水稻粒型基因克隆和调控机制研究进展

刘喜 牟昌铃 周春雷 程治军 江玲,* 万建民,



水稻粒型基因克隆和调控机制研究进展

刘喜1牟昌铃1周春雷1程治军2江玲1,*万建民1,2

(1南京农业大学 作物遗传与种质创新国家重点实验室, 南京 210095;2中国农业科学院 作物科学研究所/农作物基因资源与基因改良国家重大科学工程, 北京 100081;*通讯联系人, E-mail: jiangling@njau.edu.cn)

水稻粒型是影响其产量和品质的重要性状，阐明其遗传调控机理，有助于提高水稻单产和改良品质。水稻粒型性状主要包括粒长、粒宽、粒厚、长/宽比，属于数量性状，受胚、胚乳及母体植株等不同遗传体系的控制。随着水稻功能基因组学和重测序技术的快速发展，目前已经定位超过400个与水稻粒型相关的数量性状位点(QTL)，并已克隆了60个水稻粒型基因，涉及植物激素、泛素-蛋白酶体通路、丝裂原活化蛋白激酶(MAPK)信号通路、G蛋白信号通路及表观修饰等多个调控通路。本文对水稻粒型基因克隆及其调控机制的研究进展进行了系统总结和梳理，并对这些基因在育种上的利用价值进行了评价。

水稻；粒型；基因；调控机制

矮秆基因及杂种优势的利用实现了水稻产量的两次跨越，不过随着人口的增长，耕地面积的刚性下降，如何保证我国粮食安全仍是当前面临的重要挑战。水稻粒型不仅是重要产量性状千粒重的决定因素之一，还影响到稻米的外观品质和商品价值。在中国南方、美国和大多数亚洲国家，人们更习惯长或细长型稻米，而韩国、日本、中国北方和斯里兰卡则习惯短圆型稻米[1-3]。此外，粒型也是进化中一个易于被选择的表型，是研究水稻进化的一个重要性状[4]。因此，克隆粒型相关的基因并深入研究和阐明粒型形成的基因调控网络，可为水稻高产、优质分子育种提供重要的理论基础和基因资源。在本文中，笔者对前人的研究结果进行了系统汇总分析，以期为水稻粒型的分子育种提供帮助。

1 水稻粒型性状的遗传特点与其关键基因克隆

水稻种子的发育从双受精开始，精细胞和卵细胞结合发育成二倍体的胚，精细胞和两个极核结合发育成三倍体的胚乳，颖花中的子房壁发育成果皮，珠被发育成种皮。胚和胚乳为子代组织，其发育需要母体组织提供营养。因此，种子的体积受到胚、胚乳及母体植株等不同遗传体系的控制。

水稻的粒型性状包括粒长、粒宽、粒厚及长宽比，与粒重呈正相关[5-6]。其遗传受多基因控制[5-7]。迄今，检测到的控制粒型的QTL已超过400个，遍布水稻12条染色体。其中位于第2、第3和第5染色体上的QTL数较多，分别为58、106和55[7-9]。这3条染色体上克隆出的基因数也最多，在已克隆出的60个粒型基因中, 位于第2和第3染色体的基因各8个，第5染色体上的基因9个(图1，表1)。

2 水稻粒型基因调控机制

粒型的形成涉及复杂的遗传调控网络，自细胞增殖或伸长开始，直至完成籽粒的灌浆过程为止[8-9]。对已克隆的控制粒型的60个基因进行总结分析，发现这些基因主要涉及包括植物激素、泛素-蛋白酶体通路、MAPK信号、表观修饰、G-蛋白信号等分子路径(图2)。涉及MAPK信号、G蛋白信号和泛素-蛋白酶体通路的基因是通过控制细胞增殖，进而影响水稻粒型，而植物激素、表观修饰等通路同时影响细胞增殖和扩张，最终控制粒型。越来越多的研究表明不同粒型调控路径之间存在互作调控关系[9]。例如，G蛋白α亚基，其突变体对BR不敏感，表明参与G蛋白信号与BR信号转导途径共同调控水稻粒型[60-63]。受甲基化调控的，其突变体表现BR相关突变体的表型，表明表观修饰与BR途径存在联系[16]。

2.1 植物激素

2.1.1 油菜素内酯(BR)

