全 文 ：植物学报 Chinese Bulletin of Botany 2010, 45 (4): 492–505, www.chinbullbotany.com
doi: 10.3969/j.issn.1674-3466.2010.04.013

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收稿日期: 2009-06-17; 接受日期: 2009-10-29
基金项目: 国家自然科学基金(No.30840002, 30970223)、黑龙江省留学归国人员科学基金(No.LC08C03)、中央高校基本科研业务费专
项资金(No.DL09DA02)、东北林业大学引进人才科研启动金(No.015-602042)、中国博士后科学基金特别资助(No.200902365)和黑龙江
省留学人员科技活动项目择优资助(2009HLJLixinLi)
* 通讯作者。E-mail: lixinli1@gmail.com
植物种子贮藏蛋白质及其细胞内转运与加工
韩宝达, 李立新*
东北林业大学东北油田盐碱植被恢复与重建教育部重点实验室, 哈尔滨 150040
摘要 高等植物种子成熟过程中贮存大量的贮藏蛋白质作为种子发芽和初期生长的重要营养来源。根据溶解性不同, 种子
贮藏蛋白质可分为白蛋白、球蛋白、醇溶蛋白和谷蛋白4类。在种子胚发育过程中, 醇溶蛋白在粗面内质网合成后形成蛋白
质聚集体, 直接出芽形成蛋白体并贮存其中。白蛋白、球蛋白和谷蛋白在粗面内质网以分子量较大的前体形式合成后, 根
据各自的分选信号进入特定的运输囊泡, 经由受体依赖型运输/聚集体形式运输转运至蛋白质贮藏型液泡中, 然后经过液泡
加工酶等的剪切转换为成熟型贮藏蛋白质并贮存其中。蛋白质的合成、分选、转运和加工等过程影响种子蛋白质的品质及
含量。该文对种子贮藏蛋白质的分类和运输、加工以及这些过程对种子蛋白质品质和含量的影响进行了概述。
关键词 蛋白质液泡分选, 种子贮藏蛋白质, 液泡蛋白质的加工, 膜泡运输
韩宝达, 李立新 (2010). 植物种子贮藏蛋白质及其细胞内转运与加工. 植物学报 45, 492–505.
高等植物在种子成熟过程中合成并贮存大量的
贮藏蛋白质(seed storage protein, SSP)。贮藏蛋白
质一般占种子总蛋白含量的80%左右 (Fuji et al.,
2007)。在种子萌发及初期生长过程中, 贮藏蛋白质
逐渐被分解, 产生各种氨基酸, 为植物体的生长提供
重要的氮源、碳源和硫源 (Shewry et al., 1995;
Herman and Larkins, 1999)。某些贮藏蛋白质(如2S
白蛋白)还能够有效地抑制真菌和细菌的生长, 抵抗
植物病原体的侵染 , 在植物体防御中起重要作用
(Terras et al., 1993; Agizzio et al., 2006)。除了上述
生理学功能外, 种子贮藏蛋白质还具有重要的营养和
临床研究价值。例如, 谷类作物为人体提供必需氨基
酸(Shewry, 2007, 2009); 豆类因其总蛋白含量较高
而成为人类重要的蛋白质来源(Duranti et al., 2008)。
但是, 有些贮藏蛋白质属于食物致敏原性蛋白, 威胁
人体健康。如黄芥(Sinapis alba)、巴西果(Bertholletia
excelsa)和蓖麻(Ricinus communis)等种子中的2S白
蛋白均是致敏原性蛋白, 能与过敏病人血清中的IgE
抗体结合 , 引起过敏反应(Moreno and Clemente,
2008)。因此, 2S白蛋白类具有重要的临床研究价值。
贮藏蛋白质的合成、运输、加工和贮存等过程均会影
响种子蛋白质的含量与品质, 所以贮藏蛋白质的研究
能够为改善种子贮藏蛋白质品质和提高蛋白质含量
的分子育种工作提供重要的理论依据。
1 贮藏蛋白质研究历程
贮藏蛋白质在种子中含量丰富且具有重要的经济价
值。早在18世纪中期, 已有研究者开展了贮藏蛋白质
的研究工作(Beccari, 1745)。Osborne(1924)提取出
种子贮藏蛋白质, 并将其分为白蛋白(albumin)、球蛋
白(globulin)、醇溶蛋白(prolamin)和谷蛋白(glutelin)4
类。随后, Osborne、Danielsson、Derbyshire、Shewry
和Muntz研究小组对贮藏蛋白质的分类及结构特征进
行了系统的分析研究(Osborne, 1924; Danielsson,
1949; Boulter et al., 1967; Shewry et al., 1995;
Muntz, 1998)。
随着细胞生物学和生物化学等研究方法的不断
改进, 贮藏蛋白质的细胞内转运与加工等过程的研究
也逐步展开。例如, 1991年, Matsuoka、Neuhaus、
Bednarek和Saalbach揭示了贮藏蛋白质前体中存在
·专题论坛·
韩宝达等: 植物种子贮藏蛋白质及其细胞内转运与加工 493
液泡分选信号, 包括存在于C末端的液泡分选信号
(C-terminal vacuolar sorting determinant, ctVSD)、
序列特异性分选信号 (sequence-specific vacuolar
sorting determinant, ssVSD)和物理结构液泡分选信
号(physical structure VSD, psVSD)(Matsuoka and
Nakamura, 1991; Neuhaus et al., 1991; Bednarek
and Raikhel, 1991; Matsuoka and Neuhaus, 1999)。
随着分选信号本质的揭示, 相继发现了贮藏蛋白质液
泡分选受体。1994年, Rogers研究小组在豌豆(Pisum
sativum)中发现了第1个液泡分选受体——BP-80
