OsESV1基因对水稻淀粉合成的影响

doi:10.16819/j.1001-7216.2022.210308

摘要/Abstract

摘要：

【目的】研究水稻EARLY STARVATION1 (OsESV1)基因对水稻淀粉代谢的影响。【方法】通过CRISPR-Cas9技术获得osesv1突变体,考查osesv1的表型及胚乳淀粉的理化特性,分析OsESV1的表达特性及相关功能。【结果】 OsESV1蛋白在植物界中十分保守。osesv1突变体株高、穗长、每穗粒数低于野生型,分蘖数显著多于野生型,叶片中淀粉含量显著下降,籽粒中的直链淀粉含量上升,而总淀粉含量无明显变化。OsESV1呈组成型表达,并且具有昼夜节律表达的特征。OsESV1蛋白定位在叶绿体内且呈点状分布。酵母双杂交和双分子荧光互补实验结果表明OsESV1蛋白可以与ADP-葡萄糖焦磷酸化酶小亚基(OsAGPS) 2a和OsAGPS1互作。【结论】 OsESV1基因影响水稻叶片的淀粉合成途径,而对胚乳淀粉合成的影响不明显。

关键词: 水稻, 淀粉, 胚乳, OsESV1, 互作

Abstract:

【Objective】 It is important to reveal the role of EARLY STARVATION1 (OsESV1) in starch metabolism of rice.【Method】 The osesv1 mutants were obtained by using the CRISPR-Cas9 system. The phenotype and the physicochemical properties of endosperm starch of osesv1 were investigated. The expression pattern and function of OsESV1 were analyzed. 【Result】 The OsESV1 protein was conserved in plants. Compared with the wild type, the osesv1 mutants exhibited lower plant height, panicle length, and grain number per panicle, but higher tiller number. Though the starch contents in osesv1 leaves significantly decreased, the total starch contents of osesv1 grains did not obviously altered and the amylose contents increased. The expression of OsESV1 was constitutive and rhythmic. OsESV1 was located in chloroplasts with a dot-like distribution. Yeast two-hybrid and bimolecular fluorescence complementation(BiFC) assays showed that OsESV1 could interact with ADP glucose pyrophosphorylase small subunit (OsAGPS) 2a and OsAGPS1.【Conclusion】 OsESV1 significantly affects the starch synthesis pathway in rice leaves, while not in endosperm.

Key words: rice, starch, endosperm, OsESV1, interaction

王永祥, 燕海刚, 徐含聪, 傅玉双, 单壮壮, 胡晓晴, 张文伟, 江玲. OsESV1基因对水稻淀粉合成的影响[J]. 中国水稻科学, 2022, 36(2): 139-149.

WANG Yongxiang, YAN Hangang, XU Hancong, FU Yushuang, SHAN Zhuangzhuang, HU Xiaoqing, ZHANG Wenwei, JIANG Ling. Effects of OsESV1 on Starch Synthesis in Rice[J]. Chinese Journal OF Rice Science, 2022, 36(2): 139-149.

图/表 10

表1 实验所用引物

Table 1 Primers used in this study.

