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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (4): 1016-1027.doi: 10.3724/SP.J.1006.2023.24037

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

Cloning of genes involved in cuticular very-long-chain alkane synthesis and its interaction with BnCER1-2 in Brassica napus

BAI Cheng-Cheng**(), YAO Xiao-Yao**(), WANG Yu-Lu, WANG Sai-Yu, LI Jin-Ying, JIANG You-Wei, JIN Shu-Rong, CHEN Chun-Jie, LIU Yu, WEI Xing-Yue, XU Xin-Fu, LI Jia-Na, NI Yu*()   

  1. College of Agronomy and Biotechnology/Academy of Agricultural Sciences, Southwest University, Chongqing 400716, China
  • Received:2022-02-13 Accepted:2022-06-07 Online:2023-04-12 Published:2022-06-21
  • Contact: *E-mail: nmniyu@126.com
  • About author:**Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China(32171938);National Natural Science Foundation of China(31771694);Natural Science Foundation of Chongqing, China(cstc2021jcyj-msxmX0332);China Agriculture Research System of MOF and MARA(CARS-12)

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Abstract:

Very-long-chain (VLC) alkanes are the main components of cuticular wax in Brassica napus, which play a key role in preventing non-stomatal water loss. BnCER1-2 is the core enzyme that catalyzes the synthesis of VLC alkanes in B. napus. However, it is unclear whether BnCER1-2 protein regulates VLC alkane synthesis by interacting with other proteins. Four genes potentially involved in VLC alkane synthesis, BnCER3.a10, BnCER3.c02, BnCYTB5B.c09, and BnCER1-L2.a05, were screened previously by the transcriptome in B. napus with differential wax load. In this study, their coding sequences were cloned from B. napus. A typical fatty acid hydroxylase domain and a wax2 C-terminal domain were detected in the predicted BnCER3.a10/c02 and BnCER1-L2.a05 proteins, while cyt_b5 protein family conserved domain was in the predicted BnCYTB5B.c09. Subcellular localization showed that BnCER3.a10/c02, BnCYTB5B.c09, and BnCER1-L2.a05 were all located in the endoplasmic reticulum, which were co-located with BnCER1-2. Bimolecular fluorescence complementary (BiFC) and luciferase complementation assay (LCA) revealed that BnCER3.a10, BnCYTB5B.c09, and BnCER1-L2.a05 interacted with BnCER1-2 protein, while BnCER3.c02 did not interact with BnCER1-2. The RT-qPCR indicated that BnCER3.a10 and BnCYTB5B.c09 were mainly expressed in the stems or leaves of B. napus, and the relative expression was significantly up-regulated under drought stress, which were consistent with the expression pattern of BnCER1-2. The relative expression levels of BnCER3.a10 decreased significantly under NaCl and low temperature stresses. Further, the relative expression level of BnCER3.a10 was significantly down-regulated by MeJA and ACC, while the relative expression level of BnCYTB5B.c09 was up-regulated by ABA. The highest relative expression of BnCER1-L2.a05 was in flowers and the lowest in stems and leaves, and its expression was significantly down-regulated under drought, cold, and NaCl stresses. Further, the relative expression level of BnCER1-L2.a05 was up-regulated by SA, while down-regulated by MeJA. The relative expression levels of BnCER3.a10 and BnCYTB5B.c09 in B. napus with high wax/alkane load were significantly higher than that with low wax/alkane load, while BnCER1-L2.a05 had the opposite change. Comprehensive analysis suggested that BnCER3.a10 and BnCYTB5B.c09 may promote the biosynthesis of VLC alkanes in B. napus by interacting with BnCER1-2, while BnCER1-L2.a05 may negatively regulate the synthesis of VLC alkanes by interacting with BnCER1-2.

