旱生植物霸王ZxCER6的基因克隆及功能分析

doi:10.11686/cyxb2025122

摘要/Abstract

摘要：

角质层蜡质在旱生植物霸王抵御不良生境中发挥着重要作用。β-酮脂酰辅酶A合成酶（KCS）是催化蜡质前体物质合成的关键酶。本研究克隆得到霸王ZxKCS6/CER6的cDNA全长1548 bp，具有2个决定底物选择性的高度保守跨膜区和3个酶催化位点，属于缩合酶超家族成员。与其他高等植物同源蛋白的氨基酸序列比对发现，ZxCER6属于KCS家族中CER6亚家族，具有第224位的半胱氨酸高度保守激活位点。系统进化树分析发现，ZxCER6与拟南芥AtCER6的亲缘关系最近。表达模式分析表明，ZxCER6主要在地上部组织中表达，尤其在叶表皮中表达丰度最高；且受50 mmol·L^-1 NaCl处理的强烈诱导，其表达丰度在36 h时达到峰值。利用表皮特异性启动子驱动ZxCER6在拟南芥中表达发现，干旱处理后转基因拟南芥的地上部干鲜重、叶绿素含量、净光合速率和水分利用效率均显著高于野生型，而离体叶片失水率、叶绿素浸出率和叶片相对质膜透性均低于野生型；转基因植株地上部角质层蜡质含量，尤其是烷烃含量显著增加。表明ZxCER6的超表达可增加转基因植株表皮中烷烃的含量，减少水分散失，从而提高其抗旱性。本研究初步揭示了ZxCER6介导的蜡质积累在荒漠植物霸王抗旱性中的作用，为优良牧草及农作物抗旱性的遗传改良提供了优异的基因资源。

关键词: 霸王, 角质层蜡质, β-酮脂酰辅酶A合成酶, 抗旱性

Abstract:

Cuticular wax plays an important role in resistance to environmental stresses in the xerophyte Zygophyllum xanthoxylum. β-ketoacyl-CoA synthase （KCS） is the key enzyme catalyzing the synthesis of wax precursors. In this study， the full-length cDNA of ZxKCS6/CER6 was cloned. The 1548-bp sequence encoded a polypeptide with two highly conserved transmembrane regions that determine substrate selectivity and three enzyme catalytic sites， and it belongs to the condensation enzyme superfamily. Comparison with homologous protein sequences of other higher plants revealed that ZxCER6 belongs to the CER6 subfamily of the KCS family and has a highly conserved cysteine activation site at amino acid position 224. A phylogenetic analysis revealed a close relationship between ZxCER6 and AtCER6 of Arabidopsis thaliana. Transcript profile analysis revealed that ZxCER6 transcript levels were much higher in aboveground tissues， especially in the leaf epidermis， than in below-ground organs. Expression of ZxCER6 was strongly induced by 50 mmol·L^-1 NaCl treatment and its transcript level peaked at 36 hours of this treatment. ZxCER6 was expressed in A. thaliana under the control of an epidermal-specific promoter， and the performance of the transgenic plants was compared with that of wild type plants. Upon drought treatment， the shoot dry and fresh weight， total chlorophyll content， net photosynthetic rate， and water use efficiency of transgenic Arabidopsis were significantly higher than those of the wild type， and the water loss rate， chlorophyll leaching rate， and relative membrane permeability of detached leaves were lower than those of the wild type. The wax content， especially the alkane content， in the aboveground cuticle was significantly higher in the transgenic plants than in the wild type plants. These findings indicate that ZxCER6 can increase the proportion of alkanes in the cuticular wax on the epidermis of transgenic plants leading to reduced water loss， and consequently， improved drought resistance. The results of this study reveal that ZxCER6 mediates the accumulation of epicuticular waxes that play an important role in the drought resistance of the desert plant Z. xanthoxylum. Our findings highlight an excellent genetic resource that can be used for the genetic improvement of drought resistance in forage and crop plants.

Key words: Zygophyllum xanthoxylum, cuticular wax, β-ketoacyl-CoA synthase, drought tolerance

未丽, 邓育轩, 赵静, 刘俊良, 马克华, 王锁民. 旱生植物霸王ZxCER6的基因克隆及功能分析[J]. 草业学报, 2026, 35(1): 154-169.

Li WEI, Yu-xuan DENG, Jing ZHAO, Jun-liang LIU, Ke-hua MA, Suo-min WANG. Cloning and functional analysis of ZxCER6 from the xerophyte Zygophyllum xanthoxylum[J]. Acta Prataculturae Sinica, 2026, 35(1): 154-169.