引物 Primer	正向引物序列 Forward primer sequence (5′-3′)	反向引物序列 Reverse primer sequence (5′-3′)
Crispr-OsESV1	GGCAAGCACCTGGTACAGGGAGAG	AAACCTCTCCCTGTACCAGGTGCT
OsESV1-TI	AGCTCCTCAATCCCGTGATA	CTCATACGAAAATGTGGT
PAN580-OsESV1-GFP	TCCGGAGCTAGCTCTAGAATGGCCGCGTGCTCCAGG	CGCCCTTGCTCACCATGGATCCTTCCAGAGGCGAAGGAGGCA
1305-OsESV1-GFP	TCCGGAGCTAGCTCTAGAATGGCCGCGTGCTCCAGG	CGCCCTTGCTCACCATGGATCCTTCCAGAGGCGAAGGAGGCA
QRT-OsESV1	GTGATACTCCGCCGAAGAGA	TCGTCTCCACTCTCCCTGTA
Actin	TGCTATGTACGTCGCCATCCAG	AATGAGTAACCACGCTCCGTCA
AD-OsESV1	CATGGAGGCCGAATTCATGGCCGCGTGCTCCAGG	CGAGCTCGATGGATCCTCATTCCAGAGGCGAAGGAGG
BD-OsESV1	CATGGAGGCCGAATTCATGGCCGCGTGCTCCAG	GCAGGTCGACGGATCCTCATTCCAGAGGCGAAGGAGG
AD-OsAGPS1	GGAGGCCAGTGAATTCATGGCGATGATGGCGATG	CGAGCTCGATGGATCCTTATATGACTGTTCCGCTAG
BD-OsAGPS1	CATGGAGGCCGAATTCATGGCGATGATGGCGATG	GCAGGTCGACGGATCCTTATATGACTGTTCCGCTAG
AD-OsAGPS2a	GGAGGCCAGTGAATTCATGGCGATGGCGGCAGCCAT	CGAGCTCGATGGATCCTCATATAACTGTTCCGCTAG
AD-OsAGPS2b	CATGGAGGCCGAATTCATGAATGTATTGGCATCTAAG	CGAGCTCGATGGATCCTCATATAACTGTTCCGCTAGG
AD-OsAGPL1	CATGGAGGCCGAATTCATGCAGTTCAGCAGTGTGTTT	CGAGCTCGATGGATCCCTATATGACCTTCCCGTCC
AD-SSⅠ	CATGGAGGCCGAATTCATGGCGACGGCGGCGGGGAT	CGAGCTCGATGGATCCTTACATGACATATGGTTGATC
AD-SSⅡa	CATGGAGGCCGAATTCATGTCGTCGGCCGTCGTCGCGTC	CGAGCTCGATGGATCCTCACCATTGGTACTTGGCCTT
AD-SSⅡb	CATGGAGGCCGAATTCTTCACCTCCTCTTCGCCGCG	CGAGCTCGATGGATCCTCACCACTGGTACTTGGCCTT
AD-BEI	GGAGGCCAGTGAATTCATGCTGTGTCTCACC	CGAGCTCGATGGATCCCTCATTTGCAGTCTTC
AD-BEⅡb	CATGGAGGCCGAATTCATGGCGGCGCCGGCGTCTG	CGAGCTCGATGGATCCTCATTCCGCTGGAGCATA
p2YC-OsESV1	ATTTACGAACGATAGTTAATTAAATGGCGATGGCCGCGTGCTCCAGG	CACTGCCACCTCCTCCACTAGTTTCCAGAGGCGAAGGAGG
p2YN-OsAGPS2a	ATTTACGAACGATAGTTAATTAAATGGCGATGGCGGCAGCCAT	CACTGCCACCTCCTCCACTAGTTATAACTGTTCCGCTAGGG
p2YN-OsAGPS1	ATTTACGAACGATAGTTAATTAAATGGCGATGGCGATGATGGCGATG	CACTGCCACCTCCTCCACTAGTTATGACTGTTCCGCTA

表1 实验所用引物

Table 1 Primers used in this study.

