Chinese Bulletin of Botany ›› 2017, Vol. 52 ›› Issue (6): 713-722.DOI: 10.11983/CBB16239
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Liu Kaige, Qi Shuanghui, Duan Shaowei, Li Dong, Jin Changyu, Gao Chenhao, Liu Mingxun Chen Xuanxia*()
Received:
2016-12-05
Accepted:
2017-04-03
Online:
2017-11-01
Published:
2017-11-22
Contact:
Liu Mingxun Chen Xuanxia
Liu Kaige, Qi Shuanghui, Duan Shaowei, Li Dong, Jin Changyu, Gao Chenhao, Liu Mingxun Chen Xuanxia. Functional Analysis of Brassica napus BnTTG1-1 Gene[J]. Chinese Bulletin of Botany, 2017, 52(6): 713-722.
Primer name | Primer sequence (5′-3′) | Annotation |
---|---|---|
AtACTIN7-F | GCCCCTGAGGAGCACCCAGTT | RT-PCR |
AtACTIN7-R | CCGGTTGTACGACCACTGGCA | |
BnTTG1-1-F | GCCAGTATCCGTCCTCAACA | RT-PCR |
BnTTG1-1-R | CTCCCAGATAAGAGCCTGCG | |
BnACTIN7-F | GGAGCTGAGAGATTCCGTTG | qRT-PCR |
BnACTIN7-R | GAACCACCACTGAGGACGAT | |
BnTTG1-1-F | CTGCAGTGGTCTTCTTCGTT | qRT-PCR |
BnTTG1-1-R | GTTACAATCACATAGATGCAGAGAC | |
BnTTG1-1-Xma1-F | TATTcccgggATGGACAACTCAGCTCCAGACTC | 35S:BnTTG1-1-GFP and 35S:BnTTG1-1 |
BnTTG1-1-Spe1-R | GGactagtAACTCTAAGGAGCTGCATTTTGTTAGC |
Table 1 Sequences of primers
Primer name | Primer sequence (5′-3′) | Annotation |
---|---|---|
AtACTIN7-F | GCCCCTGAGGAGCACCCAGTT | RT-PCR |
AtACTIN7-R | CCGGTTGTACGACCACTGGCA | |
BnTTG1-1-F | GCCAGTATCCGTCCTCAACA | RT-PCR |
BnTTG1-1-R | CTCCCAGATAAGAGCCTGCG | |
BnACTIN7-F | GGAGCTGAGAGATTCCGTTG | qRT-PCR |
BnACTIN7-R | GAACCACCACTGAGGACGAT | |
BnTTG1-1-F | CTGCAGTGGTCTTCTTCGTT | qRT-PCR |
BnTTG1-1-R | GTTACAATCACATAGATGCAGAGAC | |
BnTTG1-1-Xma1-F | TATTcccgggATGGACAACTCAGCTCCAGACTC | 35S:BnTTG1-1-GFP and 35S:BnTTG1-1 |
BnTTG1-1-Spe1-R | GGactagtAACTCTAAGGAGCTGCATTTTGTTAGC |
Figure 1 qRT-PCR analysis of BnTTG1-1 expression in de- veloping seeds at different developmental stages in Brassica napus cv. ‘QINYOU Seven’ (means±SD)DAP: Days after pollination. The qRT-PCR result was normaliz- ed against the expression of BnACTIN7 as an internal control.
Figure 2 Subcellular localization of BnTTG1-1 protein fus- ed with GFP (35S:BnTTG1-1-GFP) in tobacco (Nicotiana ben- thamiana) leave cellsDAPI: 4’, 6-diamidino-2-phenylindole dihydrochloride; GFP: Green fluorescent protein; Merge: Merged picture of bright, DAPI, and GFP fields. Bars=5 μm
Figure 3 Identification of ttg1-13 35S:BnTTG1-1 transgenic plants(A) PCR-based DNA and RNA genotyping of ttg1-13 35S: BnTTG1-1 transgenic plants, AtACTIN7 was regarded as an internal control; (B) Heterologous expression of BnTTG1-1 in the ttg1-13 background fully rescued no trichomes and anthocyanins phenotypes of ttg1-13.
Figure 4 Comparison of seed coat color, seed size and se- ed weight among the wild-type (Col-0), ttg1-13, and ttg1-13 35S:BnTTG1-1 transgenic plants of Arabidopsis (means±SD)(A) Microscopic observation of mature seeds; (B) Comparison of seed size and weight of mature seeds
Figure 5 Comparison of seed storage compounds among the wild-type (Col-0), ttg1-13, and ttg1-13 35S:BnTTG1-1 trans- genic plants of Arabidopsis (means±SD)(A) The content of seed storage proteins in different lines; (B) The content of seed fatty acids in different lines. Asterisks de- note statistically signi?cant differences between the wild-type and ttg1-13 mutant (Student’s t test, P<0.05).
Figure 6 Seed germination rate and seedling establishment on MS agar medium containing 3% (w/v) Glucose and containing 100 mmol·L-1 NaCl among the wild-type (Col-0), ttg1- 13, and ttg1-13 35S:BnTTG1-1 transgenic plants of Arabidopsis(A) Seed germination rate; (B) Seedling establishment. Values are the means±SD from three independent experiments evaluating 100 seeds.
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