拟南芥LMBD2基因突变回复mur3-3表型的分子机制

Xyloglucan, the most abundant hemicelluloses in plant primary cell wall, together with cellulose and pectin, control the elongation and expansion of plant cell, which require accurate coordination of many factors. To date, the biosynthesis pathway and structure of xyloglucan are almost clear. However, molecular mechanisms of how plant regulating its xyloglucan biosynthesis during development are still unknown. The Arabidopsis mur3-3 mutant carries a T-DNA insertion in its xyloglucan-specific galactosyltransferase encoding gene MUR3, in which the xyloglucan structure and subunit composition are changed, and shows dwarf “cabbage”-like phenotype and internal membrane system in chaos. The mur3-3 mutant, therefore, provides an excellent model system in studying of xyloglucan biosynthesis regulation and plant development. Through suppressor screening of mur3-3 lines which were processed by EMS, we identified a suppressor mrs-1 which was proved to be controlled by a recessive point mutation in gene AT5G65290 (LMBD2) with re-sequencing and genetic analysis. This gene was predicted encoding a LMBR1-like protein with unknown functions. LMBR1-like genes, which are highly conserved among species, are supposed to encode membrane transport proteins or receptors with 8~9 transmembrane domains, and have got people’s great attention because of their connections with human development and diseases and transport of drugs. However, no studies focusing on this gene family are carried out in plants. This study aims to carry out a detailed functional analysis of LMBD2, and elucidate the molecular mechanisms underlying the phenotype restoration of mrs-1.

木葡聚糖是植物初生细胞壁中最丰富的半纤维素，其与纤维素、果胶质等相互交联，控制着植物细胞的伸长和扩张。当前，木葡聚糖的合成与结构已经基本得到阐明，但对于木葡聚糖调控植物生长发育的分子机制仍然不清楚。拟南芥木葡聚糖半乳糖基转移酶突变体mur3-3呈现矮化的“卷心菜”表型、内膜系统紊乱等，是一个理想的研究木葡聚糖结构与植物生长发育关系的模式体系。我们前期从mur3-3的EMS诱变群体中筛选到一个由隐性单基因控制的回复子mrs-1，利用重测序和遗传学手段证明突变基因为AT5G65290（LMBD2），编码类LMBR1蛋白质。类LMBR1基因家族在动植物中保守存在，编码含有8~9个跨膜区的膜受体或转运蛋白，因其与人类发育、疾病和药物转运等相关，受到人们的极大关注，但在植物中尚未有该类基因的研究报道。本项目拟对该基因的功能开展深入解析，阐明其使mur3-3突变体恢复野生型表型的分子机制。

木葡聚糖是植物初生细胞壁中最丰富的半纤维素，其与纤维素、果胶质等相互交联，控制着植物细胞的伸长和扩张。当前，木葡聚糖的合成与结构已经基本得到阐明，但对于木葡聚糖调控植物生长发育的分子机制仍然不十分清楚。拟南芥木葡聚糖半乳糖基转移酶突变体mur3-3呈现矮化的“卷心菜”表型，是一个理想的研究木葡聚糖结构与植物生长发育关系的模式体系。为了鉴定更多参与木葡聚糖合成与调控或者参与感知植物细胞壁结构改变的功能基因，我们利用EMS诱变创制了以mur3-3为遗传背景的突变体库，并从中筛选到一个抑制子mrs1。利用重测序和遗传学手段证明突变基因为LMBD2，功能未知。为研究该基因的功能，我们获得了lmbd2 T-DNA插入突变体，并通过表型鉴定、功能互补实验、表达分析和亚细胞定位分析等，发现LMBD2在拟南芥生长发育过程中广泛表达，且其突变体与WT无明显表型差异；LMBD2编码一个含有9个跨膜结构域的膜蛋白，在质膜、高尔基体膜和内质网膜均有定位。同时，我们测定了mrs1细胞壁木葡聚糖的结构和组成及其叶片单糖组成，发现LMBD2不能改变木葡聚糖的亚基组成。为进一步研究LMBD2的功能，我们通过Y2H和Co-IP筛选互作蛋白，发现LMBD2与YIP4B互作。因此，推测LMBD2可能为YIP4A/YIB4B/ECH蛋白复合体的一员，并在高尔基体TGN介导的果胶、半纤维素等的分泌中发挥重要作用。为进一步在遗传学上验证它们的关系，创制了lmbd2 ech、mur3-3 ech双突变体及lmbd2 yip4a yip4b三突变体。表型分析发现，lmbd2突变能部分恢复yip4a yip4b及ech的表型。综上，本项目研究了LMBD2突变使mur3-3回复野生型表型的分子机制，首次揭示了LMBD2通过与YIP4A、YIP4B和ECH等形成蛋白复合体参与细胞壁多糖的分泌，在细胞壁结构维持中发挥重要作用。

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