引物 Primer	正向引物序列 Forward primer sequence (5′-3′)	反向引物序列 Reverse primer sequence (5′-3′)
Crispr-OsESV1	GGCAAGCACCTGGTACAGGGAGAG	AAACCTCTCCCTGTACCAGGTGCT
OsESV1-TI	AGCTCCTCAATCCCGTGATA	CTCATACGAAAATGTGGT
PAN580-OsESV1-GFP	TCCGGAGCTAGCTCTAGAATGGCCGCGTGCTCCAGG	CGCCCTTGCTCACCATGGATCCTTCCAGAGGCGAAGGAGGCA
1305-OsESV1-GFP	TCCGGAGCTAGCTCTAGAATGGCCGCGTGCTCCAGG	CGCCCTTGCTCACCATGGATCCTTCCAGAGGCGAAGGAGGCA
QRT-OsESV1	GTGATACTCCGCCGAAGAGA	TCGTCTCCACTCTCCCTGTA
Actin	TGCTATGTACGTCGCCATCCAG	AATGAGTAACCACGCTCCGTCA
AD-OsESV1	CATGGAGGCCGAATTCATGGCCGCGTGCTCCAGG	CGAGCTCGATGGATCCTCATTCCAGAGGCGAAGGAGG
BD-OsESV1	CATGGAGGCCGAATTCATGGCCGCGTGCTCCAG	GCAGGTCGACGGATCCTCATTCCAGAGGCGAAGGAGG
AD-OsAGPS1	GGAGGCCAGTGAATTCATGGCGATGATGGCGATG	CGAGCTCGATGGATCCTTATATGACTGTTCCGCTAG
BD-OsAGPS1	CATGGAGGCCGAATTCATGGCGATGATGGCGATG	GCAGGTCGACGGATCCTTATATGACTGTTCCGCTAG
AD-OsAGPS2a	GGAGGCCAGTGAATTCATGGCGATGGCGGCAGCCAT	CGAGCTCGATGGATCCTCATATAACTGTTCCGCTAG
AD-OsAGPS2b	CATGGAGGCCGAATTCATGAATGTATTGGCATCTAAG	CGAGCTCGATGGATCCTCATATAACTGTTCCGCTAGG
AD-OsAGPL1	CATGGAGGCCGAATTCATGCAGTTCAGCAGTGTGTTT	CGAGCTCGATGGATCCCTATATGACCTTCCCGTCC
AD-SSⅠ	CATGGAGGCCGAATTCATGGCGACGGCGGCGGGGAT	CGAGCTCGATGGATCCTTACATGACATATGGTTGATC
AD-SSⅡa	CATGGAGGCCGAATTCATGTCGTCGGCCGTCGTCGCGTC	CGAGCTCGATGGATCCTCACCATTGGTACTTGGCCTT
AD-SSⅡb	CATGGAGGCCGAATTCTTCACCTCCTCTTCGCCGCG	CGAGCTCGATGGATCCTCACCACTGGTACTTGGCCTT
AD-BEI	GGAGGCCAGTGAATTCATGCTGTGTCTCACC	CGAGCTCGATGGATCCCTCATTTGCAGTCTTC
AD-BEⅡb	CATGGAGGCCGAATTCATGGCGGCGCCGGCGTCTG	CGAGCTCGATGGATCCTCATTCCGCTGGAGCATA
p2YC-OsESV1	ATTTACGAACGATAGTTAATTAAATGGCGATGGCCGCGTGCTCCAGG	CACTGCCACCTCCTCCACTAGTTTCCAGAGGCGAAGGAGG
p2YN-OsAGPS2a	ATTTACGAACGATAGTTAATTAAATGGCGATGGCGGCAGCCAT	CACTGCCACCTCCTCCACTAGTTATAACTGTTCCGCTAGGG
p2YN-OsAGPS1	ATTTACGAACGATAGTTAATTAAATGGCGATGGCGATGATGGCGATG	CACTGCCACCTCCTCCACTAGTTATGACTGTTCCGCTA

图1 OsESV1蛋白的保守性分析 A—OsESV1及其同源蛋白的进化树分析,蛋白名采用相应的GenBank 序列登录号;B—多氨基酸序列比对,星号表示20个氨基酸的间隔,浅红色区域为富含色氨酸(W)区,保守色氨酸残基下用“：”标注,黄色区域为富含脯氨酸(P)区。

Fig. 1. The conservation analysis of OsESV1. A, Neighbor-joining tree of OsESV1 and its homologs. The proteins are named by GenBank protein accession numbers; B, Multiple sequence alignment of OsESV1 and its homologs. Asterisks represent the interval of twenty amino acids. The tryptophan (W)-rich region is marked by light red and those conserved tryptophan residues are indicated by colons. The proline (P)-rich region is marked by yellow.

图2 osesv1突变体的表型 A—OsESV1的基因结构。深灰和浅灰方块分别表示外显子区域和UTR区域,方块之间的黑色连线表示内含子,下面为野生型和osesv1靶位点基因序列比较,连续的点表示缺失的序列,插入的碱基加粗标示,红色标注为原间隔基序(protospacer adjacent motif , PAM) TGG;B—野生型和突变体的OsESV1蛋白结构。箭头标注富含色氨酸区（Trp-rich region）和富含脯氨酸区（Pro-rich region）;C—野生型和osesv1植株,标尺为10 cm;D—野生型和osesv1幼苗在白天和夜晚结束时OsESV1基因的表达水平。数据表示为平均值±标准差(n=3)。*和**分别表示差异达0.05和0.01显著水平（t 检验）。下同。

Fig. 2. Phenotype of the osesv1 mutants. A, Structure of the OsESV1 gene. The dark gray and light gray boxes represent the exon and UTR regions, respectively. The black lines connecting boxes are introns. The corresponding sequences of the wild type (WT) and osesv1 were compared below and a succession of points indicates the deleted sequence in osesv1. The inserted base is shown in bold. TGG (PAM) is marked by red; B, The protein structure of WT and osesv1; Arrows indicate the tryptophan-rich region and proline-rich region; C, WT and osesv1 plants. Bar=10cm; D, Expression level of OsESV1 in WT and osesv1 seedlings at end of day(ED) and end of night(EN). Values are mean ± SD (n=3). * and **indicate significant difference at 0.05 and 0.01 level, respectively (t-test). The same below.

