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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (11): 1678-1689.doi: 10.3724/SP.J.1006.2020.04036

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Regulatory mechanism of the seed coat color gene BrTT1 in Brassica rapa L.

WANG Yan-Hua1,2(), JIAN Hong-Jiu1, QIU Xiao2, LI Jia-Na1,*()   

  1. 1 College of Agronomy and Biotechnology, Southwest University / Chongqing Engineering Research Center for Rapeseed, Chongqing 400715, China
    2 University of Saskatchewan, Saskatoon S7N5A8, Canada
  • Received:2020-02-17 Accepted:2020-07-02 Online:2020-11-12 Published:2020-07-15
  • Contact: Jia-Na LI E-mail:hawer313@163.com;ljn1950@swu.edu.cn
  • Supported by:
    This study was supported by the Project of China Postdoctoral Science Foundation(2019M653319);the Project of Chongqing Natural Science Foundation Postdoctoral Science Foundation(cstc2019jcyj-bsh0102);Project of Intellectual Base for Discipline Innovation in Colleges and Universities (“111” Project)(B12006)

Abstract:

Brassica rapa (B. rapa L., 2n = 20, AA) is a specie of Brassica genus, belonging to the basic species of cultivated rapeseed. China is the original center of Chinese cabbage and Brassica campestris. Compared with Brassica napus, it has a long history of origin and cultivation and rich genetic resources, which has natural and stable yellow seed resources. Dahuang has the natural yellow seed resource in B. rapa. Its seed coat color is bright yellow, the yellow seed trait can be stably inherited, and Dahuang has the advantages of large grain, high oil content and good self-adhesiveness. Sequence comparison showed that nucleotide polymorphisms were solely found in BrTT1 sequences from different seed color lines (yellow, red or brown, and black), which could be used to predict seed color phenotype. Yeast two-hybrid analysis indicated BrTT1 could interact with two other transcriptional factors R2R3-MYB (BrTT2) and WD40 (BrTTG1), and one catalytic enzyme (BrTT3). Quantitative RT-PCR analysis of transgenic B. rapa lines with the gene down-regulated by RNA interference and up-regulated by overexpression revealed that two contrasting groups of genes were regulated by BrTT1 in the biosynthesis and deposition of flavonoids pigments in the seed of B. rapa. These results further define the regulatory activity of BrTT1 in seed coat color formation in Brassica species.

Key words: Brassica rapa, BrTT1, flavonoid, yeast two-hybrid

Supplementary Table 1

Yellow- and red- and black-seeded materials in B. rapa"

编号
No.
材料名称和来源
Material name and origin
种子粒色表型
Phenotype of the seeds
编号
No.
材料名称和来源
Material name and origin
种子粒色表型
Phenotype of the seeds
Y1 大黄(中国青海)
Dahuang (Qinghai, China)
纯黄籽
Yellow seeds
R6 18B167-168 (中国青海)
18B167-168 (Qinghai, China)
红褐籽
Red or brown seeds
Y2 18B07-08 (中国青海)
18B07-08 (Qinghai, China)
纯黄籽
Yellow seeds
R7 18B171-172 (中国青海)
18B171-172 (Qinghai, China)
红褐籽
Red or brown seeds
Y3 彭波黄(中国青海)
Pengbo yellow (Qinghai, China)
纯黄籽
Yellow seeds
R8 18B141-142 (中国青海)
18B141-142 (Qinghai, China)
红褐籽
Red or brown seeds
Y4 18B001-002 (中国青海)
18B001-002 (Qinghai, China)
纯黄籽
Yellow seeds
R9 18B51-52 (中国青海)
18B51-52 (Qinghai, China)
红褐籽
Red or brown seeds
Y5 Sarson (印度)
Sarson (India)
纯黄籽
Yellow seeds
B1 浩油 11号(中国青海)
Haoyou 11 (Qinghai, China)
纯黑籽
Black seeds
Y6 18BCan01 (加拿大)
18BCan01 (Canada)
纯黄籽
Yellow seeds
B2 芦花小油菜(中国青海)
Luhua rapeseed (Qinghai, China)
纯黑籽
Black seeds
R1 18B017-018 (中国青海)
18B017-018 (Qinghai, China)
红褐籽
Red or brown seeds
B3 街子小油菜(中国青海)
Jiezi rapeseed (Qinghai, China)
纯黑籽
Black seeds
R2 18B021-022 (中国青海)
18B021-022 (Qinghai, China)
红褐籽
Red or brown seeds
B4 18B077-078 (中国青海)
18B077-078 (Qinghai, China)
纯黑籽
Black seeds
R3 18B105-106 (中国甘肃)
18B105-106 (Gansu, China)
红褐籽
Red or brown seeds
B5 麻玉小油菜(中国青海)
Mayu rapeseed (Qinghai, China)
纯黑籽
Black seeds
R4 18B131-132 (中国青海)
18B131-132 (Qinghai, China)
红褐籽
Red or brown seeds
B6 18B161-162 (中国青海)
18B161-162 (Qinghai, China)
纯黑籽
Black seeds
R5 18B165-166 (中国青海)
18B165-166 (Qinghai, China)
红褐籽
Red or brown seeds
B7 18BCan02 (加拿大)
18BCan02 (Canada)
纯黑籽
Black seeds

