关键词:小麦; MYB转录因子; 防御反应; 小麦根腐病; 转基因 Development and Characterization of TaMYB86-Overexpressing Transgenic Wheat Lines with Resistance to Common Root Rot SHAN Tian-Lei, HONG Yan-Tao, DU Li-Pu, XU Hui-Jun, WEI Xue-Ning, ZHANG Zeng-Yan* National Key Facility for Crop Gene Resources and Genetic Improvement / Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture / Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China Fund:The study was supported by the National Major Project for Developing New GM Crops (2016ZX08002-001-004) and the National Natural Science Foundation of China (31471494) AbstractWheat common root rot, mainly caused by Bipolaris sorokiniana, is a difficultly prevent soil-borne disease of wheat ( Triticum aestivum L.) worldwide. TaMYB86, a B. sorokiniana-induced wheat MYB gene ,encodes a MYB transcription factor. We constructed the TaMYB86 overexpression vector pUbi:MYC-TaMYB86 and introduced TaMYB86 into Yangmai 16 via the particle bombardment. The TaMYB86 transgenic wheat lines on generations of T0-T3 were underwent by molecular characteristics analysis and disease resistance evaluation. The PCR and quantitative RT-PCR results showed that the alien TaMYB86was introduced into three transgenic wheat lines, and the relative transcriptional level of TaMYB86was apparently higher in transgenic wheat lines than in non-transformed Yangmai 16. As Western blot results presented, the introduced MYC-TaMYB86 gene was translated into the MYC-TaMYB86 protein in the three overexpressing transgenic lines, but not in non-transformed Yangmai 16. The infection types and disease indexes of three TaMYB86 transgenic wheat lines were significantly lower than those of non-transformation Yangmai 16 ( t-test, P< 0.01). The transcript levels of 3 wheat defense genes ( PR10, PR17c, and Chit1) were significantly elevated in three transgenic wheat lines than in the non-transformed Yangmai 16. These results indicate that overexpression of TaMYB86 enhances significantly resistance to B. sorokiniana in transgenic wheat lines and TaMYB86 plays a positive role in defense response to B. sorokiniana.
Keyword: Triticum aestivum; MYB transcription factor; Defense response; Common root rot; Transgene Show Figures Show Figures
图3 过表达株系中TaMYB86的转录水平分析WT: 未转基因扬麦16; MO86-7、MO86-14、MO86-28: TaMYB86过表达株系。每个样本进行3次重复实验, 采用t检验分析差异显著性; * * P < 0.01概率水平极显著。Fig. 3 qRT-PCR analysis of TaMYB86transcriptional levels in transgenic wheat linesWT: wild type, non-transformed Yangmai 16; MO86-7, MO86-14, MO86-28: TaMYB86transgenic wheat lines. Three biological replicates for each line were averaged and statistically treated (t-test; * * P < 0.01).
图4 根腐病菌接种后TaMYB86的表达模式Y16: 未转基因扬麦16; MO86: TaMYB86过表达株系MO86-7、MO86-14、MO86-28的混合样品。每个样本进行3次重复实验。Fig. 4 Transcriptional patterns of TaMYB86 in B. sorokiniana-inoculated wheatY16: non-transformed Yangmai 16; MO86: pooled samples of transgenic wheat lines MO86-7, MO86-14, and MO86-28.
图5 转TaMYB86基因过表达株系和未转基因扬麦16的Western杂交分析WT: 未转基因扬麦16; MO86-7, MO86-14, MO86-28: TaMYB86过表达株系。箭头指示MYC-TaMYB86融合蛋白的位置。Fig. 5 Western blot pattern of TaMYB86-overexpressed transgenic lines and non-transformed Yangmai 16 using an anti-MYC antibodyWT: wild type, non-transformed Yangmai 16; MO86-7, MO86-14, MO86-28: TaMYB86transgenic wheat lines. The arrow shows the position of MYC-TaMYB86 fusion protein.
表1 Table 1 表1(Table 1)
表1 转TaMYB86基因过表达株系与未转基因扬麦16的根腐病抗性鉴定 Table 1 Common root rot response of TaMYB86-overexpressed transgenic lines and non-transformed wheat Yangmai 16
株系 Line
T1
T2
T3
IT
DI
IT
DI
IT
DI
MO86-7
1.27± 0.43* *
31.67± 10.87* *
1.51± 0.45* *
37.76± 11.33* *
1.67± 0.49* *
41.67± 12.13* *
MO86-14
2.00± 0* *
50.00± 0* *
1.50± 0.53* *
37.50± 13.18* *
1.25± 0.43* *
31.25± 10.69* *
MO86-28
1.80± 0.45* *
45.00± 11.18* *
1.54± 0.34* *
38.43± 8.42* *
1.50± 0.50* *
37.50± 12.50* *
CI12633
1.15± 0.23
28.69± 5.77
1.07± 0.09
26.85± 2.21
1.17± 0.24
29.33± 6.10
阴性植株 NTP
2.80± 0.24
70.00± 6.12
2.15± 0.28
53.75± 7.07
2.56± 0.66
64.00± 16.55
扬麦16 Yangmai 16
3.00± 0.17
75.04± 4.14
2.17± 0.46
54.17± 11.49
2.62± 0.77
65.38± 19.20
MO 86-7, MO86-14, and MO86-28 represent TaMYB86-overexpression transgenic wheat lines. Negative-transgenic plants indicate negative PCR plants. IT represents infection type. DI indicates disease index of common root rot. Response value for each line is the mean of at least 10 plants in each generation. Statistically significant differences between each line ofTaMYB86-overexpression wheat and non-transformed Yangmai 16 were determined using t-test (* * P < 0.01). NTP: negative-transgenic plants. MO86-7、MO86-14、MO86-28代表TaMYB86-过表达的转基因株系; 阴性植株表示转基因检测为阴性的植株; 扬麦16代表非转基因扬麦16。IT: 感染类型; DI: 病情指数; 每个株系对至少10株小麦进行抗病鉴定; 采用t检验分析差异显著性; * * P < 0.01概率水平极显著。
表1 转TaMYB86基因过表达株系与未转基因扬麦16的根腐病抗性鉴定 Table 1 Common root rot response of TaMYB86-overexpressed transgenic lines and non-transformed wheat Yangmai 16
图6TaMYB86过表达株系中TaMYB86和下游防卫基因的qRT-PCR分析接种根腐病菌47 d后TaMYB86过表达株系和未转基因扬麦16 (WT)植株中TaMYB86和下游防卫基因的转录表达。防卫基因包括 PR10 (CA613496)、PR17c (TA65181)和Chit1 (CA665185)。采用t检验分析差异显著性; * * P < 0.01极显著。Fig. 6 qRT-PCR analysis of the relative transcript level of TaMYB86 and three defense genes in TaMYB86-overexpressing transgenic linesThe expression of TaMYB86 and three defense genes in TaMYB86-overexpressing transgenic and non-transformed wheat Yangmai 16 (WT) inoculated with B. sorokiniana for 47 d. Defense genes include PR10 (CA613496), PR17c (TA65181) and Chit1 (CA665185). Three biological replicates of each line were averaged and statistically treated (t-test; * * P < 0.01).
4 结论通过基因枪转化、分子检测和抗根腐病鉴定, 创制、筛选出抗根腐病的TaMYB86过表达转基因小麦新种质3份, 证明TaMYB86在小麦防御根腐病过程中起正向调控作用。 The authors have declared that no competing interests exist.
作者已声明无竞争性利益关系。The authors have declared that no competing interests exist.
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