(Kirsch et al., 1994), 但该受体主要在溶解型液泡蛋
白分选中起作用。1997年, Hara-Nishimura研究小组
在南瓜(Cucurbita moschata)中发现了另一种液泡分
选受体——PV72(Shimada et al., 1997), 该受体在贮
藏蛋白质和液泡蛋白酶类的分选中发挥重要作用
(Shimada et al., 2002; Watanabe et al., 2004)。同年,
Raikhel在拟南芥(Arabidopsis thaliana)中发现了另
一液泡分选受体——AtELP/AtVSR1(Ahmed et al.,
1997), 该受体也与贮藏蛋白质分选有关。
通常, 蛋白质在内质网上合成后, 经由高尔基体
接受受体分选, 最终到达靶位点。到20世纪末, 人们
一直认为这是蛋白质运输的唯一途径。1998年, Hara-
Nishimura研究小组在种子胚发育过程中的南瓜种子
细胞中发现了前体蓄积小泡(precursor-accumulating
vesicle, PAC)(又称为小泡)从内质网出芽, 且不经由
高尔基体而直接将贮藏蛋白质前体转运到蛋白质贮
藏型液泡 (protein storage vacuole, PSV)(旧称
protein body II, PB II)中, 据此提出了高尔基体非依
赖 型 蛋 白 质 运 输 途 径 (Hara-Nishimura et al.,
1998a)。另外, Chrispeels研究小组还在发育中的豆
类子叶细胞中发现了在高尔基体出芽形成的贮藏蛋
白运输小泡——高密度小泡 (dense vesicle, DV)
(Chrispeels, 1983)。虽然PAC小泡和DV募集贮藏蛋
白质的机制尚不明确, 但二者均不需要受体的分选,
因此被称为聚集体形式运输。从此, 贮藏蛋白质运输
存在2种不同机制(即受体依赖型运输机制和聚集体
形式运输机制)的观点逐渐形成。
另外, Hara-Nishimura和Nishimura研究小组通
过检测成熟型贮藏蛋白质与贮藏蛋白质前体相对量
的变化以及酶活性在时间与空间上的变化等, 提出贮
藏蛋白质前体运输到液泡后接受液泡加工酶剪切的
理论, 并利用生物化学手段进行了验证(Hara-Nishi-
mura and Nishimura, 1987; Hara-Nishimura et al.,
1991; Shimada et al., 2003b)。此后, 贮藏蛋白质的
液泡内加工过程也得到系统地阐述。
2 贮藏蛋白质的分类
Osborne(1924)从脱脂种子提取物中逐步萃取得到了
种子贮藏蛋白质, 并根据溶解性不同将其分为溶于水
的白蛋白、溶于稀盐溶液的球蛋白、溶于乙醇/水混合
物的醇溶蛋白和溶于稀酸或稀碱的谷蛋白(Shewry et
al., 1995; Muntz, 1998; 黄荟等, 2008)。
2.1 2S白蛋白
2S白蛋白最初是根据其沉降系数(S20.W)约等于2命名
的, 其硫氨基酸含量丰富且广泛分布于单子叶和双子
叶植物种子中 , 在十字花科植物 , 尤其是油菜
(Brassica campestris)和拟南芥中已经得到深入的研
究(Youle and Huang, 1981; Moreno and Clemente,
2008)。2S白蛋白以分子量为15–20 kDa的前体蛋白
形式合成, 该前体由N末端的信号肽和前肽、成熟2S
白蛋白大亚基和小亚基的2条多肽链、两肽链间的连
接肽以及C末端的短肽构成, 经蛋白酶水解后可转换
为成熟型蛋白质。典型的成熟2S白蛋白由分子量为
3–5 kDa的小亚基和分子量为8–10 kDa的大亚基组
成 , 二者间通过二硫键连接(Ericson et al., 1986;
Shewry et al., 1995; Tai et al., 2001; Chua et al.,
2008)(图1)。
2.2 球蛋白
球蛋白是分布最广泛的贮藏蛋白质, 不仅分布在单子
叶和双子叶植物中, 在蕨类植物的孢子中也有发现
(Templeman et al., 1987)。球蛋白分子中半胱氨酸和
甲硫氨酸的含量较低, 根据沉降系数的不同可分为
7S vicilin型球蛋白和11S/12S legumin型球蛋白。球
蛋白在内质网上以大分子量的前体形式合成, 绝大多
数种类的前体由N末端的信号肽及成熟7S(或11S/
12S)球蛋白大亚基和小亚基2条多肽链组成(Simon
et al., 1985; Kagawa and Hirano, 1989; Rodin et
al.,1990; Shewry et al., 1995; Tai et al., 2001; Chua
et al., 2008)。成熟7S球蛋白是典型的三聚体蛋白,
494 植物学报 45(4) 2010


图1 贮藏蛋白质前体及其液泡内加工(Shimada et al., 2003b;
Yamada et al., 2005)

Figure 1 Precursors of storage proteins and their vacuolar
processing (Shimada et al., 2003b; Yamada et al., 2005)


分子量约为150–190 kDa(Gatehouse et al., 1982;
Shewry et al., 1995; Jin et al., 2008; Kratzer et al.,
2009)。不同种类的7S球蛋白的亚基组成不同, 主要
是由于翻译后的加工(蛋白酶水解和糖基化)不同造成
的(Shewry et al., 1995)。如豌豆的7S球蛋白最初以
分子量为47 kDa和50 kDa的前体形式合成, 经翻译
后修饰产生分子量为12.5–33 kDa的亚基(Gateho-
use et al., 1982); 而四季豆(Phaseolus vulgaris)的
