Cloning and functional analysis of ZxCER6 from the xerophyte Zygophyllum xanthoxylum

doi:10.11686/cyxb2025122

Abstract

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

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.

Figures/Tables 17

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

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

Fig.2 Structural characterization analysis of ZxCER6 protein

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

Fig.4 Phylogenetic tree analysis of CER6 in different plants

Fig.5 Expression pattern analysis of ZxCER6

Fig.6 Construction of plant expression vector

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

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

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

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

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

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

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

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

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

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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