水稻转录因子基因OsSHR2的表达特征及其在营养生长中的调控作用

doi:10.16819/j.1001-7216.2018.7037

摘要/Abstract

摘要：

【目的】水稻OsSHR2(LOC_Os03g31880)基因为拟南芥AtSHR的同源基因,与OsSHR1、OsSCR1和OsSCR2 同属于水稻GRAS转录因子家族。已有研究报道,转录因子基因SHR和SCR共同调控植物根系、叶片的发育,并参与各项生命活动。本研究旨在阐明OsSHR2在水稻中的时空表达特征及其在营养生长中的调控作用。【方法】通过生物信息学分析、表达模式分析、萌发动力学分析和水培实验验证该基因的功能。【结果】生物信息学分析发现OsSHR2、OsSHR1、OsSCR1和OsSCR2与拟南芥和其他物种的SHR亚家族和SCR亚家族成员具有很高的序列一致性;表达模式和pOsSHR2::GUS材料染色分析发现,OsSHR2在整个生长发育过程中的根系、叶片、维管组织和生殖器官中表达强烈,并集中在根尖的中柱、侧根原基和叶片及茎维管组织的中心表达,在野生型的地上部和根系中,OsSHR2受缺磷影响下调表达;对获得的OsSHR2的CRISPR-Cas9突变体osshr2进行种子萌发实验和水培实验,发现与野生型相比,osshr2的萌发时间延后,萌发率降低,在正常供磷和缺磷处理下,osshr2的地上部和根系长度显著小于野生型。【结论】OsSHR2在地上部和根系的发育、维管组织形成以及营养与生殖生长中具有重要作用,这为今后OsSHR2在分子育种等领域的应用奠定理论基础。

关键词: 水稻, OsSHR2, 时空表达特征, 营养生长, 磷

Abstract:

【Objective】OsSHR2(LOC_Os03g31880) is a homologous gene of AtSHR of Arabidopsis thaliana, which falls into GRAS transcription factor family together with OsSHR1, OsSCR1 and OsSCR2 in rice. It has been reported that the transcription factor genes SHR and SCR regulate the development of roots and leaves, and participate in various life activities. We analyzed the temporal and spatial expression patterns and the way OsSHR2 regulates vegetative growth in rice.【Method】The function of OsSHR2 was verified by bioinformatics analysis, expression pattern analysis, germination kinetic analysis and hydroponic experiments.【Result】 Biomechanical analysis showed that OsSHR2, OsSHR1, OsSCR1 and OsSCR2 had high homology with SHR subfamily and SCR subfamily in Arabidopsis thaliana and other species. The expression pattern analysis by qRT-PCR and pOsSHR2::GUS staining showed that OsSHR2 was strongly expressed in the roots, leaves, vascular tissues and reproductive organs during the whole vegetative and reproductive growth stages, and especially in the stele of the root tip, lateral root primordium and the central of leaf and stem vascular tissue. Additionally, the relative expression of OsSHR2 was down-regulated in Pi-deficiency in shoots and roots of wild type. The seed germination and hydroponic experiment analysis of CRISPR-Cas9 mutant osshr2 showed that seed germination of osshr2 was delayed and with lower germination rate than that of WT, in addition the length of shoot and root of osshr2 were significantly shorter than WT under Pi-sufficient and Pi-deficient conditions.【Conclusion】OsSHR2 plays an important role in the development of shoots and roots, the formation of vascular tissue and various activity in vegetative and reproductive growth, which lays an important theoretical basis for the application of OsSHR2 in molecular breeding.

Key words: rice, OsSHR2, temporal and spatial expression pattern, vegetative growth, phosphorus

中图分类号:

Q755
S511.01

张占田, 孙雅菲, 艾昊, 罗闻真, 冯冰, 孙文献, 徐国华, 孙淑斌. 水稻转录因子基因OsSHR2的表达特征及其在营养生长中的调控作用[J]. 中国水稻科学, 2018, 32(5): 427-436.

Zhantian ZHANG, Yafei SUN, Hao AI, Wenzhen LUO, Bing FENG, Wenxian SUN, Guohua XU, Shubin SUN. Expression Patterns and Regulation of Transcription Factor Gene OsSHR2 in Vegetative Growth in Rice[J]. Chinese Journal OF Rice Science, 2018, 32(5): 427-436.