7S球蛋白则不同, 其糖基化程度更高, 但不进行蛋白
酶水解, 由分子量为43–53 kDa的糖基化亚基组成
(Hall et al., 1977)。11S/12S球蛋白以分子量约为
50–60 kDa的前体形式合成, 经蛋白酶水解形成成熟
的α、β亚基。成熟11S/12S球蛋白通常以六聚体的形
式存在, 每个单体由1个α-β亚基对组成, 二亚基通过
二硫键连接(Staswick et al., 1984; Jung et al., 1998;
Shimada et al., 2003b; Li et al., 2006, 2007; Chua
et al., 2008) (图1)。
2.3 醇溶蛋白
与白蛋白和球蛋白相比, 醇溶蛋白的分布相对狭窄,
仅分布于谷类作物中 , 富含谷氨酰胺和脯氨酸
(Shewry et al., 1995)。醇溶蛋白分为2类: 麦类作物
醇溶蛋白和黍类作物醇溶蛋白(Shewry and Halford,
2002; Sabelli and Larkins, 2009)。麦类作物的醇溶
蛋白是一种高度多态的混合物, 分子量介于30–90
kDa之间(Shewry et al., 1995), 根据氨基酸序列不同
可分为硫富含(S-rich)、硫缺乏(S-poor)和高分子量醇
溶蛋白 3类 (Shewry et al., 1984; Shewry and
Tatham, 1990; Shewry and Halford, 2002)。在黍类
作物中, 对玉米(Zea mays)醇溶蛋白(zein)的研究较
多。根据氨基酸组分的差异, zein分为α-、β-、γ-和
δ-zein 4类, 均富含谷氨酰胺和脯氨酸, 而赖氨酸和
色氨酸的含量较低。其它氨基酸的含量随种类不同有
明显的差异: 在β和γ-zein中, 甲硫氨酸含量丰富; 在
δ-zein中, 半胱氨酸和组氨酸含量丰富(Shewry and
Tatham, 1990)。α-zein包含19 kDa和22 kDa 2种组
分, 占玉米总醇溶蛋白的75%–85%。β-zein由分子量
分别为14 kDa和16 kDa 2种组分组成, 占总醇溶蛋
白的10%–15%。γ-zein分子量为28 kDa, 占总醇溶蛋
白的5%–10%。δ-zein分子量约为14.4 kDa, 在总醇
溶蛋白中所占比例较低(Shewry and Tatham, 1990;
Shewry et al., 1995)。其它的谷类作物 , 如燕麦
(Avena sativa)和水稻(Oryza sativa)也含有少量的醇
溶蛋白。燕麦的醇溶蛋白(avenin)由分子量为20–29
kDa的蛋白质组成; 水稻的醇溶蛋白由10、13和16
kDa三组小分子量蛋白质组成 (Yamagata et al.,
1982; Ogawa et al., 1987; Chesnut et al., 1989)。
2.4 谷蛋白
谷蛋白存在于水稻和小麦(Triticum aestivum)等谷类
作物的种子中。水稻谷蛋白以分子量为57 kDa的前体
形式合成, 在内质网腔内切除信号肽后装配形成三聚
体 , 运送到PSV后 , 经蛋白酶水解为成熟的37–39
kDa的α亚基和19–20 kDa的β亚基, 并贮藏于PSV中
(Yamagata et al., 1982; Wang et al., 2009)。小麦谷
蛋白(glutenin)由低分子量的亚基和高分子量的亚基
组成, 各亚基间通过二硫键连接, 形成分子量较大的
多聚体(Muntz, 1998)。
3 贮藏蛋白质的细胞内转运
贮藏蛋白质最初在粗面内质网上以前体的形式合成,
然后根据各自的特性, 从内质网经由多条途径运输至
韩宝达等: 植物种子贮藏蛋白质及其细胞内转运与加工 495
蛋白体(protein body, PB)(旧称PB I)或蛋白质贮藏型
液泡中。
3.1 贮藏蛋白质分选信号
含有液泡分选信号的贮藏蛋白质, 在内质网中完成合
成过程后, 根据各自的分选信号进入特定的运输囊
泡, 最终被转运到PSV。到目前为止, 已发现3种液泡
分选信号: 序列特异性液泡分选信号(ssVSD)、C末端
液泡分选信号(ctVSD)和物理结构型液泡分选信号
(psVSD)。ssVSD最初是在甘薯 (Dioscorea escu-
lenta)贮藏蛋白(sporamin)的前体中发现的, 蓖麻2S
白蛋白和蓖麻蛋白 (ricin)也包含ssVSD(Matsuoka
and Nakamura, 1991; Brown et al., 2003; Jolliffe et
al., 2004)。ssVSD含有NPIXL/NPIR功能序列, 该序
列的缺失或突变均会导致贮藏蛋白分泌到细胞外空
间(Matsuoka and Nakamura, 1991; Nakamura and
Matsuoka, 1993; Matsuoka and Neuhaus, 1999;
Brown et al., 2003; Jolliffe et al., 2004)。另外, 把
sporamin N末端的NPIR序列转移到C末端, ssVSD仍
能发挥分选作用(Koide et al., 1997), 说明ssVSD序
列的保守性较其在蛋白质中的位置对于贮藏蛋白质
的正确分选更为重要。
另一种液泡分选信号为C末端液泡分选信号
(ctVSD), 最初是在大麦 (Hordeum vulgare)凝集素
(lectin)和烟草(Nicotiana tabacum)壳多糖酶(chitin-
ase)中发现的(Bednarek and Raikhel, 1991; Neu-
haus et al., 1991)。ctVSD由C末端疏水性前肽组成,
序列保守性较低, 但ctVSD必须暴露在蛋白质C末端
才有功能, 当蛋白质C末端糖基化或ctVSD后连接1
