Functions of OsPtr1 in Rice
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摘要:
目的 利用T-DNA插入突变的方法揭示候选基因P0421H01.23（命名为OsPtr1，Peptite 24-48Transporter 1）的功能表达。 方法 提取水稻明恢86成花后12 d的胚乳RNA，反转录成cDNA后，进行OsPtr1基因扩增，以胚乳特异表达Gt1为启动子，构建OsPtr1基因植物过表达载体，用农杆菌介导法将OsPtr1基因导入水稻品种日本晴，对其后代及突变体w9101进行氮代谢物测定分析；同时，构建绿色荧光蛋白基因GFP和OsPtr1基因融合表达载体，用基因枪转入洋葱表皮细胞，培养24～48 h后，用荧光共聚焦电子显微镜观察OsPtr1基因亚细胞定位。 结果 OsPtr1基因编码蛋白定位于质膜上，该基因与氮类物质的运输有关，OsPtr1功能缺失突变体种子的氨基酸和蛋白质等含氮物质积累效率降低，过表达植株种子的氨基酸和蛋白质等含氮物质含量增加。 结论 OsPtr1基因参与水稻氮类物质的跨膜运输。 Abstract:Objective Functions of a peptide transporter protein gene, OsPtr1, in rice were studied by means of T-DNA insertion mutation. Method The target RNA was extracted from the endosperm of Minghui 86 rice 12 d after flowering and reverse-transcribed into cDNA. By cloning the candidate gene P0421H01.23, named OsPtr1, and constructing the plant overexpression vector using the promoter Gt1 of endosperm-specific expressing, OsPtr1 was introduced into Nipponbare rice using an agrobacterium-mediated method. Nitrogen metabolism of the offspring and mutant w9101 was analyzed. A fusion expression vector of green fluorescent protein gene, GFP, and OsPtr1 was constructed and transferred to onion epidermal cells with a gene gun. In 24–48 h afterward, subcellular localization of OsPtr1 was examined under a fluorescence confocal electron microscope. Result OsPtr1 was localized at the membrane and possibly involved in the transport of nitrogen substances. Knockout on OsPtr1 could cause a reduction on nitrogen-containing substances, such as amino acids and proteins, in the rice grains. Conversely, an overexpressed OsPtr1 would increase the accumulation. Conclusion OsPtr1 participated in the transmembrane transport of nitrogen substances in rice. -
Key words:
- Rice /
- endosperm-specific expressing /
- peptide transporter /
- OsPtr1 /
- nitrogen-metabolism
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图 1 pCAMBIA1300-OsPtr1-hGFP酶切鉴定
1、2分别代表用Sal I、kpn I+Bam HI酶切鉴定，M为λ-14 Marker（EcoT14Ⅰ酶切）。
Figure 1. Electrophoresis of digested plasmid pCAMBIA1300-OsPtr1-hGFP
1: pCAMBIA1300-OsPtr1-hGFP/Sal I; 2: pCAMBIA1300-OsPtr1-hGFP/kpn I+Bam HI; M: λ-EcoT14 Ⅰ marker.
图 2 pCAMBIA1300-OsPtr1-hGFP载体
CaMV35S promoter为花椰菜花叶病毒（Cauliflower mosaic virus）启动子；hygromycin为潮酶素抗性标记基因；OsPtr1为肽转运子1；hGFP为绿色荧光蛋白；T-border为T-DNA边界。
Figure 2. Vector pCAMBIA1300-OsPtr1-hGFP
CaMV35S promoter is a cauliflower mosaic virus promoter; hygromycin, hygromycin-resistant marker gene; OsPtr1, a peptide transporter; hGFP, a green fluorescent protein; and T-border, border of T-DNA.
图 3 OsPtr1基因的激光共聚焦亚细胞定位
A是B中洋葱表皮细胞的明场。
Figure 3. Subcellular localization of OsPtr1 by laser scanning confocal fluorescence
A is bright field of onion epidermal cells in B.
