| Biological sciences & biotechnology |
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| Cloning and expression of a C3H-type zinc finger protein gene BoCCCH2 from Brassica oleracea var. italica. |
| JIANG Ming1*, LIU Qing’e2, ZHANG Yanru1, ZHU Qi1, GONG Xiu1, YU Keke1, ZHOU Xiuqian1 |
| (1. Ecology Key Discipline of Zhejiang Province, College of Life Sciences, Taizhou University, Jiaojiang 318000, Zhejiang, China; 2. College of Ecology, Lishui University, Lishui 323000, Zhejiang, China) |
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Abstract Brassica oleracea var. italica is an important vegetable crop worldwide, and in China, Taizhou City of Zhejiang Province is one of the major broccoli production areas. Downy mildew and grey mold rot are two common fungal diseases caused by Hyaloperonospora parasitica and Botrytis cinerea, respectively. In recent years, broccoli production in Taizhou was frequently affected by these two fungal diseases, resulting in yield and quality loss. Broccoli germplasm resources resistance to disease is scarce; therefore, molecular breeding is regarded as an effective solution to solve the problem. This is critically important to isolate genes associated with disease resistance, which will act as potential target genes for broccoli breeding.
Zinc finger proteins are kinds of important transcription factors in eukaryotic organisms, which involve in various biological activities, such as replication, transcription, translation, repair, metabolism and signaling. According to the number and order of cysteine and histidine residues, zinc finger proteins were classified into several different types, such as C2H2, C2C2, C2C2C2, C2HC and C3H. For example, C3H-type ones contain one to six typical motifs with three cysteine residues and one histidine residue. However, their functions are little known, and no gene has been reported in broccoli.
In this study, a C3H-type zinc finger protein gene BoCCCH2 was isolated from broccoli, and later the expression patterns in different organs as well as leaves infected by H. parasitica and B. cinerea were studied.
Results indicated that BoCCCH2 contained no intron, and the full length of coding sequence was 1 740 bp encoding 579 amino acids. The deduced protein sequence contained two ANK domains and two CCCH zinc finger structures, respectively, and the CCCH zinc finger types were C—X8—C—X5—C—X3—H and C—X5—C—X4—C—X3—H. Reverse transcription-polymerase chain reaction results showed that the BoCCCH2 was expressed in roots, leaves, stalks, young siliques, flower buds and flowers, with highest level in roots. Expression levels increased when challenged by both H. parasitica and B. cinerea. When infected by H. parasitica, expression levels increased after 24 h, and decreased after 72 h, while infected by B. cinerea, the highest level was detected after 6 h, and slowed down in 12 h. Homologous sequences were downloaded from NCBI (National Center for Biotechnology Information) website, including Citrus sinensis, Gossypium raimondii, Populus trichocarpa, Ricinus communis, Prunus persica, P. mume, Malus domestica, Fragaria vesca, Phaseolus vulgaris, Glycine soja, B. rapa, Camelina sativa, Capsella rubella, Arabidopsis thaliana and Eutrema salsugineum. Phylogenetic analysis results revealed that BoCCCH2 was grouped with homogeneous sequences from other Cruciferae plants with bootstrap confidence of 100%, and sequences from Leguminosae, Euphorbiaceae and Rosaceae were found on different clades.
In conclusion, these results indicate that the BoCCCH2 might play an important role in defense responses challenged by either H. parasitica or B. cinerea. Cloning and expression analysis of BoCCCH2 provide evidence for further studies on gene function.
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Published: 20 March 2016
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青花菜C3H型锌指蛋白基因BoCCCH2的克隆与表达
青花菜为材料，在克隆C3H型锌指蛋白基因BoCCCH2的基础上，研究该基因在不同器官及霜霉菌和灰葡萄孢菌侵染叶片中的表达模式。测序结果表明，BoCCCH2没有内含子，编码区全长为1 740 bp，编码579个氨基酸，推导的蛋白质具2个ANK结构域和2种CCCH锌指结构，锌指结构的类型分别为C—X8—C—X5—C—X3—H和C—X5—C—X4—C—X3—H。反转录聚合酶链反应表明：BoCCCH2在根、叶、花茎、嫩角果、花蕾和花中均有表达，其中在根中的表达量最高；经霜霉菌和灰葡萄孢菌侵染后，该基因表达量均有不同程度的增加，其中在霜霉菌侵染下，表达量在24 h后开始增加，72 h时下降，而在灰葡萄孢菌侵染下，6 h时的表达量最大，12 h时开始缓慢下降。聚类结果表明，BoCCCH2与其他十字花科植物的同源序列聚为一类，支持率达100%，而与豆科、大戟科和蔷薇科等植物的序列处于不同分支。对BoCCCH2基因的克隆和表达分析为该基因功能研究奠定了基础。
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