表2 野生型和osesv1的农艺性状

Table 2 Agronomic traits of the wild type (WT) and osesv1.

材料 Materials	株高 Plant height /cm	穗长 Length of panicle /cm	分蘖数 Tiller number	每穗粒数 Grain number per panicle	一次枝梗数 Primary rachis branch number	千粒重 1000-grain weight /g
野生型WT	94.80±3.10	19.31±0.77	26.13±8.55	127.58±9.33	8.60±0.83	22.57±1.07
osesv1-1	90.96±4.44^**	17.82±0.52^**	33.43±10.45^*	112.11±15.42^*	8.44±0.96	21.50±0.56
osesv1-2	90.47±5.40^**	17.69±0.82^**	43.08±13.85^**	113.63±10.71^**	9.00±0.95	23.01±0.85

图3 野生型和osesv1的叶片淀粉水平 A—白天和夜晚结束时野生型(WT)和osesv1叶片的碘染观察; B—白天和夜晚结束时野生型(WT)和osesv1叶片的淀粉含量测定。

Fig. 3. Starch contents in leaves of WT and osesv1. A and B show iodine staining and starch contents of WT and osesv1 leaves at end of day and end of night, respectively.

图4 osesv1种子表型及淀粉理化性质分析 A—野生型(WT)和osesv1的成熟种子表型,标尺为5 mm;B, C—野生型(WT)和osesv1种子总淀粉含量、直链淀粉含量;D—野生型和osesv1胚乳淀粉的黏度曲线。

Fig. 4. Phenotype of osesv1 grains and physicochemical properties of endosperm starch. A, Phenotype of WT and osesv1 mature seeds. Bar=5 mm; B, Total starch contents of WT and osesv1 grains; C, Amylose content of WT and osesv1 grains; D, Viscosity profiles of endosperm starch of WT and osesv1.

图5 OsESV1的表达模式分析 A—OsESV1在野生型不同组织及胚乳发育不同时期的表达水平;B—OsESV1的昼夜节律表达。黑框和白框分别表示黑夜和白天的时间轴。

Fig. 5. Expression pattern of OsESV1. A, Expression level of OsESV1 in various tissues and endosperm at different developmental stages of WT; B, Diurnal expression pattern of OsESV1. The black and white boxes represent the timelines of night and day, respectively.

图6 OsESV1的亚细胞定位 A—OsESV1-GFP融合蛋白在水稻原生质体中的表达,pAN580-GFP空载体作为对照,标尺为20 μm;B—OsESV1-GFP融合蛋白在烟草表皮细胞中的表达,p1305-GFP空载体作为对照,标尺为20 μm。

Fig. 6. Subcellular localization of OsESV1. A, Transient expression of OsESV1-GFP fusion protein in rice protoplasts. The empty vector pAN580-GFP was used as control. Bars=20 μm; B, Transient expression of OsESV1-GFP fusion protein in tobacco epidermal cells. The empty vector p1305-GFP was used as control. Bars=20 μm.

图7 酵母双杂实验表明OsESV1可以与OsAGPS2a和OsAGPS1互作二缺：SD-Leu/-Trp;四缺：SD-Leu/-Trp/-His/-Ade。

Fig. 7. Yeast two-hybrid assays show that OsESV1 can interact with OsAGPS2a and OsAGPS1. DDO(double dropout supplements), SD-Leu/-Trp; QDO(quadruple dropout supplements), SD-Leu/-Trp/ -His/-Ade.

图8 双分子荧光互补实验证明OsESV1可以与OsAGPS2a和OsAGPS1在烟草表皮细胞中互作

Fig. 8. BiFC assays show that OsESV1 can interact with OsAGPS2a and OsAGPS1 in tobacco epidermal cells. Bars = 10 μm.

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