个或多个甘氨酸均会导致蛋白质的分泌(Bednarek
and Raikhel, 1991; Neuhaus et al., 1991; Matsuoka
and Neuhaus, 1999; Koide et al., 1999; Vitale and
Hinz, 2005; Jolliffe et al., 2005)。此外, 巴西果2S白
蛋白C末端含有IAGF功能序列的16个氨基酸、豆类
7S球蛋白 (phaseolin)的AFVY序列、大豆 (Glycine
max) 球 蛋 白 (β-conglycinin)α 亚 基 C 末 端 的
PLSSILRAFY序列、大豆球蛋白(glycinin)A1aB1b亚基
C末端的10个氨基酸以及苋属(Amaranthus)植物11S
球蛋白C末端的KISIA序列均属于ctVSD(Saalbach et
al., 1996; Frigerio et al., 1998; Nishizawa et al.,
2003; Maruyama et al., 2006; Petruccelli et al.,
2007)。
除了上述2种液泡分选信号外, 某些贮藏蛋白质
还含有物理结构型液泡分选信号(psVSD)。psVSD的
具体特征目前尚不清楚, 可能是由多个结构域组成的
“信号斑(signal patch)”, 如豌豆11S球蛋白的正确
分选需要完整的α链存在(Saalbach et al., 1991)。
psVSD发挥作用依赖于贮藏蛋白质内在序列的完整
性, 这种完整性保证了蛋白质分子能够形成正确的构
象, 从而形成“信号斑”(Saalbach et al., 1991; Vitale
and Chrispeels, 1992; Muntz, 1998; Matsuoka and
Neuhaus, 1999; Hwang, 2008)。
3.2 贮藏蛋白质分选受体
参与液泡分选过程的跨膜受体称为液泡分选受体
(vacuolar sorting receptor, VSR)。一些可溶性液泡蛋
白质, 如某些贮藏蛋白质和水解酶, 通过自身液泡分
选信号与VSR相互作用, 然后被分选进入PSV中。

3.2.1 南瓜PV72/拟南芥AtVSR1/豌豆BP-80
PV72是Hara-Nishimura研究小组在南瓜种子胚发育
过程中的种子细胞的PAC小泡上发现的I型整合膜蛋
白, 该膜蛋白由腔内区、跨膜区和胞质部分组成, 在
腔内区的C末端含有3个表皮生长因子样(EGF-like)
重复功能序列(Shimada et al., 1997)。在Ca2+存在的
条件下, PV72能够与南瓜2S白蛋白前体中含有液泡
分选信号的C末端相互作用。亚细胞定位表明, PV72
在高尔基体上也有分布, 由此可以推测PV72在高尔
基体和PAC小泡间循环, 负责募集运输到高尔基体
的贮藏蛋白质前体并将其转运至 PAC小泡中
(Shimada et al., 2002)。此外, PV72的腔内部分也可
结合溶解型液泡水解酶――半胱氨酸蛋白酶AtALEU
的前体 , 在该酶分选进入溶解型液泡中起作用
(Watanabe et al., 2004), 因此, PSV和溶解型液泡分
选受体很可能通过相同的识别机制结合分选信号。
PV72在拟南芥中有7个同源蛋白质 , 分别命名为
AtVSR1–7(Shimada et al., 2003a)。AtVSR1–7中与
PV72同源性最高的是AtVSR1, 主要分布于高尔基体
和前液泡体(prevacuolar compartment, PVC)上。在
Ca2+存在的条件下, AtVSR1能够与12S球蛋白的C末
端肽链结合, 在拟南芥种子胚发育过程中的种子细胞
中负责将贮藏蛋白质从高尔基体转运至PVC(Ahmed
496 植物学报 45(4) 2010
et al., 1997; Li et al., 2002; Shimada et al., 2003a;
Fuji et al., 2007)。贮藏蛋白质释放到PVC后, AtVSR1
返回高尔基体进行下一个蛋白质分选循环。与贮藏蛋
白质相比, AtVSR受体的数量相对较少, 因此AtVSR
受体的循环利用机制很重要。AtVSR的循环受
Retromer 复 合 体 的 调 控 。 MAIGO1(MAG1) 和
AtVPS35是Retromer复合体中的成员 , 参与受体
AtVSR从PVC回收到高尔基体的循环利用过程
(Shimada et al., 2006; Yamazaki et al., 2008)。
BP-80是PV72的另一同源物, 最初是在豌豆种
子胚发育过程中的种子子叶细胞的网格蛋白有被小
泡(clathrin-coated vesicle, CCV)上发现的, 因其分
子量约等于80 kDa而命名(Kirsch et al., 1994)。
BP-80能够与包含NPIR液泡分选信号的半胱氨酸蛋
白酶前体(proaleurain)相互作用。在纯化的网格蛋白
有被小泡中, BP-80含量丰富, 而在负责贮藏蛋白质
运输的DV中含量却很低(Kirsch et al., 1994; Hinz et
al., 1999), 因此, BP-80很可能在蛋白质的溶解型液
泡转运过程中起重要作用。

3.2.2 RMR受体(receptor homology region-trans-
membrane domain-ring H2 motif protein)
Jiang 等 (2000) 在 烟 草 种 子 PSV 内 晶 体
(crystalloid)的系统发生研究中发现了一个新型跨膜
蛋白, 将其命名为RMR。RMR由信号肽、N端腔内区
域、跨膜区和胞质ring H2 finger结构域组成。腔内区
可能与货物分子相互作用, 而胞质部分可能与运输相
关因子相互作用(Jiang et al., 2000; Park et al.,