图/表 17

表1 所用引物信息

Table 1 Primer information used in the experiment

引物名称Primer name	引物序列Primer sequence （5′-3′）	目的Purpose
3F-CER6	ACGAGGCGGAGACTGTTATTT	基因克隆 Gene cloning
3FN-CER6	ATCCCAATTCAAATGCTGTCG
5R-CER6	CTTCATACACGCAACGATAGG
5RN-CER6	TCATCAGCTCCTTTGTGGGTC
WF-CER6	AACAGTCCACTGCCTTCAACAGTAC
WR-CER6	GCCATCCACCAATCAACCTTAT
P	CTAATACGACTCACTATAGGGC
NP	AAGCAGTGGTATCAACGCAGAGT
QF-ZxCER6	CGTTGCGTGTATGAAGAAGAGG	qRT-PCR
QR-ZxCER6	TATGGCTTGATTTTCGGGTTG
QF-ZxACTIN	TTTTCCAGCCATCCCTTGTT
QR-ZxACTIN	TGCAGTGATCTCCTTGCTCATAC
VF AtCER6（HindⅢ）	TGTTGGCCCAAGCTTCTTCGATATCGGTTGTTGACGAT	植物表达载体构建 Construction of plant expression vector
VR AtCER6	CAAGATTTGAGGCATCGTCGGAGAGTTTTAATGTATAAT
VF ZxCER6	ATGCCTCAAATCTTGCCCGATTTCT
VR ZxCER6（SacⅠ）	GGGAAATTCGAGCTCCTACAGCTTGACAACTTCAGGAAT

图1 ZxCER6的克隆与cDNA全长及对应蛋白序列A： ZxCER6基因的PCR扩增产物PCR amplification product of ZxCER6 gene； M： DL 2000 DNA marker；下同The same below. 1： cDNA扩增产物cDNA amplified product. B： ZxCER6的cDNA全长序列Full cDNA sequence of ZxCER6. 红色方框处为起始密码子（ATG）和终止密码子（TAG）。The start codon （ATG） and the stop codon （TAG） were marked in red boxes.

Fig.1 Cloning full-length cDNA and corresponding protein sequence of ZxCER6

图2 ZxCER6蛋白结构特征分析A： ZxCER6蛋白的跨膜域ZxCER6 protein transmembrane domain； B： ZxCER6蛋白保守结构域ZxCER6 protein conserved domain.

Fig.2 Structural characterization analysis of ZxCER6 protein

图3 ZxCER6与其他高等植物CER6的氨基酸序列多重比对CmCER6：甜瓜Cucumis melo CER6 （XP_008446598.1）； CsCER6：黄瓜Cucumis sativus CER6 （XP_004135090.1）； EgCER6：巨桉Eucalyptus grandis CER6 （XP_010066726.1）； GmCER6：大豆Glycine max CER6 （XP_003555901.1）； PmCER6：梅Prunus mume CER6 （XP_008243224.1）； PtCER6：毛果杨Populus trichocarpa CER6 （XP_002311457.1）； TcCER6：可可Theobroma cacao CER6 （XP_007044356.2）； ZxCER6：霸王Z. xanthoxylum CER6. 下同The same below. 黄色方框表示CER6的2个跨膜区（TM1、TM2），红色星号表示高度保守激活位点Cys224。Transmembrane domain （TM1， TM2） was indicated with yellow box. A highly conserved catalytically residue was indicated with red asterisk.

Fig.3 Amino acid sequence alignment of ZxCER6 with other CER6 from higher plants

图4 不同植物CER6系统进化树分析VrCER6：三裂叶绿豆Vigna radiata var. radiata CER6； FvCER6：林地草莓Fragaria vesca subsp. vesca CER6； MdCER6：苹果Malus domestica CER6； JcCER6：麻疯树Jatropha curcasx CER6； HbCER6：橡胶树Hevea brasiliensis CER6； PeCER6：胡杨Populus euphratica CER6； CcCER6：克莱门柚Citrus clementina CER6； GrCER6：雷蒙德氏棉G. raimondii CER6； DzCER6：榴莲Durio zibethinus CER6； SiCER6：芝麻Sesamum indicum CER6； AtCER6：拟南芥A. thaliana CER6； PdCER6：海枣P. dactylifera CER6； MaCER6：小果野芭蕉M. acuminate CER6； ZmCER6：玉米Zea mays CER6； GbCER6：银杏Ginkgo biloba CER6.

Fig.4 Phylogenetic tree analysis of CER6 in different plants

图5 ZxCER6的表达模式分析A：组织特异性表达模式Tissue specific expression pattern； ML：中层叶Middle leaves； MLE：中层叶表皮Epidermis of middle leaves； UL：上层叶Upper leaves； C：子叶Cotyledons； S：茎Stems； R：根Roots. B： 50 mmol·L-1 NaCl处理下表达模式Expression patterns under 50 mmol·L-1 NaCl. 不同小写字母表示不同部位或不同处理时间下差异显著（P<0.05）。Different lowercase letters indicate significant difference among different tissues or different treatments time （P<0.05）.