Key words: Brassica napus, cuticular wax, protein interaction, very-long-chain alkanes

Table 1

Primers used in BiFC"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 酶切位点
Restriction site		 重组载体
Vector
FBnCER1-2 TCTGAGGAGGATCTTCCCGGGATGGCTACGAAACCAGGCATCC Sma I nYFP
RBnCER1-2 AGGGCATGCCTGCAGGTCGACTCAGGGGTATTGGAAGTGATGTGG Sal I
FBnCER3.a10 TCTAGGAGCTCGGTACCCGGGATGGTAGCTTTTTCAGCTTGGCCT Sma I cYFP
RBnCER3.a10 ATCGTATGGGTACATACTAGTTCTTGTGAGTGAAGAAACAGAGCGAA Spe I
FBnCER3.c02 TCTAGGAGCTCGGTACCCGGGATGGTTACTTTATCAGTCTGGCCTTGG Sma I cYFP
RBnCER3.c02 ATCGTATGGGTACATACTAGTATTAGTGAGTGAAGACACAGAGCTAAGGC Spe I
FBnCYTB5B.c09 TCTAGGAGCTCGGTACCCGGGATGGGCGGAGACGGCAAAGT Sma I cYFP
RBnCYTB5B.c09 ATCGTATGGGTACATACTAGTAGAAGAAGAAGGAGTCTTGGTGTAAGAACG Spe I
FBnCER1-L2.a05 TCTAGGAGCTCGGTACCCGGGATGGCGTCGAGACCAGGTTTTC Sma I cYFP
RBnCER1-L2.a05 ATCGTATGGGTACATACTAGTGTAACCATTGCTCAATCCTTTGGTCTC Spe I

Table 2

Primers used in LCA"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 酶切位点
Restriction site		 重组载体
Vector
FBnCER1Bi AACACGGGGGACGAGCTCGGTACCATGGCTACGAAACCAGGCAT Kpn I nLUC
RBnCER1Bi GGACGCGTACGAGATCTGGTCGACGGGGTATTGGAAGTGATGTGG Sal I
FBnCER3.a10Bi TACGCGTCCCGGGGCGGTACCATGGTAGCTTTTTCAGCTTGGCCT Kpn I cLUC
RBnCER3.a10Bi ACGAAAGCTCTGCAGGTCGACTCATCTTGTGAGTGAAGAAACAGAGCG Sal I
FBnCER3.c02Bi TACGCGTCCCGGGGCGGTACCATGGTTACTTTATCAGTCTGGCCTTGG Kpn I cLUC
RBnCER3.c02Bi ACGAAAGCTCTGCAGGTCGACATTAGTGAGTGAAGACACAGAGCTAAGGC Sal I
FBnCYTB5.c09Bi TACGCGTCCCGGGGCGGTACCATGGGCGGAGACGGCAAAGT Kpn I cLUC
RBnCYTB5.c09Bi ACGAAAGCTCTGCAGGTCGACTCAAGAAGAAGAAGGAGTCTTGGTGTA Sal I
FBnCER1-L2.a05Bi TACGCGTCCCGGGGCGGTACCATGGCGTCGAGACCAGGTTTTC Kpn I cLUC
RBnCER1-L2.a05Bi ACGAAAGCTCTGCAGGTCGACCTAGTAACCATTGCTCAATCCTTTGGTCT Sal I

Table 3

Primers used in subcellular localization"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 酶切位点
Restriction site
FBnCER3.a10S GCTGCGGCAGCGGCCGAATTCATGGTAGCTTTTTCAGCTTGGCCT EcoR I
RBnCER3.a10S TTATCTAGATCCGGTGGATCCTCATCTTGTGAGTGAAGAAACAGAGCG BamH I
FBnCER3.c02S GCTGCGGCAGCGGCCGAATTCATGGTTACTTTATCAGTCTGGCCTTGG EcoR I
RBnCER3.c02S TTATCTAGATCCGGTGGATCCTCAATTAGTGAGTGAAGACACAGAGCTA BamH I
FBnCYTB5.c09S GCTGCGGCAGCGGCCGAATTCATGGGCGGAGACGGCAAAGT EcoR I
RBnCYTB5.c09S TTATCTAGATCCGGTGGATCCTCAAGAAGAAGAAGGAGTCTTGGTGTAAGAA BamH I
FBnCER1-L2.a05S GCTGCGGCAGCGGCCGAATTCATGGCGTCGAGACCAGGTTTTC EcoR I
RBnCER1-L2.a05S TTATCTAGATCCGGTGGATCCCTAGTAACCATTGCTCAATCCTTTGGTC BamH I

Table 4

Primers for RT-qPCR"

