水稻磷脂酰丝氨酸合酶SUI1在烟草叶肉细胞中的定位

doi:10．3969/j．issn．1001G7216．2015．04．002

摘要/Abstract

摘要：

在克隆水稻编码磷脂酰丝氨酸合酶的短穗颈基因 SUI1的基础上,利用Gateway方法分别构建了N端和C端两种GFP融合载体pMDC45-SOVT和 pMDC201-SOV。利用农杆菌侵染烟草叶片的方法,观察SUI1蛋白在烟草叶肉细胞中的瞬时表达情况,结果显示GFP-SUI1定位于质膜和核上,而SUI1-GFP定位于质膜和核膜上,并且两种融合蛋白在质膜上的分布都是不连续的。由于N端融合可能改变了目标蛋白的折叠方式及跨膜方式,从而影响蛋白的正确定位,因而SUI1蛋白主要定位于烟草叶肉细胞的质膜和核膜中。

关键词: 水稻, SUI1, 亚细胞定位

Abstract:

SUI1 encodes a phosphatidylserine synthase. And its mutant sui1-1 exhibites a shortened uppermost internode and apartly sheathed panicle. On this basis, two vectors pMDC45-SOVT and pMDC201-SOV were constructed by the Gateway system, which responds to the N-terminal and C-terminal fusion GFP. To observe the subcellular localization of SUI1, tobacco leaves have been infected by Agrobacterium with destination vectors. The results showed that GFP-SUI1 fusion proteins positioned in the plasma membrane and nucleus, while SUI1-GFP were localized in the plasma membrane and the nuclear membrane. Moreover, the distribution of the two fusion proteins were not continuous in the plasma membrane. We speculate that SUI1 protein was localized in the plasma membrane and nuclear membrane of tobacco leaf cells because the target protein folding and transmembrane manner may be changed by the N-terminal fusion, affecting the correct position of the protein.

Key words: rice, SUI1, subcellular localization

中图分类号:

赵娟, 康书静, 饶玉春, 邱振楠, 徐杰, 胡江, 张光恒, 曾大力, 郭龙彪, 钱前, 朱丽. 水稻磷脂酰丝氨酸合酶SUI1在烟草叶肉细胞中的定位[J]. 中国水稻科学, 2015, 29(4): 343-349.

Juan ZHAO, Shu-jing KANG, Yu-chun RAO, Zhen-nan QIU, Jie XU, Jiang HU, Guang-heng ZHANG, Da-li ZENG, Long-biao GUO, Qian QIAN, Li ZHU. Subcellular Localization Analysis of Phosphatidylserine Synthase (OsSUI1) in Tobacco Mesophyll Cells[J]. Chinese Journal OF Rice Science, 2015, 29(4): 343-349.

图/表 4

表1 构建载体过程中用到的引物序列

Table 1 Markers used for carrier construction.

引物名称 Primer	引物序列(5'-3') Sequence(5'-3')	产物 Product/bp	退火温度 T_m/℃
SOV2F	CACCATGGAGGTCAATGGTCATCACA	1282	60
SOV2R	TCATAGCCTTTTCCTTATCATTGTTCGGT
SOV3	TAGCCTTTTCCTTATCATTGTTCGGTAAA	1279	60
35SPF	TCATTGCGATAAAGGAAAGGC	2200 /1500	55

图1 载体PCR扩增鉴定(A,泳道1和2是以35SPF/SOV2R为引物扩增目的载体pMDC45-SOVT和M-DL2000(TAKARA)。B,泳道1~5是以35SPF/SOV3R为引物扩增目的载体pMDC201-SOV; M-DL5000(TaKaRa)。)

Fig. 1. Verification of vectors by PCR amplification. (A, lanes 1 and 2, PCR analysis of pMDC45-SOVT by primers 35SPF/SOV2R; M, DL2000(TaKaRa). B, lanes 1 to 5, PCR analysis of pMDC201-SOV by primers 35SPF/SOV3R; M, DL5000(TaKaRa).)

图2 载体酶切鉴定(泳道1-Hind Ⅲ-SacⅠ酶切空目的载体pMDC45; 泳道2-HindⅢ-SacⅠ酶切目的载体pMDC45-SOVT; 泳道3-SacⅠ酶切空目的载体pMDC201; 泳道4-SacⅠ酶切目的载体pMDC201-SOV。M1-DL5000(TaKaRa); M2-1 kb DNA ladder(TaKaRa)。)

Fig. 2. Verification of vectors by restriction enzyme digestion. (Lane 1, pMDC45 digested by HindⅢ-SacⅠ; Lane 2, pMDC45-SOVT digested by HindⅢ-SacⅠ; Lane 3, pMDC201 control digested by SacⅠ; Lane 4, pMDC 201-SOV digested by SacⅠ. M1,DL5000(TaKaRa); M2, 1 kb DNA ladder(TaKaRa).)

图3. SUI1蛋白在烟草叶肉细胞中的定位(A-pMDC45空载体; B-GFP-SUI1融合蛋白定位; C-pMDC201空载体; D-SUI1-GFP融合蛋白定位。)

Fig. 3. Subcellular localization of SUI1 fusion protein in tobacco mesophyll cells. (A, Localization of pMDC45; B, Localization of GFP-SUI1; C, Localization of pMDC201; D, Localization of SUI1-GFP.)

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