扁果枸杞表皮蜡质转运相关基因<i>LbABCG11</i>的克隆及其表达特征分析

doi:10.7668/hbnxb.20194241

摘要/Abstract

摘要：

为研究ATP结合转运蛋白G亚家族蛋白11(ABCG11)在旱生经济灌木扁果枸杞中的功能,以扁果枸杞为试验材料,利用逆转录(RT-PCR)技术和RACE法克隆了扁果枸杞LbABCG11基因的cDNA序列,通过生物信息学分析该序列特征,并用实时荧光定量PCR 法(qRT-PCR)研究了 LbABCG11在盐处理、渗透胁迫和脱落酸(ABA)处理下不同器官的转录水平。结果表明,LbABCG11基因全长为2 971 bp,开放阅读框长2 130 bp,编码710个氨基酸,有6个跨膜区,预测蛋白质分子质量为79.39 ku,理论等电点8.42,脂肪指数为89.63,不稳定系数为35.52,平均亲水性为0.070,其编码蛋白质分子式为C₃₅₉₀H₅₅₇₇N₉₃₅O₁₀₂₇S₃₅,属于性质稳定的碱性疏水蛋白,含有核苷酸结合域(NBD)以及跨膜结构域(TMD),并以NBD-TMD的形式排列,是一种 WBC型(WBC)半分子转运蛋白。该蛋白质二级结构主要由α-螺旋及无规则卷曲组成,分别占45.21%和33.66%,与三级结构预测结果相符。系统进化树分析发现,LbABCG11蛋白与茄科番茄SlABCG11和辣椒CbABCG11的亲缘关系较近,与拟南芥AtABCG11、藜麦CqABCG11同源性较低。LbABCG11基因在叶、茎、根中均表达,其表达量受NaCl、渗透胁迫和ABA 诱导,表明LbABCG11参与旱生植物盐和干旱等多种胁迫的调控。

关键词: 扁果枸杞, 蜡质转运, LbABCG11, 基因克隆, 生物信息学, 表达模式

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

袁惠君, 张瑞艳, 关玉晨, 余诗曼, 徐琰莹, 马倩国, 鲍婧婷. 扁果枸杞表皮蜡质转运相关基因LbABCG11的克隆及其表达特征分析[J]. 华北农学报, 2023, 38(6): 45-54. doi: 10.7668/hbnxb.20194241.

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.

http://www.hbnxb.net/CN/Y2023/V38/I6/45

图/表 10

表1 本研究所用引物序列

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

图1 LbABCG11基因核心片段(A)、5'-RACE(B)和3'-RACE(C)克隆

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

图2 扁果枸杞LbABCG11序列

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

图3 LbABCG11亲水/疏水性预测分析

Fig.3 Hydrophilic/hydrophobicity analysis of LbABCG11

图4 LbABCG11蛋白二级结构(A)、跨膜区(B)、三级结构(C)预测

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

图5 LbABCG11蛋白与其他植物ABCG11同源氨基酸序列多重比较

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

图6 LbABCG11的系统进化分析

Fig.6 Phylogenetic tree analysis of LbABCG11

图7 盐处理下LbABCG11在扁果枸杞不同器官中的表达模式分析不同小写字母表示处理间差异达显著水平(P<0.05)。图8—9同。

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.

图8 渗透处理下LbABCG11在扁果枸杞不同器官中的表达模式分析

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

图9 ABA处理下LbABCG11在扁果枸杞不同器官中的表达模式分析

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

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扁果枸杞表皮蜡质转运相关基因LbABCG11的克隆及其表达特征分析

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

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扁果枸杞表皮蜡质转运相关基因LbABCG11的克隆及其表达特征分析

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

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