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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (12): 2394-2406.doi: 10.3724/SP.J.1006.2021.04259

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

Identification and relative expression analysis of CBF gene family in Brassica napus L.

XIE Pan1,4(), LIU Wei1, KANG Yu1, HUA Wei1, QIAN Lun-Wen1,2,3, GUAN Chun-Yun1,2,3,*, HE Xin1,2,3,*   

  1. 1Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China, Hunan Agricultural University, Changsha 410128, Hunan, China
    2Oil Crops Research, Hunan Agricultural University, Changsha 410128, Hunan, China
    3Hunan Branch of National Oilseed Crops Improvement Center, Changsha 410128, Hunan, China
    4College of Landscape Architecture and Art Design, Hunan Agricultural University, Changsha 410128, Hunan, China
  • Received:2020-11-28 Accepted:2021-04-14 Online:2021-12-12 Published:2021-05-21
  • Contact: GUAN Chun-Yun,HE Xin E-mail:xiepan@hunau.edu.cn
  • Supported by:
    Science and Technology Innovation Program of Hunan Province(2020RC2057);National Natural Science Foundation of China(31801398)

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Abstract:

Low temperature is an important environmental stress factor affecting plant growth and development. The ICE1-CBF-COR [inducer of CBF expression1-c-repeat (CRT)-binding factors-cold responsive] plays an important role in the signaling pathway in response to low temperature stress in plants. To explore the evolution of CBF gene in Brassica napus L. and its function in response to low temperature stress, 11, 6, and 6 CBF genes were detected from Brassica napus, Brassica rapa, and Brassica oleracea, respectively. In addition, the molecular characteristics, protein conserved domains, phylogenetic tree, gene structure, and chromosome distribution were systematically investigated. The results showed that 11 BnaCBF genes could be divided into two subgroups including Subgroup I (CBF1/2/3) and Subgroup II (CBF4). Transcriptome sequencing revealed that all the CBF genes of Brassica napus were induced by low temperature, among which four genes in subgroup Ib responded rapidly and durably to cold stress, and two genes in Subgroup II responded weakly to cold stress, however, the relative expression level of CBF genes in roots was significantly higher than that in leaves, which were involved in salt stress and osmotic stress responses. Further analysis demonstrated that the CBF gene family in Brassica napus responded more strongly to freezing stress, especially BnaA08G30930D, BnaCnng49280D, BnaAnng34260D, BnaC07g39680D in Subsection I, and BnaA10g07630D, BnaC09G28190D in Subsection II. This study lays a foundation for further understanding the biological function of the CBF family gene and its response to low temperature, especially freezing stress in Brassica napus., and provides a reference for bioinformation research of CBF family genes in other species.

Key words: Brassica napus L., CBF, low temperature, chilling stress, relative expression level

Table 1

Primers for qRT-PCR used in this study"

基因名称
Gene name		 正向引物序列
Forward sequence (5°-3°)		 负向引物序列
Reverse sequence (5°-3°)
BnaCBF4-RT GAGACCACGGAAGAGAACGG AGTCGTTATGATTCCATCCAAGTT
BnaCBF1-C09-RT CGACTATGGTTGAGGCAGTGAAA AAGTCGTTATGATTCCATCCAAGTT
BnaCBF3-RT CCAAGCCGAGTCAGCAAAAT GAGAAACTCAGGTAAATGGGTGTG
BnaCBF1-Cnn-RT GAGACGACGGTGGAGGGTG AAGTCATCATTATGTCCCCATTGTA
BnaC05g34300D/BnaACTIN GGTTGGGATGGACCAGAAGG TCAGGAGCAATACGGAGC

Table 2

BnaCBF gene information"

基因名称
Gene ID		 核苷酸长度Nucleotide length (bp) 氨基酸长度
Amino
Acid
length		 等电点预测
Isoelectric point prediction		 分子量Molecular weight (kD) 内含子数目
Number of introns		 外显子数目Number of exons 亚细胞定位
Subcellular localization		 染色体定位
Chromosome location		 白菜同源基因Homologous gene in Brassica rape 甘蓝同源基因Homologous gene in Brassica oleracea
BnaA08g30950D 666 221 4.97 24.56 2 3 核酸Nuclear chrA08_random:1781546..1782397 Bra010460 Bo8g050800
BnaUnng01150D 651 216 5.34 23.81 1 3 核酸Nuclear chrUnn_random:1448150..1448983
BnaA08g30910D 654 217 5.13 24.02 2 3 核酸Nuclear chrA08_random:1768075..1768896 Bra010461
BnaA08g30930D 570 189 4.73 20.57 2 3 核酸Nuclear chrA08_random:1771443..1772198 Bra010463 Bo8g050740
BnaCnng49280D 576 191 4.58 20.83 2 3 核酸Nuclear chrCnn_random:48753744..48754496
BnaA03g13620D 645 214 4.58 20.82 0 1 核酸Nuclear chrA03:6228484..6229128 Bra022770 Bo3g024240
BnaC03g71900D 645 214 5.03 23.79 0 1 核酸Nuclear chrC03_random:417520..418164
BnaAnng34260D 645 214 5.21 23.83 0 1 核酸Nuclear chrAnn_random:39020794..39021438 Bra019162 Bo7g110320
BnaC07g39680D 648 215 5.23 23.81 0 1 核酸Nuclear chrC07:40421468..40422115
BnaA10g07630D 663 220 5.52 24.55 0 1 核酸Nuclear chrA10:6107845..6108507 Bra028290 Bo9g104770
BnaC09g28190D 663 220 6.14 24.5 0 1 核酸Nuclear chrC09:30491719..30492381

