海岛棉CAD和CAD-Like基因家族的鉴定、表达及其对大丽轮枝菌的响应

doi:10.3864/j.issn.0578-1752.2023.19.005

摘要/Abstract

摘要：

【目的】肉桂醇脱氢酶（CAD）是木质素合成途径中的关键酶，在增强植物机械强度、抵御病原菌入侵等方面发挥重要作用。鉴定海岛棉GbCAD、GbCAD-LIKE（GbCADL）基因家族成员，并对其表达特征及其在黄萎病抗性中的作用进行分析，为棉花抗黄萎病机制的解析及抗病育种提供参考。【方法】利用生物信息学方法对海岛棉GbCAD、GbCADL基因家族成员进行鉴定，并对其染色体定位、系统发育关系、基因结构、启动子顺式元件的预测进行系统分析。通过获得公开释放的转录组数据和实时荧光定量聚合酶链式反应（qRT-PCR）分析GbCAD基因及GbCADL基因的表达特征。利用病毒诱导的基因沉默（VIGS）技术对GbCAD基因及GbCADL基因进行功能分析。【结果】从海岛棉中鉴定到25个GbCAD基因（分布在10条染色体）和34个GbCADL基因（分布在17条染色体）。GbCAD基因分为3个亚组，GbCADL基因分为4个亚组，同一个组内基因含有相似的外显子-内含子结构和保守结构域。GbCAD基因和GbCADL基因具有不同的转录表达特征，其启动子中含有多种激素和胁迫响应元件。转录组数据和qRT-PCR显示，GbCAD10A/D、GbCADL4A/D、GbCADL5A/D、GbCADL6A/D、GbCADL7A/D的表达受大丽轮枝菌诱导，尤其是GbCAD10A/D、GbCADL4A/D、GbCADL6A/D、GbCADL7A/D的表达在大丽轮枝菌处理后显著增加。利用VIGS技术，将显著受大丽轮枝菌诱导表达的GbCAD10A/D、GbCADL4A/D、GbCADL6A/D、GbCADL7A/D分别在棉花中沉默，分析VIGS株系对大丽轮枝菌抗性的变化。结果显示，与对照植株相比，VIGS株系对大丽轮枝菌的抗性显著降低。二氨基联苯胺（DAB）染色结果显示，在未受到黄萎病侵染时，VIGS植株和对照植株的染色程度无显著差异；在大丽轮枝菌处理6 h后，与对照植株相比，沉默株系GbCAD10A/D、GbCADL4A/D、GbCADL6A/D、GbCADL7A/D均表现出较深的着色，表明在沉默株系中有较高的活性氧累积。茎的切片结果显示，大丽轮枝菌处理后，TRV:GbCAD10A/D、TRV:GbCADL4A/D、TRV:GbCADL6A/D、TRV:GbCADL7A/D沉默株系的茎维管组织表现出明显的深褐色，表明其对大丽轮枝菌的抗性显著降低。【结论】抑制GbCAD10A/D、GbCADL4A/D、GbCADL6A/D、GbCADL7A/D的表达可以显著降低棉花对大丽轮枝菌的抗性。

关键词: 海岛棉, 肉桂醇脱氢酶, 基因家族, 表达特征, 基因沉默, 大丽轮枝菌

Abstract:

【Objective】Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin synthesis pathway, which plays an important role in enhancing plant mechanical strength and resisting pathogen invasion. The aim of this study is to identify CAD and CAD-Like (CADL) gene family members in Gossypium barbadense and to analyze their expression characteristics and their role in Verticillium wilt resistance, which provides reference for the mechanism elucidation and disease resistance breeding of cotton against Verticillium wilt. 【Method】The CAD and CADL gene family members in G. barbadense genome were identified by bioinformatics method, and their chromosomal location, phylogenetic relationship, gene structure and promoter cis-element prediction were systematically analyzed. The expression characteristics of GbCAD and GbCADL were analyzed by obtaining publicly released transcriptome data and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Functional analysis of GbCAD and GbCADL genes was performed by viral-induced gene silencing (VIGS) technique. 【Result】A total of 25 GbCAD and 34 GbCADL genes were identified from G. barbadense and distributed on 10 and 17 different chromosomes, respectively. GbCAD and GbCADL genes are divided into 3 and 4 subgroups, respectively. The genes in the same group contain similar exon-intron structures and conserved domains. GbCAD and GbCADL genes have different transcriptional expression characteristics, and the promoters of GbCAD and GbCADL genes contain various hormone response elements and stress response elements. Transcriptome data and qRT-PCR showed that the expressions of GbCAD10A/D, GbCADL4A/D, GbCADL5A/D, GbCADL6A/D, and GbCADL7A/D were induced by Verticillium dahliae, especially the GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, and GbCADL7A/D indicated significant increased expressions under V. dahliae treatment. The genes of GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, and GbCADL7A/D were respectively silenced in cotton by virus-induced gene silencing (VIGS) technology, to analyze the changes of VIGS plant lines against V. dahliae treatment. The results showed that, compared with the control plants, the VIGS plant lines indicated significant decreased resistance to V. dahliae. The results of diaminobenzidine (DAB) histochemical stain displayed that, both control and VIGS plants showed similar normal phenotype without V. dahliae addition; after 6 h treatment of V. dahliae, the VIGS plant lines silencing GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, GbCADL7A/D expressions demonstrated a deeper brown coloring, indicating a higher reactive oxygen species (ROS) accumulation in the VIGS plant lines. The results of stem sectioning showed that, the stem vascular tissues of VIGS plant lines TRV:GbCAD10A/D, TRV:GbCADL4A/D, TRV:GbCADL6A/D, and TRV:GbCADL7A/D showed obvious dark brown enrichment after V. dahliae treatment, indicating the significant decreased resistance to V. dahliae. 【Conclusion】 Suppressing the expressions of GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, GbCADL7A/D could significantly reduce the cotton resistance to V. dahliae.

Key words: Gossypium barbadense, cinnamyl alcohol dehydrogenase, gene family, expression characteristics, gene silencing, Verticillium dahliae

张钰佳, 崔凯文, 段力升, 曹爱萍, 谢全亮, 沈海涛, 王斐, 李鸿彬. 海岛棉CAD和CAD-Like基因家族的鉴定、表达及其对大丽轮枝菌的响应[J]. 中国农业科学, 2023, 56(19): 3759-3771.

ZHANG YuJia, CUI KaiWen, DUAN LiSheng, CAO AiPing, XIE QuanLiang, SHEN HaiTao, WANG Fei, LI HongBin. Identification and Expression of CAD and CAD-Like Gene Families from Gossypium barbadense and Their Response to Verticillium dahliae[J]. Scientia Agricultura Sinica, 2023, 56(19): 3759-3771.

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