(bào)道了所編碼基因的克隆及其影響次生壁纖維素合成的效應(yīng),。近期，中科院遺傳與發(fā)育生物研究所周奕華課題組與李家洋課題組及中國(guó)水稻所錢(qián)前課題組合作，從BC1的生化特性入手,，利用生物化學(xué),、細(xì)胞生物學(xué)、遺傳學(xué),、生物物理學(xué),、植物化學(xué)等多種手段，以充分的證據(jù)證明BC1蛋白是一個(gè)磷脂酰肌醇（GPI）錨定的膜蛋白,，切除GPI錨定的BC1定位在細(xì)胞壁,、尤其是次生壁上。BC1蛋白的N端有一個(gè)碳水化合物結(jié)合結(jié)構(gòu)域（CBM）,。體外生化分析和體內(nèi)遺傳學(xué)實(shí)驗(yàn)證實(shí),，該結(jié)構(gòu)域可特異結(jié)合晶體化纖維素，是一個(gè)重要的功能域,。此外還發(fā)現(xiàn),，BC1蛋白的細(xì)胞壁定位需要GPI和CBM結(jié)構(gòu)域共同發(fā)揮作用。雖然BC1與CESAs基因高度共表達(dá),，但遺傳學(xué)證據(jù)表明BC1的作用位置與CESA不同,，可能在CESA蛋白合成纖維素葡聚糖鏈后發(fā)揮作用。利用廣角X衍射(WAXS)方法分析了突變體和相關(guān)材料中纖維素的高級(jí)結(jié)構(gòu),，證實(shí)該基因突變可導(dǎo)致纖維素微晶尺寸顯著變小,，而過(guò)量表達(dá)BC1可以提高纖維素的相對(duì)結(jié)晶度。表達(dá)CBM中特定芳香族氨基酸點(diǎn)突變的BC1變異形式可使轉(zhuǎn)基因材料的纖維素微晶大小改變,，并與其體外結(jié)合纖維素能力相符,。
這一發(fā)現(xiàn)首次證明了纖維素的組裝需要蛋白的參與。因此,，BC1可作為改良植物纖維素性質(zhì)的關(guān)鍵元件,，其應(yīng)用前景令人期待。
該項(xiàng)工作已于8月22日在PLoS Genetics雜志上在線發(fā)表(DOI：10.1371/journal.pgen.1003704),。周奕華課題組劉立鋒和上官科科為該論文的共同第一作者,。
該研究得到了“973”、國(guó)家自然科學(xué)基金和轉(zhuǎn)基因?qū)ｍ?xiàng)的資助,。（生物谷Bioon.com）
doi:10.1371/journal.pgen.1003704
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Brittle Culm1, a COBRA-Like Protein, Functions in Cellulose Assembly through Binding Cellulose Microfibrils
Lifeng Liu equal contributor, Keke Shang-Guan equal contributor, Baocai Zhang, Xiangling Liu, Meixian Yan, Lanjun Zhang, Yanyun Shi, Mu Zhang, Qian Qian, Jiayang Li, Yihua Zhou mail
Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.
