Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (1): 152-163.doi: 10.3864/j.issn.0578-1752.2021.01.011

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Functional Analysis of BraERF023a Under Salt and Drought Stresses in Cauliflower (Brassica oleracea L. var. botrytis)

LI Hui1(),HAN ZhanPin1,HE LiXia2(),YANG YaLing1,YOU ShuYan2,DENG Lin2,WANG ChunGuo2   

  1. 1College of Horticulture and Landscape, Tianjin Agricultural University, Tianjin 300384
    2College of Life Sciences, Nankai University, Tianjin 300071
  • Received:2020-04-12 Accepted:2020-06-29 Online:2021-01-01 Published:2021-01-13
  • Contact: Hui LI,LiXia HE E-mail:lihui@tjau.edu.cn;helx@mail.nankai.edu.cn

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Abstract:

【Objective】In our previous study, Ethylene-responsive factors (ERFs) involved in abiotic stress responses in cauliflower were identified. In the present study, one of these ERF transcription factors with unknown function, named BraERF023a, was cloned. The expression profiles and function of BraERF023a under salt and drought stresses were further explored. 【Method】BraERF023a was cloned by RT-PCR in cauliflower. The sequence characteristics of BraERF023a were analyzed by biosoftwares such as MEGA 6. The expression profiles of BraERF023a under salt and drought stresses were explored by qRT-PCR. BraERF023a overexpression vector was then constructed and genetically transformed into Arabidopsis. The phenotypes and survival rate of overexpression BraERF023a transgenic Arabidopsis under salt and drought stresses were observed and analyzed.【Result】Sequence analysis confirmed that BraERF023a coding region was 597 bp in length and encoded a protein containing 198 amino acids with an AP2 conserved domain. BraERF023a was highly conserved in Brassica. The expression level of BraERF023a significantly increased under both salt and drought stresses in cauliflower. Functional analysis indicated that comparative with the wild-type controls, overexpression BraERF023a transgenic Arabidopsis lines exhibited better growth vigor and higher plant survival rate under salt or drought stress.【Conclusion】 BraERF023a played important roles in positive response to salt and drought stresses in cauliflower. Overexpression of BraERF023a in Arabidopsis could significantly improve the tolerance of transgenic lines to salt and drought stresses.

Key words: cauliflower, ERF transcription factor, BraERF023a, salt stress, drought stress

Table 1

Primers used for BraERF023a cloning, vector construction and qRT-PCR assay"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)
BraERF023a-F ATGCTGAGTACAGTGAGTGAAACC
BraERF023a-R TCACAGACACGCCATGAACTG
Nco I-BraERF023a-F CCATGGATGCTGAGTACAGTGAGTG
BstE II-BraERF023a-R GGTCACCTCACAGACACGCCAT
qBraERF023a-F CCACCACTTACCAACGAAC
qBraERF023a-R AAGATCCGAGCCAAATCCG
35S-F AACAGAACTCGCCGTAAAG
BraActin-F GCTCCTCTTAACCCAAAGGC
BraActin-R CACACCATCACCAGAATCCAGC
AtActin-F GGTAACATTGTGCTCAGTGGTGG
AtActin-R AACGACCTTAATCTTCATGCTGC

Fig. 1

Sequence analysis of BraERF023a in cauliflower Sequences marked by the black lines indicated the AP2 domain, and the boxes indicated the conserved fourteenth and nineteenth amino acids in the AP2 domain of ERFs, respectively"

Fig. 2

Expression profiles of BraERF023a under salt (a) and drought (b) stresses detected by qRT-PCR ** indicated the difference is significant at the 0.01 level. The same as below"

Fig. 3

Identification of BraERF023a in Agrobacterium tumefaciens and transgenic lines in Arabidopsis a: Identification of Agrobacterium tumefaciens with BraERF023a by PCR used the specific primers and combined primers, respectively. M: DNA marker, 1-6 indicated PCR identification used the specific primers, 1′-6′ indicated PCR identification used the combined primers. b: Identification of individual Arabidopsis plant with BraERF023a by PCR used the specific primers and combined primers, respectively. M: DNA marker, 1-10 indicated PCR identification of individual plants used the specific primers, 1′-10′ indicated PCR identification of individual plant used the combined primers. c: Expression levels of BraERF023a in differential overexpression BraERF023a transgenic Arabidopsis lines detected by qRT-PCR, CK: Wild-type control, L3/L4/L6/L8/L9 indicated the five independent transgenic lines"

Fig. 4

Phenotypes of overexpression BraERF023a transgenic Arabidopsis at different developmental stages a, b and c indicated the phenotypes of 3-week-old, 4-week-old and 8-week-old overexpression BraERF023a transgenic Arabidopsis and the control (CK). L4/L8/L9 indicated three independent overexpression BraERF023a transgenic lines"

Table 2

Phenotypic analysis of overexpression BraERF023a transgenic Arabidopsis"

表型Phenotype 对照CK L4 L8 L9
株高 Stem length (cm) 43.125±2.780c 56.750±3. 862a 51.125±1.0307ab 49.625±3.0923b
抽薹时间 Bolting time (d) 26.00±2.4495b 32.00±2.4495a 29±1.9148a 31.00±1.2583a
茎粗 Stem diameter (mm) 1.2375±0.0727b 1.7800±0.2191a 1.6300±0.1551a 1.7125±0.1059a
主茎 Stem (number) 5.75±0.9574c 6.75±1.0708ab 6.25±0.5000b 7.775±0.5774a
一级分枝 Primary branch (number) 13.75±2.630b 15.00±2.449ab 16.00±3.742a 13.75±5.132b
二级分枝 Secondary branch (number) 5.00±0.816a 5.00±1.155a 5.75±2.602a 6.00±1.15a
果荚长 Pod length (mm) 18.000±1.0475a 17.153±1.3553a 17.600±1.2035a 18.028±0.8587a

Fig. 5

Phenotypes of overexpression BraERF023a transgenic Arabidopsis under salt stress a: The phenotypes of overexpression BraERF023a transgenic Arabidopsis (L4, L8, L9) and control before salt stress (200 mmol·L-1 NaCl); b: The phenotypes of overexpression BraERF023a transgenic Arabidopsis (L4, L8, L9) and control under salt stress (200 mmol?L-1 NaCl) for three weeks"

Fig. 6

Phenotypes of overexpression BraERF023a transgenic Arabidopsis under drought stress L4, L8, L9 indicated three independent overexpression BraERF023a transgenic lines; CK indicated wild-type control"

Fig. 7

Survival rate of overexpression BraERF023a transgenic Arabidopsis under drought stress L4, L8 and L9 indicated three independent overexpression BraERF023a transgenic lines; CK indicated wild-type control"

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