Fig.5 Expression pattern analysis of ZxCER6

图6 植物表达载体的构建A：植物表达载体pBIB-BASTA-pAtCER6：：ZxCER6构建示意图Schematic diagram of plant expression vector pBIB-BASTA-pAtCER6：：ZxCER6 construction. B： pBIB-BASTA-35S-GWR-FLAG基础载体Hind Ⅲ和Sac Ⅰ双酶切电泳图谱pBIB-BASTA-35S-GWR-FLAGHind Ⅲand Sac Ⅰ double enzyme electrophoresis； 1， 2：双酶切电泳条带The electrophoretic bands of double enzyme. C： AtCER6和ZxCER6分别加入Hind Ⅲ和Sac Ⅰ酶切位点电泳图谱AtCER6 and ZxCER6 join Hind Ⅲ and Sac Ⅰ enzyme electrophoresis； 1， 2： AtCER6电泳条带AtCER6 electrophoretic bands； 3， 4： ZxCER6电泳条带ZxCER6 electrophoretic bands. D： AtCER6和ZxCER6菌液PCR电泳图谱PCR electrophoresis of AtCER6 and ZxCER6 strains.

Fig.6 Construction of plant expression vector

图7 转基因拟南芥中ZxCER6 的RT-PCR检测WT：野生型拟南芥植株Wild type Arabidopsis plants； OE5， OE8， OE12， OE19：转基因拟南芥株系Transgenic Arabidopsis lines； AtActin2：内参基因Internal gene. 下同The same below.

Fig.7 RT-PCR detection of ZxCER6 in transgenic Arabidopsis

图8 不同干旱周期下野生型和ZxCER6转基因拟南芥的生长状态图A和B分别为第1个干旱周期对照及处理；图C和D分别为第2个干旱周期对照及处理。A： The first drought period control； B： The first drought period； C： The second drought period control； D： The second drought period.

Fig.8 The growth state of wild type and ZxCER6 transgenic Arabidopsis under different drought periods

图9 不同干旱周期下野生型和ZxCER6转基因拟南芥植株株高A：第1个干旱周期The first drought period； B：第2个干旱周期The second drought period. 不同小写字母表示野生型和不同转基因株系在不同处理间差异显著（P<0.05）。Different lowercase letters indicate that WT and different transgenic lines have significant differences among different treatments （P<0.05）. 下同The same below.

Fig.9 The plant height of wild type and ZxCER6 transgenic Arabidopsis under different drought periods

图10 第2个干旱周期下野生型和ZxCER6转基因拟南芥植株地上部干鲜重

Fig.10 Shoot dry and fresh weight of wild type and ZxCER6 transgenic Arabidopsis in the second drought periods

图11 第2个干旱周期下野生型和ZxCER6转基因拟南芥叶片叶绿素含量和光合参数分析

Fig.11 Analysis of chlorophyll content and photosynthesis index between wide type and ZxCER6 transgenic Arabidopsis in the second drought period

图12 野生型和ZxCER6转基因拟南芥叶片的角质层透性正常条件（A、B）和第2个干旱周期下（C、D）野生型和ZxCER6转基因拟南芥叶片失水率（A、C）及叶绿素浸出率（B、D）。Water loss rate （A， C） and chlorophyll leaching rate （B， D） of wild type and ZxCER6 transgenic Arabidopsis leaves under control （A， B） and the second drought period （C， D）. 数值为平均值±标准误（n=5）。Data are mean±standard error （n=5）.

Fig.12 Cuticle permeability of wide type and ZxCER6 transgenic Arabidopsis leaves

图13 野生型和ZxCER6转基因拟南芥叶片相对含水量

Fig.13 Relative water content of wide type and ZxCER6 transgenic Arabidopsis leaves

图14 野生型和ZxCER6转基因拟南芥叶片相对质膜透性

Fig.14 Relative membrane permeability of wide type and ZxCER6 transgenic Arabidopsis leaves

图15 野生型和ZxCER6转基因拟南芥莲座叶中角质层蜡质含量及不同碳链长度烷烃组分含量正常条件（A、B）和第2个干旱周期下（C、D）野生型和ZxCER6转基因拟南芥莲座叶角质层蜡质含量（A、C）及不同碳链长度烷烃组分含量（B、D）。Wax amount （A， C） and amounts of different chain alkane constituents （B， D） on rosette leaves of wild type and ZxCER6 transgenic Arabidopsis under control （A， B） and the second drought period （C， D）. 不同小写字母表示各株系之间差异显著（P<0.05）。下同。Different lowercase letters indicate significant difference among the lines （P<0.05）. The same below.

Fig.15 Wax amount and amounts of different chain alkane constituents on rosette leaves of wide type and ZxCER6 transgenic Arabidopsis

图16 野生型和ZxCER6转基因拟南芥花序茎角质层蜡质含量及不同碳链长度烷烃组分含量正常条件（A、B）和第2个干旱周期下（C、D）野生型和ZxCER6转基因拟南芥花序茎角质层蜡质含量（A、C）及不同碳链长度烷烃组分含量（B、D）。Wax amount （A， C） and amounts of different chain alkane constituents （B， D） on inflorescence stems of wild type and ZxCER6 transgenic Arabidopsis under control （A， B） and the second drought period （C， D）.

Fig.16 Wax amount and amounts of different chain alkane constituents on inflorescence stems of wide type and ZxCER6 transgenic Arabidopsis

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