关键词:转基因小麦; NADP苹果酸酶; 净光合速率; 气孔导度 Photosynthetic Characteristics of Transgenic Wheat Expressing Maize C4-Type NADP-ME Gene WANG Yong-Xia1,2, DU Xin-Hua1,2, XU Wei-Gang1,2,*, QI Xue-Li2, LI Yan2, WANG Hui-Wei2, HU Lin2 1Nanjing Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China
2 Henan Provincial Laboratory of Wheat Biology / Wheat Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
Fund:This study was supported by the National GMO Program of China (2011ZX08002-003) and the China Agriculture Research System (CARS-3-1-9) AbstractTo explore the physiological characteristics of the transgenic wheat expressing maize C4-type NADP-ME, we introduced NADP-ME into the C3 crop wheat by using particle bombardment transformation. Two transgenic wheat lines (10T(9)-1-1, 10T(9)-225-4) and parental control (Zhoumai 23) were used to study molecular characteristics and photosynthesis property, to reveal the mechanism. The results showed that the NADP-ME sequence was integrated into wheat genome, and the transcription and translation were exactly same as expect. The enzyme activity of NADP-ME in flag leaf in transgenic plants were increased significantly than untransformed plants, for instance it was increased 1.33 and 1.13 times on the 7th day after flowering. Net photosynthetic rate ( Pn) of flag leaf in transgenic plants obviously decreased when compared to the untransformed plants. On the 7th day after anthesis, Pn of transgenic wheat decreased by 17.26% and 10.35%. The yield and 1000-grain weight were decreased than the control. Utilization efficiency on strong light utilizing and ability of CO2 assimilation in transgenic line 10T(9)-225-4 were significantly declined, photosynthesis rate was also decreased. Stomatal opening rate and stomatal conductance were significant decrease, malic acid content of transgenic wheat reducing 5.6% while pyruvate level is raised by 17.1%, and Pn of transgenic wheat can be restored by feeding with exogenous malate. Those results indicated that the transgenic wheat expressing maize NADP-ME gene showed lower photosynthetic characteristics than the control, the reason was maybe the decrease of stomatal aperture caused by decline of malic acid content.
Keyword:Transgenic wheat; NADP-dependent malic enzyme; Net photosynthetic rate; Stomata conductance Show Figures Show Figures
图2 转基因小麦及其对照(WT)不同生育期旗叶NADP-ME基因的相对表达量 内参基因为β -actin。* * 表示转基因植株与对照之间差异显著(P< 0.01)。Fig. 2 Relative expressions of NADP-MEin flag leaves of transgenic wheat lines and wild type (WT) at different stages Gene β -actinwas used as the internal reference. * * indicates significant difference between the transgenic plant and WT (P< 0.01).
图3 T3代转基因株系Western杂交(A)和杂交图片灰度分析(B)结果 以WT的表达蛋白含量为1。Fig. 3 Western blot (A) and gray level analysis (B) of T3 transgenic lines The expression protein content of WT was 1.
图4 不同生育期转基因小麦及对照(WT)旗叶NADP-ME酶活性(A)和净光合速率(B)的变化 * 和* * 分别表示转基因植株与对照在P< 0.05和P< 0.01水平差异显著。Fig. 4 Dynamic changes of NADP-ME enzyme activity(A) and photosynthetic rate (B) in flag leaves of transgenic wheat lines and the wild type (WT) at different stages * and * * indicate significant difference between the transgenic plant and WT at P< 0.05 and P< 0.01 level, respectively.