2005; Hinz et al., 2007)。RMR主要分布于PVC, 在
高尔基体中分布较少(Hinz et al., 2007)。免疫共沉淀
和体外结合实验表明, RMR腔内区域在酸性条件下能
够与菜豆(Phaseolus vulgaris)球蛋白(phaseolin)C末
端的信号肽相互作用(Park et al., 2005)。因此, RMR
很可能是贮藏蛋白质的受体, 不过这一观点还需要通
过对RMR功能缺失型突变体的分析来验证。
3.3 贮藏蛋白质的细胞内转运
高等植物的种子贮藏蛋白质贮存于2种不同的细胞区
室, 一种是蛋白体(protein body, PB), 另一种是蛋白
质贮藏型液泡(PSV)(图2)。PB主要贮存谷类作物, 如
玉米和水稻等的醇溶蛋白。醇溶蛋白在粗面内质网上
合成后, 在BiP等分子伴侣的协助下装配形成蛋白质
聚集体, 直接形成PB。在玉米和水稻的胚乳中, PB与
内质网并未完全脱离, 电镜下观察玉米胚乳细胞可发
现内质网膜的末端与PB连接(Larkins and Hurkman,
1978; Ogawa et al., 1987; Li et al., 1993a, 1993b;
Herman and Larkins, 1999)。在转基因水稻中, 醇溶
蛋白与绿色荧光蛋白 (green-fluorescent protein,
GFP)的融合蛋白在种子胚乳中形成PB, 在叶片和根
中形成类似于PB的高电子密度结构 (Saito et al.,
2009)。在小麦中, PB直接从内质网上出芽形成独立
的结构, 最初形成的PB直径约为1–2 μm, 随后PB体
积增大且合并现象显著, 并最终在细胞中形成蛋白质
基质(Herman and Larkins, 1999; Loussert et al.,
2008)。PSV主要贮存谷蛋白、球蛋白和白蛋白, 如
水稻谷蛋白、小麦谷蛋白、蓖麻2S白蛋白以及大豆和
豌豆的11S球蛋白等, 这些蛋白在内质网上以前体的
形式合成后, 根据各自的信号序列被分选和运输, 并
最终进入PSV中(Yamagata et al., 1982; Okita and
Rogers, 1996; Brown et al., 2003; Maruyama et al.,
2006; von Lüpke et al., 2008; Tosi et al., 2009)。
贮藏蛋白质运输至PSV的机制大体上可分为2类
(图2), 即受体依赖型运输(receptor-mediated sort-
ing)和聚集体形式运输(aggregation sorting) (Hara-
Nishimura et al., 2004; Harasaki et al., 2005; Jolliffe
et al., 2005; Vitale and Hinz, 2005; Robinson et al.,
2005; Li et al., 2006; Fuji et al., 2007; Hwang,
2008), 二者的主要区别在于是否需要受体。在受体
依赖型运输中, 贮藏蛋白质在内质网上合成后被运输
至高尔基体, 在那里经过受体的分类进入特定的运输
囊泡, 然后被运输至PSV。体外分析表明, 蓖麻2S白
蛋白和蓖麻蛋白分子的ssVSD信号能够与VSR/BP-
80样蛋白质相互作用; 免疫电镜和细胞组分分析表
明, 蓖麻种子中的2S白蛋白和蓖麻蛋白均通过高尔
基体转运至PSV, 在转运过程中二者具有相同的定
位, 因此它们很可能在反面高尔基网络(trans-Golgi
network, TGN)由VSR/BP-80受体分选, 然后转运到
PVC(Jolliffe et al., 2004)。另外, 一些含有ctVSD的
贮藏蛋白质也通过受体依赖型途径运输, 如拟南芥
12S球蛋白在高尔基体与AtVSR1结合后, 经由PVC
转运到PSV。受体在运输中起重要作用, 如AtVSR1
的缺失会导致大量贮藏蛋白质分泌到细胞外空间
韩宝达等: 植物种子贮藏蛋白质及其细胞内转运与加工 497


图2 种子发育过程中贮藏蛋白质细胞内运输机制的多样性
Y: AtVSR1受体

Figure 2 Diversity of intracellular transport mechanisms of
storage proteins during seed maturation
Y: AtVSR1 receptor

(Shimada et al., 2003a; Fuji et al., 2007)。调控
AtVSR1循环利用的因子, 如MAG1和AtVPS35缺失,
也会导致贮藏蛋白质分泌到细胞外空间(Shimada et
al., 2006; Yamazaki et al., 2008)。在受体依赖型分
选中, 除了受体起关键作用外, 还有许多相关因子也
起决定性作用。例如, Li等(2006)分离出的MAG2因
子可调控贮藏蛋白质从内质网的运出。当MAG2缺失
时, 贮藏蛋白质前体不能有效地从内质网运出, 致使
由前体组成的新型细胞结构——MAG2 Body形成。此
外, 调控内体和种子胚胎形成的KATAMARI2 (KA-
M2)因子、调节液泡形成以及种子成熟的VAMP727/
SYP22 SNARE复合体、调控TGN和PSV间运输的
VTI12/SYP4 SNARE复合体等均在受体依赖型运输
中起重要作用(Bassham et al., 2000; Sanderfoot et
al., 2001; Tamura et al., 2007; Sanmartin et al.,
2007; Ebine et al., 2008)。
贮藏蛋白质运输的另一类机制是聚集体形式运
输, 是指贮藏蛋白质在内质网腔或高尔基体囊里形成
蛋白质聚集体, 不需要受体的分类直接从内质网或高