油菜素内酯(BR)是调控植物生长发育的重要激素[97]。已报道一些BR合成和信号基因的突变会影响水稻籽粒体积，如、、、、等(表1)。这些基因突变使籽粒变短、植株矮化、叶倾角变小。其中，和编码细胞色素P450加氧酶，正向调节细胞伸长，参与油菜素内酯的合成[10-11,47-48]。编码与拟南芥同源的BR信号激酶，包含一个激酶结构域和一个C端的TPR结构域，是BR信号的正向调控因子，是磷酸化底物，此外，的TPR结构域与其激酶结构域互作，阻止与()互作[50]。Feng等[81]克隆了控制粒型的基因它编码一个类BAHD酰基转移酶，抑制细胞伸长，参与介导油菜素内酯稳态的调控，其突变引起籽粒变长、叶倾角变大。Jiang等[46]克隆了一个控制器官体积的基因，它编码一个与有33%同源性的LRR激酶结构域蛋白，该基因突变，导致细胞分裂速度下降，细胞数目减少，器官变小，突变体内源BR的含量降低，根、胚芽鞘组织伸长对BR的响应增强。Hu等[26]克隆了一个水稻粒长基因，编码生长调节因子，促进细胞分裂和扩张，从而调节粒长和粒宽。Che等[27-28]研究发现/与BR信号转导通路的负调控因子直接互作，其转录激活活性受抑制，其表达受调节。

图1 已克隆的控制水稻粒型的基因在12条染色体上的分布

Fig. 1. Distribution of cloned genes for grain shape on the 12 chromosomes in rice.

表1已克隆的控制水稻粒型的基因

Table 1. Cloned genes for grain shape in rice.

水稻粒型是由5个主要的信号途径调控，包括激素、泛素-蛋白酶体通路、MAPK信号、表观修饰与G蛋白信号转导。这些调控因子通过影响细胞增殖和扩张来控制粒型。

Fig. 2. Major pathways of rice grain shape regulation.

2.1.2 生长素(IAA)和细胞分裂素(CK)

是一个生长素响应基因，是生长素响应和转运的正调控因子，促进颖壳细胞伸长与扩张，调控植物的器官体积[30]。控制水稻粒重及灌浆速率的主效QTL，编码IAA-葡萄糖水解酶，能水解IAA-葡萄糖成游离的IAA和葡萄糖，在胚乳发育过程中调控生长素平衡[64]。编码一个生长素响应因子，负向调节细胞伸长，受生长素和BR诱导，与编码油菜素内酯受体基因的启动子结合，直接影响的表达，过表达导致转基因植株小粒、矮秆、窄叶和叶倾角增大[69]。

调控水稻粒长、穗粒数和芒长的基因，编码一个表皮模式因子类蛋白EPFL1，通过激活和的表达降低植株內源细胞分裂素的含量, 从而调控粒长、穗粒数和芒发育[86]。

2.2 泛素-蛋白酶体

泛素-蛋白酶体途径(ubiquitin-26S proteasome pathway)主要由泛素活化酶(ubiquitin-activating enzyme, E1)、泛素结合酶(ubiquitin-conjugating enzyme, E2)、泛素蛋白连接酶(ubiquitin protein ligase, E3)和26S蛋白酶体组成。泛素化过程先是E1激活泛素分子，并把激活的泛素连接到E2上，此过程需要ATP提供能量，E3识别靶蛋白，促进E2将泛素转移到靶蛋白上，泛素化水平达到一定程度，靶蛋白就被运输到26S蛋白酶体进行降解[98]。控制水稻粒宽的基因，编码一个环型的E3泛素连接酶，与拟南芥和小麦同源[23-25]。将其底物锚定到蛋白酶体进行降解，从而负调节细胞分裂。该基因突变使得本应降解的底物不能被特异识别降解，从而激活颖花外壳细胞的分裂，进而增加颖花外壳的宽度，另外灌浆速率也得到了提高，胚乳也随之增大，最终使粒宽、粒重以及产量都得到提高[23]。可能通过参与泛素蛋白酶体途径来调控粒型[52-53]，但最新的结果表明，该基因是通过影响BR信号途径基因的磷酸化来实现对粒型的调控(刘家范等，未发表资料)。编码一个有功能的U-box E3泛素连接酶，且与存在遗传互作，上位于[45]。促进细胞分裂，突变体对BR不敏感，表现为粒变短、第2节间变短、叶片直立，这说明水稻存在-介导的BR信号途径[45]。调控水稻抽穗期和粒重的基因(), 编码一个含有泛素相关结构域的蛋白，正向调控细胞增殖，与泛素通路上各组分的基因存在共表达，说明其可能通过泛素途径调控水稻抽穗期和粒重[70]。