图 4 Gt1-OsPtr1过表达载体酶切鉴定
1、2分别为用Hind III、Bam HI单酶切载体，M为λ-14 Marker（EcoT14Ⅰ酶切）。
Figure 4. Identification on over-expression vector of Gt1-OsPtr1 by enzyme-cutting
1: Gt1-OsPtr1/Hind III; 2: Gt1-OsPtr1/Bam HI, M: λ-EcoT14 Ⅰ marker.
图 5 转过表达OsPtr1基因水稻植株Southern blot分析
1~6分别为Ptr1g10、Ptr1g15、Ptr1g19、Ptr1g21、Ptr1g29、Ptr1g5。
Figure 5. Southern blot analysis on transgenic rice lines of over-expressed OsPtr1
1–6: Ptr1g10，Ptr1g15，Ptr1g19，Ptr1g21，Ptr1g29, and ptr1g5, respectively.
图 6 突变体 w9101的氨基酸含量
*和**分别表示在P＜0.05和P＜0.01水平上差异显著。图7同。
Figure 6. Amino acid content of mutant w9101
* and ** were significantly different at P＜0.05 and P＜0.01, respectively. Same for below.
图 7 突变体w9101总氨基酸含量和总蛋白质含量分析
Figure 7. Contents of total amino acids and protein in mutant w9101
表 1 OsPtr1基因水稻植株氨基酸含量和蛋白质含量
Table 1. Contents of amino acids and protein in transgenic rice plantlets of over-expressed OsPtr1 （单位：g·hg−1）
指标
Indices水稻材料 Rice materials ptr9101g15 ptr9101g21 ptr9101g29 日本晴
Nipponbare(WT)天门冬氨酸 Asp 0.92±0.02 ab 0.96±0.01 a 0.87±0.01 b 0.78±0.02 c 丝氨酸 Ser 0.52±0.02 a 0.54±0.02 a 0.49±0.01 ab 0.44±0.02 b 谷氨酸 Glu 1.68±0.03 b 1.76±0.02 a 1.62±0.03 c 1.45±0.02 d 甘氨酸 Gly 0.46±0.03 a 0.47±0.02 a 0.44±0.03 ab 0.40±0.03 b 丙氨酸 Ala 0.57±0.02 a 0.58±0.02 a 0.57±0.02 a 0.51±0.02 b 胱氨酸 Cys 0.12±0.03 a 0.13±0.02 a 0.12±0.01 a 0.10±0.02 a 甲硫氨酸 Met 0.22±0.01 a 0.17±0.01 b 0.13±0.02 c 0.12±0.02 c 酪氨酸 Tyr 0.46±0.03 a 0.33±0.02 b 0.28±0.02 c 0.27±0.02 c 脯氨酸 Pro 0.36±0.02 a 0.37±0.02 a 0.34±0.03 ab 0.30±0.02 b 组氨酸 His 0.25±0.04 a 0.26±0.03 a 0.24±0.03 ab 0.21±0.04 b 精氨酸 Arg 0.98±0.02 a 0.89±0.04 b 0.77±0.03 c 0.71±0.04 d 苏氨酸 Thr 0.36±0.02 a 0.37±0.03 a 0.34±0.03 ab 0.30±0.04 b 缬草氨酸 Val 0.56±0.02 ab 0.59±0.02 a 0.55±0.02 ab 0.51±0.01 b 异亮氨酸 Ile 0.39±0.01 a 0.39±0.01 a 0.37±0.02 a 0.33±0.01 b 亮氨酸 LEeu 0.84±0.01 a 0.86±0.02 a 0.80±0.02 a 0.72±0.02 b 苯丙氨酸 Phe 0.54±0.01 a 0.57±0.02 a 0.54±0.02 a 0.47±0.01 b 赖氨酸 Lys 0.39±0.01 a 0.40±0.01 a 0.36±0.012 ab 0.31±0.01 b 氨基酸总量 Amino acid contents 9.61±0.05 a 9.55±0.05 a 8.83±0.05 b 7.95±0.04 c 蛋白质含量 Protein contents 10.10±0.40 a 9.69±0.06 bc 9.42±0.04 c 8.02±0.07 d 不同小写字母表示不同水稻材料之间差异显著（P＜0.05）。
Data with different lowercase letters indicate significant difference (P＜0.05).
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