图/表 6

图1 不同物种SHR和SCR同源基因的生物信息学分析 A–拟南芥和水稻的SHR和SCR同源基因氨基酸和核苷酸的序列一致性分析;B–通过基因家族分析网站InterPro(http://www.ebi.ac.uk/ interpro/)进行拟南芥与水稻的SHR和SCR同源基因GRAS保守结构域和保守基序VHIID的位置预测;C–不同物种SHR和SCR同源基因的进化树分析。

Fig. 1. Bioinformatics analysis of SHR and SCR homologous genes of different species. A, Sequence homology analysis of amino acids and nucleic acid of SHR and SCR homologous genes in Arabidopsis thaliana and rice; B, Prediction of the positions of SHR and SCR homologous GRAS conserved domains and conserved motifs in Arabidopsis thaliana and rice by InterPro (http://www.ebi.ac.uk/interpro/); C, Phylogenetic tree analysis of SHR and SCR homologous genes of different species.

图2 OsSHR1和OsSHR2在水稻中的时空表达模式分析

Fig. 2. Temporal and spatial expression pattern of OsSHR1 and OsSHR2 in rice.

图3 pOsSHR2::GUS材料的GUS染色鉴定 A–种子根;B–种子根及侧根;C–新叶;D–新叶的放大图;E–叶原基;F–叶舌;G–茎节;H–茎及叶鞘;I–茎;J–茎基部;K–颖壳及小穗轴;L–颖花;M–子房和柱头;N–胚(萌发后3 d)。A~E,J：水稻苗期;F,G,I,K~N：水稻灌浆期;H：分蘖期。A~J中标尺为2 mm;K~N中标尺为0.5 mm。

Fig. 3. Identification of OsSHR2 promoter-driven tissue-specific GUS staining. A, Seed root; B, Seed root and lateral root; C, Young leaf; D, The enlarged view of the young leaf; E, Leaf primordium; F, Ligule; G, Node; H, Stem and leaf sheath; I, Stem; J, Basal stem; K, Husk and rachilla; L, Spikelet; M, Ovary and stigma; N, Embryo(three days after germination). A-E, J: Seedling stage; F, G, I, K-N: Grain filling stage; H, Tilling stage. A-J, Bar=2 mm; K-N, Bar=0.5 mm.

图4 WT和突变体osshr2的萌发及水培下的表型 A–萌发表型(萌发后3 d、5 d和8 d),标尺为5 cm;B–水培表型(萌发后14 d),标尺为5 cm;C–材料地上部和根系长度统计(萌发后3 d、5 d、8 d和14 d);DAG–萌发后天数。

Fig. 4. Phenotype and statistics of seed germination and hydroponics experiments of WT and osshr2. A, Germination phenotype(3, 5 and 8 days after germination), bar=5 cm; B, Hydroponics phenotype(14 days after germination), bar=5 cm; C, Shoot and root length (3, 5, 8 and 14 days after germination); DAG, Day after germination.

图5 OsSHR1和OsSHR2在水稻正常供磷与缺磷条件下的相对表达量 A–OsSHR1和OsSHR2在水稻地下部的相对表达量;B–OsSHR1和OsSHR2在水稻地上部的相对表达量。

Fig. 5. Relative expression level of OsSHR1 and OsSHR2 under Pi-sufficient and Pi-deficient conditions in rice. A, The relative expression of OsSHR1 and OsSHR2 in the roots; B, The relative expression of OsSHR1 and OsSHR2 in the shoots.

图6 WT和突变体osshr2在正常供磷和缺磷条件下的表型 A和B–缺磷3 d;C和D–缺磷1周;E和F–缺磷2周;G–缺磷2周的表型, 标尺为20 cm。

Fig. 6. Phenotype of the wild type(WT) and osshr2 under Pi-sufficient and Pi-deficient conditions. A and B, Pi-deficient lasting three days; C and D, Pi-deficient lasting one week; E and F, Pi-deficient lasting two weeks; G, The phenotype of material under Pi-sufficient and Pi-deficient lasting two weeks, bar=20 cm. c410, osshr2-c410; c207, osshr2-c207.

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