表2 转NADP-ME基因小麦和非转基因对照的产量及农艺性状比较 Table 2 Comparison of yield related traits betweenNADP-ME transgenic wheat lines and WT
株系 Line
株高 Plant height (cm)
分蘖数 Spike number per plant
小穗数 Number per spike
单株产量 Grain yield per plant (g)
单穗粒数 Grain number per spike
千粒重 1000-grain weight (g)
WT
67.05± 3.03
8.60± 1.31
19.50± 1.28
19.03± 3.72
42.49± 2.62
50.06± 2.76
10T(9)-1-1
68.12± 3.11
8.60± 2.06
18.50± 1.15
16.92± 2.35*
40.62± 4.54
45.03± 2.43*
10T(9)-225-4
67.17± 1.97
8.86± 2.00
18.64± 0.98
17.03± 2.27*
40.26± 3.97
45.10± 2.17*
Data are shown as mean ± SD of 30 individual plants. * indicates significant difference between the transgenic lines and WT at P < 0.05. WT: wild. 数据为30个单株重复的平均值± 标准差。* 表示转基因株系与对照差异显著(P< 0.05)。
表2 转NADP-ME基因小麦和非转基因对照的产量及农艺性状比较 Table 2 Comparison of yield related traits betweenNADP-ME transgenic wheat lines and WT
图5 转基因株系和对照(WT)不同时期旗叶净光合速率日变化曲线Fig. 5 Diurnal variation of photosynthetic rate of flag leaves in transgenic line and wild type (WT) at different stages
图6 转基因株系和对照(WT)不同时期旗叶Gs、Tr和Ci平均日变化曲线 图中数值均为4个生育期测定数值的平均值。Fig. 6 Average diurnal variation of Gs, Tr, and Ci of flag leaves in transgenic line and wild type (WT) at different stages Data are the mean of four growth stages.
表3 Table 3 表3(Table 3)
表3 转NADP-ME小麦不同生育时期的单位日光合总量 Table 3 Diurnal photosynthesis cumulative Pn in NADP-ME transgenic wheat lines at different growth stages (× 105 μ mol m-2)
材料 Line
抽穗期 Heading
开花期 Anthesis
花后第7天 7th day after anthesis
花后第15天 15th day after anthesis
平均 Average
WT
8.30± 0.63
7.83± 0.89
9.07± 0.57
8.48± 0.68
8.42± 0.66
10T(9)-225-4
7.63± 6.28
7.38± 0.75
7.80± 0.55*
7.88± 0.89
7.68± 0.73
Data are shown as Mean ± SD of 10 individual plants. * indicates significant difference between the transgenic lines and WT at P < 0.05. WT: wild type (Zhoumai 23). 数据为10个单株重复的平均值± 标准差。* 表示转基因株系与对照差异显著(P< 0.05)。
表3 转NADP-ME小麦不同生育时期的单位日光合总量 Table 3 Diurnal photosynthesis cumulative Pn in NADP-ME transgenic wheat lines at different growth stages (× 105 μ mol m-2)
图7 转基因小麦旗叶净光合速率对光照强度(A)和胞间CO2浓度(C)的响应曲线Fig. 7 Response curves of photosynthesis to PPDF (A) and Ci (C) in transgenic wheat
表4 Table 4 表4(Table 4)
表4 转NADP-ME小麦和非转基因对照苹果酸、丙酮酸含量比较 Table 4 Comparison of malic acid and pyruvate acid content betweenNADP-ME transgenic wheat and WT
株系 Line
苹果酸含量 Malic acid content (mg g-1)
丙酮酸含量 Pyruvate acid content (mg g-1)
WT
0.45± 0.07
0.12± 0.02
10T(9)-225-4
0.41± 0.07*
0.15± 0.01*
Data are shown as mean ± SD of 5 individual flag leaves. * indicates significant difference between the transgenic lines and WT at P < 0.05. WT: wild type (Zhoumai 23). 数据为5片旗叶重复的平均值± 标准差。* 表示转基因株系与对照差异显著(P< 0.05)。WT: 受体对照(周麦23).
表4 转NADP-ME小麦和非转基因对照苹果酸、丙酮酸含量比较 Table 4 Comparison of malic acid and pyruvate acid content betweenNADP-ME transgenic wheat and WT
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