尔基体出芽形成运输小泡 , 然后直接到达PSV
(Hara-Nishimura et al., 1998a; Robinson and Hinz,
1999; Hinz et al., 1999; Shimada et al., 2002,
2003a; Fuji et al., 2007)。参与聚集体形式分选的运
输囊泡主要包括内质网起源的PAC小泡和高尔基体
起源的DV。PAC小泡主要成分是贮藏蛋白质前体, 直
径为200–400 nm, 具有高电子密度的核心, 外周由
低电子密度层包围, 一些PAC小泡内还包含小的囊
泡状结构(Hara-Nishimura et al., 1998a)。PAC小泡
从内质网出芽, 将贮藏蛋白质前体直接转运到PSV。
在运输过程中, PAC小泡还可接受高尔基体内的糖基
化蛋白(Hara-Nishimura et al., 1998a; Jolliffe et al.,
2005)。除南瓜外, PAC小泡在蓖麻、大豆和水稻中也
有发现(Hara-Nishimura et al., 1998a; Mori et al.,
2004; Takahashi et al., 2005)。这表明PAC小泡依赖
型运输途径是高等植物种子细胞中普遍存在的机制。
DV最早是在豆类种子细胞中发现的 (Chrispeels,
1983)。随后, 在水稻(Krishnan et al., 1986)、豌豆
(Craig and Goodchild, 1984)和拟南芥(Hinz et al.,
2007)中也有发现。DV没有衣被蛋白(coat protein,
COP)包被, 直径约为100–200 nm, 因其含有高电子
密度的嗜锇性组分而得名(Hohl et al., 1996; Hinz et
al., 1999; Robinson and Hinz, 1999; Hillmer et al.,
2001)。DV最初在高尔基体顺面膜囊的边缘形成, 随
着从顺面膜囊向反面膜囊的转移, 其密度逐渐增大,
最终从高尔基体TGN出芽 , 将贮藏蛋白质转运至
PVC, 再转运至PSV(Robinson and Hinz, 1999;
Hillmer et al., 2001; Robinson et al., 2005; Hinz et
al., 2007; Hwang, 2008)。在种子胚发育这个特定时
期内, 随着大量的贮藏蛋白质在内质网上不断合成,
高效的运输方式可减少细胞的能量消耗, 降低运输成
本, 而聚集体形式运输无疑满足了这种需求。
4 贮藏蛋白质的加工
贮藏蛋白质前体被转运到PSV后, 经过液泡加工酶
(vacuolar processing enzyme, VPE)的剪切转换为
成熟型的贮藏蛋白质。这些加工过程可能会改变蛋白
质的构象, 以利于其在PSV内的沉积。
4.1 液泡加工酶
液泡加工酶最初是在南瓜种子胚发育过程中的种子
中发现的(Hara-Nishimura and Nishimura, 1987)。随
后, 从蓖麻种子中也提取出了VPE(Hara-Nishimura
et al., 1991)。该酶属于半胱氨酸蛋白酶类, 具有严格
498 植物学报 45(4) 2010
的天冬酰胺 (Asn, N)和天冬氨酸 (Asp, D)特异性
(Shimada et al., 2003b; Yamada et al., 2005)。根据
cDNA推导的氨基酸序列表明, VPE由4部分组成: N
末端信号肽、N末端前肽、成熟VPE部分和C末端前
肽(Shimada et al., 2003b)。VPE前体在内质网中切
除信号肽后, 以非活化状态的前体形式运输到PSV
中, PSV的酸性环境使VPE前体发生自身剪切, 成为
有活性的成熟酶(Hiraiwa et al., 1997, 1999; Kuroy-
anagi et al., 2002; Nakaune et al., 2005)。 植物VPE
可分为3个亚家族 , 即种子型、营养器官型和新型
VPE(Hara-Nishimura et al., 1998b; Yamada et al.,
2005; Nakaune et al., 2005)。研究得比较广泛的VPE
有αVPE、βVPE和γVPE。βVPE主要存在于种子中,
在贮藏蛋白质的加工过程中起主要作用。拟南芥
βVPE缺失突变体中, 大量贮藏蛋白质以前体形式蓄
积在PSV中(Kinoshita et al., 1995a, 1995b, 1999;
Shimada et al., 2003b)。拟南芥βVPE在水稻及太阳
花(Helianthus annuus)中的同源物对谷蛋白和2S白
蛋白的加工也起重要作用(Molina et al., 2008; Wang
et al., 2009)。αVPE和γVPE主要存在于营养器官中,
在贮藏蛋白质的加工过程中也起一定作用。在βVPE
缺失的突变体中, αVPE和γVPE能够对贮藏蛋白质前
体进行剪切, 但不能完全弥补βVPE缺失造成的影响,
结果仍然有大量的贮藏蛋白质前体蓄积。在αvpe-1/
βvpe-3/γvpe-1三重突变体中无法检测到VPE活性 ,
该三重突变体种子中绝大多数贮藏蛋白质前体不能
正确剪切(Shimada et al., 2003b)。另一VPE家族成
员δVPE在发育中的种子和萌发种子的种皮中表达,
是新型亚家族成员 , 具有半胱氨酸蛋白酶 -1活性
(Gruis et al., 2002)。αvpe/βvpe/ γvpe/δvpe四重突变
体中, 所有贮藏蛋白质前体内保守的天冬酰胺残基位
点均未被VPE剪切(Gruis et al., 2004)。 除了参与贮
藏蛋白质加工剪切外, αVPE、γVPE和δVPE还参与细
胞程序性死亡(Kinoshita et al., 1999; Hatsugai et al.,