Fig. 1

Multiple alignment of CBF proteins of Brassica napus, Brassica rapa, Brassica oleracea, and Arabidopsis Black: the same bases among 26 sequences; Red: the same bases among 18-25 sequences; Blue: the same bases among 9-17 sequences; White: the same bases among 1-8 sequences."

Fig. 2

Systematic comparative analysis of CBF of Brassica napus, Brassica rapa, Brassica oleracea, and Arabidopsis chromosomes A: Neighbor-Joining phylogenetic tree; B: gene structures; C: MEME motifs analysis."

Fig. 3

Distribution of the CBF genes of Brassica napus, Brassica rapa, Brassica oleracea, and Arabidopsis chromosomes The numbers of chromosomes are labeled on the left side of each chromosome. Chr is the chromosome of Arabidopsis; A and C are the chromosomes from Brassica rapa, Brassica oleracea; chrA and chrC are the chromosomes of the A-subgenomes and C-subgenomes from Brassica napus, respectively; Random means genes are randomly distributed to the specific chromosome, and chrAnn and chrCnn are unanchored scaffolds that cannot be mapped to a specific chromosome from the A-subgenomes and C-subgenomes, respectively, chrUnn is also an unanchored scaffold."

Fig. 4

Heat map of relative expression level of BnaCBF genes in the different tissues and organs The expression levels of each gene (FPKM values) in each sample are indicated by different colored rectangles. Red block indicates relative higher expression level. Blue block indicates relative lower expression level."

Fig. 5

Heat map of the BnaCBF genes under different abiotic stresses and plant hormone treatments The relative expression levels of each gene (FPKM values) in each sample are indicated by different colored rectangles. Red block indicates relative higher expression level, blue block indicates relative lower expression level. Leaf_3h, Leaf_6h, and Leaf_12h indicates normal growth leaf of 3, 6, and 12 hours, respectively; Hot_3h, Hot_6h, and Hot_12h indicates 40℃ treatment growth leaf of 3, 6, and 12 hours, respectively; ABA_3h, ABA_6h, and ABA_12h indicates abscisic acid treatment growth leaf of 3, 6, and 12 hours, respectively; MeJA_3h, MeJA_6h, and MeJA_12h indicates methy jasmonate treatment growth leaf of 3, 6, and 12 hours, respectively; ET_3h, ET_6h, and ET_12h indicates Ethylene treatment growth leaf of 3, 6, and 12 hours, respectively; SA_3h, SA_6h, and SA_12h indicates salt stress treatment growth leaf of 3, 6, and 12 hours, respectively; Root_3h, Root_6h, and Root_12h indicates normal growth root of 3, 6, and 12 hours, respectively; NaCl_3h, NaCl_6h, and NaCl_12h indicates sodium chloride treatment growth root of 3, 6, and 12 hours, respectively; PEG_3h, PEG_6h, and PEG_12h indicates drought stress treatment growth root of 3, 6, and 12 hours, respectively."

Fig. 6

Heat map of the BnaCBF genes under different low-temperature treatments HX17_MA: mock A, HX17 material under normal growth conditions; HX17_CA: cold A, HX17 material treated at 4℃ after 14 days of domestication; HX17_FA: freezing A, HX17 material treated at -4℃ after 14 days of domestication. HX17_MB: mock B, HX17 material under normal growth condition; HX17_CB: cold B, HX17 material treated at 4℃; HX17_FB: freezing B, HX17 material treated at -4℃. HX58_MA: Mock A, HX58 material under normal growth conditions; HX58_CA: cold A, HX58material treated at 4℃ after 14 days of domestication; HX58_FA: freezing A, HX58 material treated at -4℃ after 14 days of domestication. HX58_MB: mock B, HX58 material under normal growth condition; HX58_CB: cold B, HX58 material treated at 4℃; HX58_FB: freezing B, HX58 material treated at -4℃."

Fig. 7

Relative expression patterns of BnaCBF genes under cold and MeJA treatments Treatments are the same as those given in Fig. 5."

Fig. 8

Relative expression patterns of BnaCBF genes under cold stresses treatments 17_MA: mock A, HX17 material under normal growth conditions; 17_CA: cold A, HX17 material treated at 4℃ after 14 days of domestication; 17_FA: freezing A, HX17 material treated at -4℃ after 14 days of domestication. 17_MB: mock B, HX17 material under normal growth condition; 17_CB: cold B, HX17 material treated at 4℃; 17_FB: freezing B, HX17 material treated at -4℃."

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