Supplementary Table 2

Primers sequences for vector construction"

引物名称
Primer name
正向引物
Forward primer (5°-3°)
反向引物
Reverse primer (5°-3°)
Yb-F/-R CCGGAATTCATGTTTTCATCACTCTCAAACCACT CGCGGATCCTTAAAAGTGCGTTTCAGAGACAGA
T2-F/-R CCGGAATTCATGATGAGAAAGAGAGAAAGTAGTA CGCGGATCCCTAACAATTAAAGTCCCAGAGACAA
T3-F/-R CCGGAATTCATGATTCTGGAGGAGAAAGATGCAT CCCATCGATTTAGCTATCTGAACGTTTTGGATCG
T7-F/-R CCGGAATTCATGGTGATGGGTACACCGTCTTCGT CGCGGATCCTCAAACAGGAACGCTGTGCAAGACC
T8-F/-R CCGGAATTCATGGATGAATTAAGTATTATACCGT CGCGGATCCCTAGAGTTTATTATTATATATGATT
T18-F/-R CCGGAATTCATGGGTAGCAAGATGCTGTTCAGTT CGCGGATCCTTAGCTTGCTTCTGGCAATGTGATC
TG1-F/-R CCGGAATTCATGGACAACTCAGCTCCGGACTCCT CGCGGATCCTCAAACTCTAAGGAGCTGCATTTTG
TG2-F/-R CCGGAATTCATGGAGGTGAAAGAGAGTAAGAGAG CGCGGATCCTCAAATGGCTTGATTAGAATGTTGT
T10-F/-R CCGGAATTCATGACAAGCACTGATCAGACCACCG CGCGGATCCTTAGGCCCATCGGTTTGTCTTAAAG
T12-F/-R CCGGAATTCATGGGGAGGAAGACGTGGTTCGACG CGCGGATCCTCAGAAGAGATAATCAAGTATCTGA
T5-F/-R CCGGAATTCATGAGACATTCTTGCTGTTATAAAC CGCGGATCCCTAAAGGGACTGACCAAAAGAGACA
F/R-TT1 ATGTTTTCATCACTCTCAAACCACT TTAAAAGTGCGTTTCAGAGACAGA
gCb-F/-R CACCATGTTTTCATCACTCTCAAACCACT AAAGTGCGTTTCAGAGACAGA
F35S3ND/CB-R GGAAGTTCATTTCATTTGGAGAG AAAGTGCGTTTCAGAGACAGA
FBTT1I GGATCCGACGTCGAGATCTATAAACATTCCC

Supplementary Table 3

Primer sequences for differential expression analysis"

引物名称
Primer name
正向引物
Forward primer (5°-3°)
反向引物
Reverse primer (5°-3°)
28S ATAACCGCATCAGGTCTCCAAG CCTCAGAGCCAATCCTTTTCC
BrTT1 TCGCTACAACAATCTTCAGATGCACA TCCTGCACCCTTCAACGCAGC
BrTT2 AGCTGGTCTCAAGAGGTGTGGCA AGCCTCCCAGCTATCAACGACC
BrTT3 CAGGATGGATGTATTTCATGTCG TGTGCCTCGTTACGAGTGATAG
BrTT4 GACTACTACTTCCGCATCACCAACAG GCCTAGCTTAGGGACTTCAACAACC
BrTT5 CTTCCTCGGTGGCGCAGGTG ACACAGTTCTCCGTTACTTTCTCTGA
BrTT6 CGAGAAAGAGGCACTCACCAATG TGAACCTCCCGTTGCTCAGATA
BrTT7 GTGGTTGCCGCCTCTAAATC CTAGCTCGCGCATGAGTGTTC
BrTT8 GGCTGAAGAGGCTGCGTCGG GTGCTGTGCAAGCCCTCGCT
BrTT10 GCGACTGTGCCAAGAAACGGT CCCCACGTGAGATGTCTATCAAAGTG
BrTT12 GCTCCACAGAGACATACGAGCCG ACGGTGACGAAGCTGAGCATGTA
BrTT16 TGCTCACATCGGTCTCATCGTCT GCTCGTGTGGAGGAATGGAGGC
BrTT18 AAGAAGAGCTCGAGAGCATCAAC GCATACTTCTCCTTCTCTTCCAC
BrTTG1 TCCTCCGGCGACTTCCTCCG GCTGCGTCTCCACCACGGAC
BrTTG2 AAACCTAAAGCAAAGCTTGTCTCCCA ACTTCCTTTGACTTGCTTCTGTCCGT
BrBAN TTAACTGGGCATACCCAATCTC TGCATTTCTTTCCGGGTAATC