2004; Hara-Nishimura et al., 2005; Kuroyanagi et
al., 2005; Lam, 2005; Nakaune et al., 2005; Bon-
neau et al., 2008)。
4.2 贮藏蛋白质前体在液泡内的加工
PSV中贮藏蛋白质的加工过程包括去除前体N末端和
C末端的前肽以及切除链间的连接肽, 最终形成具有
正确构象的成熟蛋白质(图1)。拟南芥2S白蛋白前体
(分子量为15–18 kDa)在内质网中切除信号肽后被运
输到PSV, 由VPE剪切形成2个亚基, 再由半胱氨酸
蛋白酶(cysteine protease, AP)修整形成成熟型小亚
基和大亚基 , 二者通过二硫键连接(Ericson et al.,
1986; Shewry et al., 1995; Tai et al., 2001; Shimada
et al., 2003b; Yamada et al., 2005; Chua et al.,
2008)。拟南芥12S球蛋白前体(分子量为49–54 kDa)
在内质网中切除信号肽后, 分子内部形成二硫键。运
输到PSV后, 在VPE的作用下产生α亚基和β亚基, 二
者也通过二硫键连接(Muntz, 1998; Shimada et al.,
2003b; Gruis et al., 2004; Li et al., 2006)。水稻谷蛋
白首先以前体(分子量为59 kDa)形式在内质网膜上合
成, 去除信号肽并在高尔基体中修饰后形成分子量为
57 kDa的前体, 然后由VPE剪切形成分子量为37–39
kDa的 α亚基和分子量为 19–20 kDa的 β亚基
(Yamagata et al., 1982; Wang et al., 2009)。
5 贮藏蛋白质品质的影响因素
贮藏蛋白质合成后, 在分子伴侣的协助下正确折叠并
被运输到PSV, 经过VPE等的加工剪切形成正确的构
象而沉积。每个环节中涉及的相关因子突变均可能导
致贮藏蛋白质以不正确的构象(或前体)形式沉积或错
误地分泌到细胞外部, 最终导致种子整体蛋白质品质
和含量下降。
Takemoto等(2002)分离得到的水稻esp2突变体
中, 由于蛋白二硫键异构酶(protein disulfide isom-
erase, PDI)的缺失, 导致种子细胞中谷蛋白前体与
醇溶蛋白不能正常分离, 两者间形成异常的链间二硫
键, 共同沉积在形态异常的蛋白体中(图3)。在Li等
(2006)分离出的mag2和mag1突变体种子细胞中, 贮
藏蛋白质前体异常蓄积, 蛋白质品质受到严重影响。
mag2突变体中, 由于运输受阻, 贮藏蛋白质前体滞
留在内质网中并形成MAG2 Body, 内质网分子伴侣
免疫球蛋白重链结合蛋白(immunoglobulin heavy-
chain-binding protein, BiP)和PDI含量也异常上升(Li
et al., 2006); mag1由于育性低下, 导致种子产量也
明显下降(Shimada et al., 2006)。在atvsr1、atvps35、
gfs和vamp727 syp22等突变体种子细胞中, 贮藏蛋
白质被分泌到细胞外, 形成高电子密度的细胞外空间
韩宝达等: 植物种子贮藏蛋白质及其细胞内转运与加工 499


图3 贮藏蛋白质运输通路及其对蛋白质品质的影响
PDI: 蛋白二硫键异构酶

Figure 3 Transport pathways of storage proteins and their
effects on protein qualities
PDI: Protein disulfide isomerase


(Shimada et al., 2003a; Fuji et al., 2007; Yamazaki
et al., 2008; Ebine et al., 2008)。kam2突变体种子中
贮藏蛋白质前体蓄积并被分泌到细胞外空间, 种子胚
胎的形成也受到影响(Tamura et al., 2007)。在VPE
缺失突变体中, 贮藏蛋白质以前体的形式蓄积在PSV
中(Shimada et al., 2003b; Yamada et al., 2005)。总
之, 成熟贮藏蛋白质的形成要经历合成、细胞内运输
和液泡内加工等过程, 任何一个环节出现问题均可能
影响种子蛋白质的品质和含量。
6 问题与展望
种子贮藏蛋白质不仅具有重要的生理学功能, 而且具
有重要的经济价值, 因此, 贮藏蛋白质的研究早在18
世纪已经开始。目前, 关于贮藏蛋白质的基本特性研
究已取得长足进展, 对PSV内的加工过程也有了一定
程度的了解。随着一些模式植物(如拟南芥和水稻等)
全基因组测序的完成, 贮藏蛋白质的运输过程研究也
由最初的形态描述阶段转向分子机制研究。目前, 在
植物细胞中已经鉴定出一些贮藏蛋白质细胞内转运
相关基因, 如MAG2、MAG1、AtVSR1、AtVPS35、
GFS、SYP22和KAM2等, 并进行了初步的特征和功
能分析, 为阐明蛋白质运输通路的分子机制奠定了基
础。然而, 关于这些新型因子的作用机制还不十分清
楚。例如, MAG2在哺乳动物和酵母中的同源蛋白
RINT-1和Tip20p以复合体的形式在内质网与高尔基
体间的膜泡运输中发挥作用 (Sweet and Pelham,
1993; Lewis et al., 1997; Hirose et al., 2004;
Arasaki et al., 2007; Aoki et al., 2009); 作为同源蛋
白, MAG2在植物细胞中与哪些因子形成复合体, 它
的上下游因子有哪些尚不清楚。另外, RINT-1在细胞
分裂间期参与囊泡运输, 进入分裂期后, 它又对调控
G2/M checkpoint起重要作用(Xiao et al., 2001); 而
MAG2是否像RINT-1一样具有双重功能也是一个值
