| 柑橘CCD基因家族鉴定及CcCCD4a对果肉颜色的影响 |
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| 引用本文： | 张亚飞,彭洁,朱延松,杨胜男,王旭,赵婉彤,江东.柑橘CCD基因家族鉴定及CcCCD4a对果肉颜色的影响[J].中国农业科学,2020,53(9):1874-1889. |
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| 作者姓名： | 张亚飞 彭洁 朱延松 杨胜男 王旭 赵婉彤 江东 |
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| 作者单位： | 1 西南大学柑桔研究所,重庆 400712;2 中国农业科学院柑桔研究所,重庆 400712 |
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| 基金项目： | 国家重点研发计划(2018YFD1000101);教育部双一流学科建设项目 |
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| 摘 要： | 【目的】研究类胡萝卜素裂解双加氧酶（CCD）基因家族在柑橘基因组中的分布、结构及进化,对CcCCD4a在果肉颜色形成过程中的表达及其在不同果肉颜色的柑橘种质中的基因型进行研究,为开发用于果肉颜色的分子辅助育种标记奠定基础。【方法】根据已报道的CCD,采用同源比对法检索柑橘基因组中的CCD家族基因（CcCCD）。采用生物信息学软件构建系统进化树,进行亚细胞定位预测,预测蛋白质的相对分子质量与等电点（pI）等理化性质,预测保守motif,绘制家族基因Scaffold定位图。利用实时荧光定量PCR技术（qRT-PCR）分析CcCCD4a在柑橘果实颜色发育过程中的表达模式,利用测序技术鉴定30个柑橘品种的CcCCD4a基因型,采用Tassel软件进行单倍型分析。【结果】从克里曼丁橘（Citrus clementina）基因组中鉴定出14个CcCCD基因家族成员,可将其分为5个亚家族,即CcCCD1、CcCCD4、CcCCD7、CcCCD8和CcNCED。该家族蛋白理论等电点分布在6.05—8.53,编码氨基酸数目介于412—611个;亚细胞定位预测结果显示该基因家族成员主要位于叶绿体和细胞质中;聚类分析发现,CCD8亚家族与其他家族成员遗传距离较远,柑橘中各CCD均能在其他物种中找到同源基因;Scaffold定位分析发现,14个CCD家族成员成员分布在除5号Scafflod外的所有Scafflod上,且分布不均匀。对10个柑橘品种在4个时期的果肉色泽进行表型鉴定,随着果实趋于成熟,果肉的色调角（h）逐步下降,果肉颜色逐步加深;CcCCD4a在不同柑橘品种中相对表达量存在显著差异,果肉颜色为浓橙红色的品种CcCCD4a表达量显著低于果肉为橙色或浅橙黄色的品种(P<0.05),CcCCD4a相对表达量与色调角呈显著正相关(P<0.05);对30个柑橘品种进行测序分析,发现单倍型hap-1、hap-4和hap-5为果肉浓橙红色品种优势单倍型。【结论】‘克里曼丁’橘包含14个CCD基因家族成员,各成员均含有RPE65保守结构域,并定位于细胞的不同位置,分布在不同的Scaffold上。CcCCD4a参与柑橘果肉颜色的形成,其基因相对表达量与果肉色调角呈显著正相关,可作为潜在的柑橘果实颜色的辅助育种标记,尤其是单倍型hap-1、hap-4、hap-5与果肉红色的关联度较高,对颜色育种的早期杂种群体筛选有一定帮助。
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| 关 键 词： | 柑橘 CcCCD基因家族 CcCCD4a 果肉颜色 基因表达 单倍型 |
| 收稿时间： | 2019-01-10 |
Genome Wide Identification of CCD Gene Family in Citrus and Effect of CcCCD4a on the Color of Citrus Flesh |
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| YaFei ZHANG,Jie PENG,YanSong ZHU,ShengNan YANG,Xu WANG,WanTong ZHAO,Dong JIANG.Genome Wide Identification of CCD Gene Family in Citrus and Effect of CcCCD4a on the Color of Citrus Flesh[J].Scientia Agricultura Sinica,2020,53(9):1874-1889. |
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| Authors: | YaFei ZHANG Jie PENG YanSong ZHU ShengNan YANG Xu WANG WanTong ZHAO Dong JIANG |
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| Institution: | 1 Citrus Research Institute, Southwest University, Chongqing 400712;2 Citrus Research Institute of Chinese Academy of Agricultural Sciences, Chongqing 400712 |
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| Abstract: | 【Objective】To reveal the distribution, structure and evolution of carotenoid cleavage dioxygenas gene family in the citrus genome (CcCCD), this study were performed to develope marker-assisted selection of flesh color in citrus breeding program, bioinformatics predication, expression analysis and genotype of CcCCD4a in flesh color development and different germplasm accessions. 【Method】The CCD gene family of Citrus clementina genome were identified by homologous search according to previously reported CCD in other plant species. Phylogenetic analysis, subcellular localization prediction, relative molecular weight, theoretical isoelectric point (PI), conserved motif prediction, and scaffold location were studied by bioinformatics methods. Real-time fluorescence quantitative PCR (qRT-PCR) was used to study the expression of CcCCD4a in 10 citrus accessions during the flesh color development period. Haplotype analysis was performed by Tassel software after sequencing of CcCCD4a in 30 citrus varieties. 【Result】Fourteen CCD family genes were found in the Citrus clementina genome, and these genes could be divided into five subfamily, namely, CcCCD1, CcCCD4, CcCCD7, CcCCD8 and CcNCED. Its theoretical isoelectric point were 6.05 to 8.53 and these CCD family genes encoded 412-611 amino acids. The subcellular localization prediction indicated that CcCCD genes mainly were located in chloroplast and cytoplasm. Phylogenetic analysis showed that CCD genes in citrus were also found in other plant species. Obviously, CCD8 subfamily had farther genetic distance with other CCD. Scaffold localization analysis showed that 14 CcCCD members were unevenly distributed in all scaffolds except scaffold 5. The phenotyping of flesh color in 10 citrus varieties demonstrated that the hue angle of flesh color was decreased along with fruit maturing. The relative expression of CcCCD4a in different citrus varieties was significantly different. The expression of CcCCD4a in the flesh color of orange red was significantly lower than that in the flesh color of orange or light orange yellow (P<0.05). There was a significant positive correlation between the relative expression of CcCCD4a and the hue angle during fruit ripening. Genotyping of CcCCD4a in 30 citrus varieties revealed that hap-1, hap-4 and hap-5 were dominant haplotype at orange red flesh varieties. 【Conclusion】The whole genome of Citrus clementina contained 14 members of CCD gene family. All these CCD gene family members contained RPE65 conserved domain, but they were located in different cell components and unevenly distributed at different scaffolds. CcCCD4a was involved in the development of citrus flesh color, and there was a significant positive correlation between its relative expression and the hue angle. Therefore, CcCCD4a could be used as potential marker for citrus fruit color breeding. Especially, hap-1, hap-4 and hap-5 had a high correlation with the phenotypes of orange red flesh, which might be helpful for selecting candidate hybrids in early stage of citrus breeding program. |
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| Keywords: | citrus CCD gene family CcCCD4a flesh color gene expression haplotype |
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