2.3 MAPK信号

丝裂原活化蛋白激酶(MAPK)是一系列细胞内级联反应的成分，能响应多种胞外刺激，MAPK信号转导以三级激酶级联的方式进行，首先MAPKKK(MAP kinase kinase kinase)受有丝分裂原刺激磷酸化而激活，在此基础上MAPKKK进而磷酸化激活MAPKK(MAP kinase kinase)，最后由MAPKK 磷酸化MAPK，使其活化进而转入核内，从而参与细胞生长、发育、分裂和分化等多种发育过程，生物体内重要的信号转导途径之一[99]。()编码一个水稻丝裂原活化蛋白激酶激酶，促进细胞细胞分裂，该基因突变导致籽粒变小，株高变矮，穗形直立、密穗[19]。()编码一个与拟南芥同源的丝裂原活化蛋白激酶()，具有磷酸化活性，该基因突变导致籽粒变小、千粒重降低，植株矮化，节间缩短，叶片直立。与互作，可能位于下游[71]。BR信号途径与MAPK信号途径在功能上存在交叉(Crosstalk)，如突变体对BR敏感性降低，且其内源BR水平降低，可能也参与影响BR稳态以及信号途径，在种子生长发育中可能作为MAPK通路和BR信号间的连接因子[71]。在拟南芥中BR信号负调控因子磷酸化，进而抑制其下游MAPK活性[100]。水稻的同源基因是否能磷酸化, 进而抑制活性，这需要进一步研究。

2.4 表观修饰

在单子叶植物如水稻胚乳是发生基因组印迹的主要部位。基因组印迹通过抑制靶基因的表达调控胚乳发育，从而控制种子体积[101-102]。甲基化在调控表达中发挥重要作用，研究发现水稻半显性突变体植株中表现出叶倾角增大和籽粒变小，是由于基因()启动子区域低甲基化[16]。编码一个H3K36甲基转移酶，其表达下调导致多种缺陷，包括植株矮化、节间缩短、叶片直立和种子变小。转录组分析发现，功能丧失，导致包括、和等参与油菜素内酯合成及信号通路的相关基因表达下调[17-18]。编码一个新型的类GNAT蛋白，促进细胞分裂，调控粒长。具有组蛋白乙酰转移酶活性()，提高该基因表达会导致转基因植株中组蛋白H4的乙酰化水平提高，增加种子颖壳细胞数目并加速籽粒灌浆，从而增加粒重和产量[65]。

2.5 G-蛋白信号

植物中G蛋白以异源三聚体的形式存在，在多个信号通路中起重要调控作用[104]。在拟南芥中，Gα()和Gβ()影响叶片和花的发育，而Gγ()影响种子和器官发育，功能缺失突变体产生小粒[105-106]。水稻G蛋白α亚基功能缺失突变体表现矮秆小粒表型，对BR的敏感性降低，表明介导的异源三聚体G蛋白与BR信号转导途径共同调控水稻粒型[60-63]。G蛋白β亚基()表达量的降低也会导致水稻出现籽粒变小的表型[32-33]。但G蛋白γ亚基()功能的缺失却促进细胞伸长，进而产生长粒[34-38]，说明水稻中G蛋白γ亚基和α亚基、β亚基的功能是不同的。同时，G蛋白γ亚基对粒型调控作用在水稻与拟南芥中是相反的，说明水稻与拟南芥中G蛋白γ亚基可能具有不同的作用因子。

我国进入经济新常态以来，随着经济发展越来越接近发达经济体，技术创新和技术转移与扩散在影响产业升级时的角色逐渐发生转变。根据以上研究结果，本文围绕着经济新形势下我国如何优化金融结构以更好地促进产业结构升级这一现实课题，提出以下几点建议：

2.6 其他通路

MicroRNA在水稻粒型调控中起重要作用。过表达的水稻植株出现小粒的表型，对油菜素内酯敏感性降低。研究发现通过降解靶基因，调控BR生物合成，最终影响植株叶夹角和种子大小[57]。能够通过下调的表达，促进油菜素内酯的信号转导，进而促进幼穗分枝、增加种子体积，提高水稻产量[58]。

和都是非典型的不结合DNA的碱性螺旋-环-螺旋蛋白, 都是水稻籽粒长度的正向调节子。通过与拮抗因子形成异源二聚体抑制的功能，从而正向调节水稻粒长[29,31]。编码包含SBP结构域的转录因子，该基因的高表达促进细胞分裂和籽粒灌浆，增加水稻粒宽及产量[83]。编码植物特有的转录因子，其高表达促进细胞伸长，进而产生大粒。5′-UTR的一个串联重复序列通过影响转录和翻译从而改变其表达，正向调控水稻颖壳的细胞体积，进而增加粒长和产量[75]。此外，可以结合和的启动子，调控其表达，但具体的遗传互作关系仍然未知。