得探索的问题。再如, KAM2不仅参与蛋白质的运输,
而且还调节内涵体以及胚胎的形成(Tamura et al.,
2007), 这些功能有怎样的内在联系？也是一个十分
有趣的问题。由于贮藏蛋白质的细胞内转运是一个持
续的动态过程, 运输途径比较复杂且相互间有交叉,
贮藏蛋白质的细胞内转运全貌尚不清晰, 如贮藏蛋白
质合成的基因水平的调控、运输小泡的形成、囊泡运
输方向的决定、各细胞器间的顺行和逆行运输、膜泡
运输所需膜和功能蛋白等的循环以及运输小泡与靶
细胞器间相互识别机制等, 以上问题在很大程度上还
不清楚。
此外, 对于植物来说, 蛋白质运输与许多生命活
动密切相关。例如, 在植物的生长发育和生殖、激素
介导的信号转导以及生物和非生物因素胁迫响应等
过程中, 膜泡运输均起到关键作用(Lukowitz et al.,
1996; Lauber et al., 1997; Leyman et al., 1999;
Leshem et al., 2006; Bao et al., 2008; Hardham et
al., 2008)。在上述过程中, 蛋白质运输保证了各种细
胞器蛋白质时间和空间上的正确定位, 以维持细胞正
常的生命活动(Geldner, 2004; Surpin and Raikhel,
2004; Leshem et al., 2006; Robatzek, 2007)。在细
胞和分子水平阐释蛋白质运输与这些生命活动的相
关性, 将是极具挑战性的课题。
致谢 感谢东北林业大学阎秀峰教授和戴绍军教授
对本文提出的宝贵修改意见。
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Seed Storage Proteins and Their Intracellular Transport and
Processing
Baoda Han, Lixin Li*
Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field (SAVER), Ministry of Education, Northeast For-
estry University, Harbin 150040, China
Abstract Higher plants accumulate large quantities of storage proteins in seeds as sources of nutrition for germination
and early growth during seed maturation. Seed storage proteins are classified into four groups, albumins, globulins,
prolamins and glutelins, on the basis of their solubility. During seed embryo development, prolamins form aggregates after
being synthesized on the endoplasmic reticulum (ER) and bud to form a protein body and are deposited in it. Albumin,
globulin and glutelin proteins are synthesized as large precursors on the ER and then are sorted into various vesicles
according to their sorting signals. The precursors are transported into protein storage vacuoles (PSVs) by recep-
tor-mediated sorting and/or aggregation sorting mechanisms. Finally, the precursors are processed into mature forms by
vacuolar processing enzymes and are deposited in PSVs. All the processes of synthesis, sorting, transport and process-
ing affect the quality and quantity of seed proteins. We give an overview of the classification, transport and processing of
the seed storage proteins and the effects of these processes on the quantity and quality of seed proteins.
Key words protein vacuolar sorting, seed storage proteins, vacuolar protein processing, vesicular transport
Han BD, Li LX (2010). Seed storage proteins and their intracellular transport and processing. Chin Bull Bot 45, 492–505.
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* Author for correspondence. E-mail: lixinli1@gmail.com
(责任编辑: 孙冬花)