水稻蔗糖转运蛋白OsSUT4参与蔗糖转运的功能研究

doi:10.16819/j.1001-7216.2020.0316

摘要/Abstract

摘要： 目的植物蔗糖的跨膜运输主要借助蔗糖转运蛋白（sucrose transporter,SUT）进行。在已鉴定出的5个水稻SUT编码基因中,OsSUT4的作用尚不清楚。方法采用CRISPR/Cas9基因编辑技术构建ossut4缺失变体,出现株高降低、分蘖数增加及产量下降等表型。结果通过进一步分析,发现ossut4突变体水稻叶片蔗糖、淀粉积累,反馈抑制光合速率;因源端光合同化物装载受阻,导致库端籽粒蔗糖供应不足,灌浆延迟。对OsSUT4进行亚细胞和组织化学定位,发现OsSUT4在细胞质膜上,且在萌发种子的胚、胚芽鞘的维管束、小穗颖壳、花药、颖果的糊粉层中都有表达,该现象表明OsSUT4具备转运蔗糖的特征。结论综上所述,OsSUT4在水稻蔗糖源端装载以及籽粒库端卸载等生理过程发挥重要作用。

关键词: 水稻, CRISPR/Cas9, 基因编辑, 蔗糖转运, 蔗糖转运蛋白

Abstract:

【Objective】The transmembrane transport of sucrose is mainly mediated by sucrose transporters, among which five SUT coding genes have been identified inrice. However, the role of OsSUT4 in the sucrose transport is unclear. 【Method】CRISPR/Cas9 techniques were used to construct ossut4-deletedmutants. The ossut4 mutant was featured by lower plant height and grain yield as well as higher tiller number as compared withwildtype plants.【Result】Sucrose and starchaccumulationin the leaf of the ossut4 mutants lead to slower net photosynthetic rate; insufficient sugar supply results in the postponeof grain filling. Base on the subcellular and histochemical localization,OsSUT4 was located on the cytoplasmic membrane. Tissue localization showed that OsSUT4 was expressed in the embryo of germinating seed, vascular bundles of coleoptile, glume shell, anthers at flowering stage and aleurone layer of caryopsis atfilling stage. 【Conclusion】OsSUT4 plays an important role in sucrose transport.

Key words: rice, CRISPR/Cas9, gene editing, sucrose transport, sucrose transporter

中图分类号:

Q786

李孟珠, 王高鹏, 巫月, 任怡, 李刚华, 刘正辉, 丁艳锋, 陈琳. 水稻蔗糖转运蛋白OsSUT4参与蔗糖转运的功能研究[J]. 中国水稻科学, 2020, 34(6): 491-498.

Mengzhu LI, Gaopeng WANG, Yue WU, Yi REN, Ganghua LI, Zhenghui LIU, Yanfeng DING, Lin CHEN. Function Analysis of Sucrose Transporter OsSUT4 in Sucrose Transport in Rice[J]. Chinese Journal OF Rice Science, 2020, 34(6): 491-498.

图/表 10

图1 ossut4突变体株系与野生型的序列比对结果

Fig.1. Results of sequence alignment between the ossut4 mutant lines and the wild-type.

W=	A	; 1)
W=	(1+Be^-kt)^1/N	; 1)
对方程(1)求导,得到灌浆速率R：
R=	AkBe^-kt	; 2)
R=	N(1+Be^-kt)^(N+1)/N	; 2)

表1 荧光定量PCR引物序列

Table 1 Primer sequences of RT-PCR.

基因名称 Gene name	正向引物 Forward primer (5'→3')	反向引物 Reverse primer(5'→3')
Actin	CAATCGTGAGAAGATGACCC	GTCCATCAGGAAGCTCGTAGC
OsSUT1	GCTTTCAACCAGGGTGTCAG	ACTTTCCGGCACATTGGTTC
OsSUT3	ATCTGTGATCGTCATCGGGTTC	GCACGGTGTTGTTGGAGAAGT
OsSUT4	CTCGTGCCCTTTTAGCTGAC	AACGTTTCCAACAGCCATCC
OsSUT5	TTCGCCTTCATCTGTGGAGT	GCCTTTGATGGAGTTTCGCA

图2 OsSUT4突变对水稻株高的影响

Fig. 2. Effects of OsSUT4 mutation on plant height in rice.

表2 OsSUT4突变对株高和产量构成的影响

Table 2 Plant height and yield components ofossut4 mutant and wild type.

株系名称 Name of line	株高 Plant height	分蘖数 Tiller number	每穗粒数 Grain number per panicle	结实率 Seed setting rate/%	一次枝梗数 Number of primary rachis branches	二次枝梗数 Number of secondary rachis branches	千粒重 1000-grain weight
WT	87.5 a	12.2 b	87.6 a	84.0a	11.0a	10.0a	25.6a
sut4-15-1	82.4bc	12.4 a	66.9 b	73.3 b	8.8b	7.4c	24.7 b
sut4-15-2	81.8 c	12.2 a	68.4 b	65.8 c	8.9 b	7.8 c	24.8b
sut4-46	83.3 b	12.0 a	68.5 b	72.8b	8.8b	8.9b	23.5 c

图3 OsSUT4突变对叶片蔗糖转运载体相对表达量的影响

Fig. 3. Effects of OsSUT4 mutation on the relative expression level of sucrose transporters in rice leaves.

图4 OsSUT4突变对水稻蔗糖含量、淀粉含量、净光合速率和SPAD值的影响 A-白天结束和夜晚结束时蔗糖含量;B-白天结束和夜晚结束时淀粉含量;C-净光合速率;D-SPAD值。平均值±标准误(n=3)。不同字母代表株系之间差异达0.05显著水平(P<0.05, 邓肯检验)。

Fig.4. Effects ofOsSUT4mutation on sucrose, starch contents, photosynthetic rate and SPAD value in rice leaves. A, Sucrose content at the end of day and night; B, Starch content at the end of day and night; C, Net photosynthetic rate; D, SPAD value. Values are Mean±SE (n=3). Different letters above bars indicate significant difference(P<0.05) among the lines (Duncan’s test).

图5 OsSUT4突变对水稻花粉活力的影响 A-花粉染色;B-花粉可染率。平均值±标准误(n=5)。不同字母代表株系之间差异达0.05显著水平(P<0.05, 邓肯检验)。

Fig. 5. Effects of OsSUT4 mutation on pollen vigor in rice. A, Pollen staining; B, Pollen dyeability rate. Values are Mean±SE(n=5). Different letters above bars indicate significant difference(P<0.05) among the lines(Duncan’s test).

图6 OsSUT4突变对水稻籽粒灌浆的影响

Fig.6. Effects of OsSUT4 mutation on grain filling in rice.

图7 水稻蔗糖转运蛋白OsSUT4的亚细胞定位(A-D)和组织定位(E-K) A-GFP绿色荧光;B-明场;C-质膜标记OSMCA1-RFP;D-融合荧光;E-萌发的种子;F-叶片;G-叶鞘;H-根;I、J-小穗;K-颖果。

Fig.7. Sub-cellular localization(A-D) of OsSUT4 and tissue expression(E-K) of pOsSUT4:GUS transgenic rice. A, cDNA-GFP; B, Bright field; C, Membrane marker OSMCA1-RFP; D, Merged; E, Germinated seed; F, Leaf; G, Leaf sheath; H, Root; I&J, Spikelet; K, Caryopsis.

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