引物名称
Primer name		 上游引物序列
Forward sequence (5°-3°)		 引物名称
Primer name		 下游引物序列
Reverse sequence (5°-3°)		 扩增片段大小
Size (bp)
qFBnCER3.a10 AGATTCGGGTTCCAATACTT qRBnCER3.a10 TAACACCAATCTTATCAGCCCTA 94
qFBnCYTB5.c09 GGATTTCCTCATCAAGATCCTTCA qRBnCYTB5.c09 GGAGTCTTGGTGTAAGAACG 87
qFBnCER1-L2.a05 TGTCTTTGAGAGAAACCGC qRBnCER1-L2.a05 GTTGATAGATTCTTTACACTGCTG 135

Fig. 1

Structure domain of BnCER3.a10/c02, BnCYTB5B.c09, and BnCER1-L2.a05 A: fatty_acid_hydroxylase domain Wax2 C-terminal domain; B: wax2 C-terminal domain; C: cyt_B5 protein conserved domain. The proteins involved in amino acid sequence alignment include Arabidopsis thaliana CER1 (AT1G02205), CER1-L1 (AT1G02190), CER1-L2 (AT2G37700), and CER3 (AT5G57800). The CYTB5 proteins aligned are from Arabidopsis thaliana (AT5G48810), Brassica oleracea (XP_013609519.1), Brassica carinata (KAG2316461.1), Capsella rubella (XP_006279708.1), and Raphanus sativus (XP_018457867.1), respectively."

Fig. 2

Phylogenetic tree of BnCER3.a10/c02, BnCYTB5B.c09, BnCER1-L2.a05, and other related proteins from Brassicaceae species The amino acid sequences involved in phylogenetic analysis include AtCER1 (AT1G02205), AtCER1-L1 (AT1G02190), AtCER1-L2 (AT2G37700), AtCER3 (AT5G57800), and AtCYTB5 (AT5G48810) from Arabidopsis thaliana, BrCER1 (XP_009118866.2), BrCER1-L2 (XP_009143457.1), BrCER3 (XP_033137694.1), and BrCYTB5B (XP_009151679.1) from Brassica rapa, BoCER1 (QCO76034.1), BoCER1-L2 (XP_013632332.1), BoCER3 (XP_013621098.1), and BoCYTB5B (XP_013597870.1) from Brassica oleracea. (XP_013597870.1)。"

Fig. 3

Subcellular localization of BnCER3.a10/c02, BnCYTB5B.c09, and BnCER1-L2.a05 in tobacco leaf Bar: 20 μm."

Fig. 4

Interaction analysis of BnCER1-2 with BnCER3.a10, BnCYTB5B.c09, and BnCER1-L2.a05 protein by using BiFC The combination “pC1300-YN-OsHAL3 + pC2300-OsHAL3-YC” is used as the positive control, while the combination “pC1300-YN-BnCER1-2 + pC2300-OsHAL3-YC”, “pC1300-YN-OsHAL3 + pC2300-BnCER3.a10-YC”, “pC1300-YN-OsHAL3 + pC2300-BnCYTB5B.c09-YC”, and “pC1300-YN-OsHAL3 + pC2300-BnCER1-L2.a05-YC” are used as the negative controls, respectively. Bar: 20 μm."

Fig. 5

Interaction of BnCER1-2 with BnCER3.a10, BnCYTB5B.c09, and BnCER1-L2.a05 protein using LCA A: the interaction detection between BnCER3.a10 and BnCER1-2; B: the interaction detection between BnCYTB5B.c09 and BnCER1-2; C: the interaction detection between BnCER1-L2.a05 and BnCER1-2. The combination of NLuc empty vector with CLuc vector containing target gene, the combination of CLuc empty vector with NLuc vector containing target gene, and the combination of CLuc empty vector with NLuc empty vector were used as the negative controls, respectively."

Fig. 6

Relative expression profiles of BnCER3.a10, BnCYTB5B.c09, and BnCER1-L2.a05 in Brassica napus A: the specific relative expression of organs and tissues; B: the differential expression in Brassica napus with high load wax and low load wax; C: induced expression by abiotic stress and hormone. SA: salicylic acid; MeJA: methyl jasmonic acid; ACC: 1-aminocyclopropanecarboxylic acid; ABA: abscisic acid. *: P < 0.05."

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