Cloning and Expression Analysis of Epidermal Wax Transport Related LbABCG11 Gene in Lycium barbarum ssp.Bianguo

doi:10.7668/hbnxb.20194241

Abstract

Abstract:

To investigate the function of ATP-binding cassette transporter subfamily G11(ABCG11)in xerophytic economic shrub Lycium barbarum ssp. Bianguo,using the Lycium barbarum ssp. Bianguo as the material. The cDNA sequence of Lycium barbarum ssp. Bianguo LbABCG11 gene was obtained by RT-PCR and RACE method, the sequence patterns were analyzed by bioinformatics software. Quantitative Real-time PCR(qRT-PCR)was used to analyze the expression patterns of LbABCG11 in different organs of Lycium barbarum ssp. bianguo under salt treatment,osmotic stress and abscisic acid(ABA)treatments.The results showed that LbABCG11 gene contained 2 971 bp in full length and a 2 130 bp open reading frame,encoding 710 amino acids.The protein contained six transmembrane regions, the calculated molecular weight, isoelectric point, adipose index, instability index and average hydropathicity was 79.39 ku,8.42, 89.63, 35.52, 0.070, the encoded protein chemical formula was C₃₅₉₀H₅₅₇₇N₉₃₅O₁₀₂₇S_35. LbABCG11 is a stable alkaline hydrophobic protein.The protein contained a nucleotide-binding domain(NBD)and a transmembrane domain(TMD),which were arranged in the form of NBD-TMD.LbABCG11 was a white-brown complex (WBC)half-size molecular transporter.The secondary structure of the protein was mainly composed of α-helix and random coil, accounting for 45.21% and 33.66%, respectively, which was consistent with the predicted results of the tertiary structure. Phylogenetic analysis showed that LbABCG11 had closer genetic relationship with ABCG11 from Solanum lycopersicum and Capsicum barbarum, but has low homology with ABCG11 from Arabidopsis thaliana and Chenopodium quinoa. LbABCG11 gene was expressed in some organs of Lycium barbarum ssp. bianguo,including leaves,stems and roots.The LbABCG11 expression level was induced by NaCl,osmotic stress and ABA,manifesting that LbABCG11 was involved in the stress response of xerophytes.

Key words: Lycium barbarum ssp.Bianguo, Epidermal wax transport, LbABCG11, Gene cloning, Bioinformatics, Expression pattern

YUAN Huijun, ZHANG Ruiyan, GUAN Yuchen, YU Shiman, XU Yanying, MA Qianguo, BAO Jingting. Cloning and Expression Analysis of Epidermal Wax Transport Related LbABCG11 Gene in Lycium barbarum ssp.Bianguo[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(6): 45-54. doi: 10.7668/hbnxb.20194241.

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Figures/Tables 10

Tab.1 The primer sequences in this study

引物类型 The type of primers	引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
核心片段引物	P1	CTTGATGARCCRACYAGTGGHCTTGA
Primers of intermediate fragment	P2	TTCCVACRTTRAGATAGATAGTTCC
5'-RACE引物	GSP1	GATTACGCCAAGCTTCCTGACTTCCTCCAGAATCTAGCACTG
Primers of 5'-RACE	NGSP1	GATTACGCCAAGCTTGCCGAAGCACTGTCAAGACCACT
3'-RACE引物	GSP2	GATTACGCCAAGCTTAGCCTCAATTCACCAGCCGAGCA
Primers of 3'-RACE	NGSP2	GATTACGCCAAGCTTCTGCATTGGAACTATCTATCTCAACGTCGG
qRT-PCR引物	ABCG11-1	GCCGAGCAGTGAAGTGTTTG
Primers of qRT-PCR	ABCG11-2	AACCGTAGCTTCATCGACCC
ACT基因引物	ACT1	CTATGAGTTGCCAGATGGACAG
Primers of ACT gene	ACT2	TGGCTGGAAAAGGACTTCTG

Fig.1 PCR products of the core fragment(A),5'-RACE(B) and 3'-RACE(C) of LbABCG11

Fig.2 Sequence of LbABCG11 in L.barbarum ssp.Bianguo

Fig.3 Hydrophilic/hydrophobicity analysis of LbABCG11

Fig.4 Secondary structure(A),transmembrane(B) and tertiary structure(C) prediction of LbABCG11

Fig.5 Amino acid sequence alignment of LbABCG11 with ABCG11 from other plants

Fig.6 Phylogenetic tree analysis of LbABCG11

Fig.7 Relative expression of LbABCG11 in different developmental tissues of L.barbarum ssp.Bianguo under NaCl treatment Different lowercase letters indicate significant differences between treatments(P<0.05).The same as Fig.8—9.

Fig.8 Relative expression of LbABCG11 in different developmental tissues of L.barbarum ssp.Bianguo under osmose treatment

Fig.9 Relative expression of LbABCG11 in different developmental tissues of L.barbarum ssp.Bianguo under ABA treatment

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