Kitagawa等[59]克隆了一个粒型基因，它编码一个由819个氨基酸组成的kinesin-13家族成员蛋白，与拟南芥高度同源，该基因突变导致细胞长度变小，从而使突变体表现出籽粒圆而小，株高降低。Li等[87]发现编码一种具有转录调控活性的类驱动蛋白，通过调控水稻中GA的合成来调节细胞伸长, 矮秆突变体表现出短小的根、茎、穗和种子。这些结果说明驱动蛋白在水稻粒型调控中也起到重要作用。

3 粒型基因间互作及其在水稻高产优质育种中的应用

粒型性状是由多基因控制的数量性状，不同粒型基因之间存在着不同程度的互作。编码一个含有泛素相关结构域蛋白，作为一个重要的上游调控蛋白促进抽穗和粒重相关基因的表达。在突变体中，、和等基因的表达降低，其中基因的表达量降低最为明显[70]。Gao等[107]研究发现和在水稻粒长调控上具有累加效应。和对的表达为正调控，同时，又能够抑制的转录，从而降低其表达水平。可以掩盖对粒长的影响，而又可以掩盖对粒宽的影响[38]。

粒型基因的克隆不仅有助于揭示水稻粒型性状形成的遗传机制，也为水稻分子标记辅助选择育种提供了理论依据和技术基础。Song等[23]研究发现携带的NIL，比其原始亲本丰矮占1号的粒长、粒宽、粒厚及千粒重分别提高了6.6%、26.2%、10.5%、49.8%，单株籽粒产量提高19.7%，且不影响株型、籽粒灌浆以及稻米的外观、蒸煮和食用品质。Hu等[26]以9311和武运粳7号为背景，分别构建了携带宝大粒等位基因的两套近等基因系(NIL)，发现在以9311为背景的NIL()中粒长、粒宽和粒厚分别提高21.0%、17.6%、11.1%，千粒重增加40.26%, 最终小区产量增加14.26%。在以WYG7为背景的NIL()中，粒长、粒宽和粒厚分别提高27.3%、12.0%、3.6%，千粒重增加59.89%，小区产量增加13.1%，虽然株高和穗长明显提高，剑叶、倒2叶和倒3叶变长，但分蘖数、一次枝梗数和二次枝梗数、粒宽等均无明显差异[28]。Zhang等[41]将来自于大粒品种N411隐性基因导入到9311中，发现NIL-千粒重提高了37.03%，穗长增加11.76%，小区产量提高16.2%，而穗粒数、分蘖数、抽穗期和株高并未受到影响。Song等[65]利用杂交、回交筛选获得了含有来自籼稻Kasalath的粒型基因的遗传背景为粳稻日本晴的NIL-，相比日本晴，谷粒产量增加15.8%。Wang等[72]研究发现，相比日本晴，遗传背景含的NIL籽粒长宽比增加，淀粉颗粒更大更致密，稻米垩白度和垩白率显著降低，外观品质改善，但千粒重、单株产量、整精米率、直链淀粉含量、胶稠度和蛋白质含量不受影响。这说明可以用于改良水稻谷粒外观品质，而不影响其产量和蒸煮食用品质。

4 展望

谢辞：感谢农业部长江中下游粳稻生物学与遗传育种重点实验室、江苏省植物基因工程技术研究中心、江苏省现代作物生产中心、长江流域杂交水稻协同创新中心的支持！

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Research Progress on Cloning and Regulation Mechanism of Rice Grain Shape Genes

LIU Xi1, MOU Changling1, ZHOU Chunlei1, CHENG Zhijun2, JIANG Ling1,*, WAN Jianmin1,2

(1State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China;2National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China;*Corresponding author,E-mail: jiangling@njau.edu.cn)

Grain shape is an important trait that affects the yield and quality of rice, so it is necessary tounderstand the genetic regulation mechanism of grain shape to improve rice yield and quality. Grain shape is characterized by a combination of grain length, grain width, grain thickness, and grain length-to-width ratio,belonging to quantitative traits, which is controlled by different genetic systems, such as embryo, endosperm and maternal plant. With the rapid development of rice functional genomics and re-sequencing technology, more than 400 QTLs related to rice grain shape have been located at present, and at least 60 genes associated with rice grain traits have been identified. Some rice grain shape regulation pathways were identified, including phytohormones, the ubiquitin-proteasome pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, epigenetic modification, the G protein signaling pathways. In this review, we systematically summarize and sort out the research progress of the cloning and functional analysis of rice grain shape genes, and evaluate the utilization value of rice grain shape genes in rice breeding for high yield and good quality.

rice(L.); grain shape; gene;regulation of mechanism

10.16819/j.1001-7216.2018.7016

S511.032; S511.2+2

A

1001-7216(2018)01-0001-11

2017-02-07;

2017-04-20。

国家自然科学基金重点项目(91535302)。