Fig. 1

BrTT1 homologous genes amino acid sequences alignment"

Fig. 2

Nucleotide polymorphisms of BrTT1 genes from different seed color lines A: phenotypes of B. rapa lines (black, reddish-brown, and yellow) with different seed colors; B: nucleotide sequences of B. rapa BrTT1 from the three lines with different seed color. TT1-B was BrTT1-B sequence amplified from the black seeds; TT1-R was BrTT1-R sequence amplified from the reddish-brown seeds; TT-Y was BrTT1-Y sequence amplified from the yellow seeds. Nucleotide polymorphisms were highlighted by color, the line below the consensus sequence represents the intron region. Bar = 5000 μm."

Fig. 3

Expression analysis of BrTT1 in the three types of B. rapa lines B1: a black seed line; R2: a reddish-brown seed line; Y2: a yellow seed line. DAF: days after flowering."

Fig. 4

Protein-predictive analysis of BrTT1 in B. rapa"

Table 1

Prediction of genetic information for interaction with BrTT1 in B. rapa"

基因ID
Gene ID
蛋白类型
Protein type
打分值
Score
基因ID
Gene ID
蛋白类型
Protein type
打分值Score
Bra027457 BrTT3 0.996 Bra005210 BrTTG2-A, WRKY44 0.814
Bra035532 BrTT2 0.992 Bra023112 BrTTG2-B 0.814
Bra009770 BrTTG1 0.909 Bra026408 Uncharacterized protein 0.607
Bra037887 BrTT8 0.894 Bra013652 BrTT18 0.568
Bra009312 BrTT7 0.885 Bra016108 Uncharacterized protein 0.568
Bra040822 Uncharacterized protein 0.882 Bra009101 Uncharacterized protein 0.568
Bra003361 BrTT12 0.881 Bra039487 Uncharacterized protein 0.434
Bra007142 BrTT5 0.822 Bra035364 Uncharacterized protein 0.434
Bra037510 BrTT10-2 0.821 Bra006205 Uncharacterized protein 0.379
Bra020720 BrTT10-1A 0.821 Bra021170 Uncharacterized protein 0.365

Fig. 5

Screening for interactors of BrTT1 by a yeast two-hybrid system Yeast cells were co-transformed and plated on -Leu/-Trp/-Ade/-His plates, with pGADT7-T/pGBKT7-53 as the positive control and pGADT7-T/pGBKT7-laminC as the negative control. X-α-gal activity was determined by colony-lift filter assay; the color intensity of the colonies depicts the qualitative binding strength of the interaction."

Supplementary Fig. 1

PCR amplification of a transgene fragment in transgenic plants with BrTT1 down-regulated by RNAi using genomic DNA isolated from the seedlings as templates The primer of F35S3ND/FBTT1I ( Supplementary Table 2) was used for the amplification of a fragment size at about 1.0 kb. + is a positive control."

Supplementary Fig. 2

PCR amplification of a transgene fragment in transgenic plants over-expressing BrTT1 using genomic DNA isolated from the seedlings as templates The primer of F35S3ND/CB-R (Supplementary Table 2) was used for the amplification of a fragment size at about 1.0 kb. + is a positive control."

Fig. 6

Expression of BrTT1 in RNAi and over-expression lines A: differential expression analysis of BrTT1 in seeds of the transgenic lines; O1-O4: over-expression lines; Ri1-Ri4: RNAi expression lines; B: non-transgenic lines as the control; DAF: days after flowering. B: left, middle and right represent seed color phenotype in transgenic lines of over-expression, RNAi expression, and the non-transgenic black seed lines, respectively. Bar = 5000 μm."

Fig. 7

Expression analysis of genes involved in the flavonoid biosynthesis in BrTT1-RNAi and over-expression lines O1-O4: over-expression lines; Ri1-Ri4: RNAi expression lines; DAF: days after flowering."

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