地涌金莲MlCCD8b基因的克隆与表达分析
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中央级公益性科研院所基本科研业务费专项资金(CAFYBB2019ZB007)


Cloning and Expression Analysis of MlCCD8b in Musella lasiocarpa
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    摘要:

    该研究以地涌金莲(黄色苞片型YN01和红色苞片型RD05)为材料,采用RACE技术克隆获得地涌金莲CCD8b基因的cDNA全长,进行氨基酸序列比对及系统进化树构建,并采用实时荧光定量PCR技术检测MlCCD8b基因在不同地涌金莲类型和不同组织中的表达模式。结果表明:(1)序列分析显示,MlCCD8b的ORF全长1 671 bp,编码556个氨基酸,存在1个类胡萝卜素加氧酶家族的典型保守结构域RPE65,推测其相对分子量为61 574.26 Da,等电点6.61,亚细胞定位于叶绿体基质中的类囊体上,所编码的MlCCD8b蛋白为亲水性蛋白。(2)同源对比分析及构建系统进化树发现,地涌金莲MlCCD8b蛋白与小果野蕉亚种、凤梨等单子叶植物的CCD8b蛋白遗传关系最近。(3)荧光定量PCR检测结果表明,MlCCD8b在吸芽数量多的黄色苞片型YN01的所有组织中均有表达,而在吸芽数量少的红色苞片型RD05中,其苞片内未能检测到MlCCD8b的表达,但其他组织中皆有表达;MlCCD8b在2种类型地涌金莲中呈现一致的组织表达特异性,即在花序轴中的表达量最高,其次是吸芽芽点、根尖和叶片,在苞片中的表达量最低或不表达。(4)2种类型地涌金莲同一组织部位比较结果显示,RD05的花序轴、吸芽芽点、根尖和叶片中的MlCCD8b相对表达量分别是YN01的4.47、4.67、2.09和1.10倍。(5)利用高效液相色谱串联质谱法测得RD05根尖部位的5脱氧独脚金醇含量是YN01的15.57倍,与MlCCD8b在根尖的表达趋势一致。研究认为,MlCCD8b基因可能通过调控独角金内酯的合成,促进或抑制地涌金莲吸芽的萌生。该研究结果可为MlCCD8b基因的生物学功能研究提供依据,并为今后通过分子辅助育种调控MlCCD8b的表达,从而控制地涌金莲吸芽数量提供理论支持。

    Abstract:

    In this study, the full length cDNA of CCD8b was cloned from Musella lasiocarpa using RACE technique, and was designated as MlCCD8b (GenBank accession number MW013147),amino acid sequence alignment and phylogenetic tree were constructed. The expression patterns of MlCCD8b gene in different types and tissues of M. lasiocarpa were detected by realtime fluorescent quantitative PCR. The results showed that: (1) the full length cDNA was 2 052 bp, containing an ORF of 1 671 bp in length, encoding 556 amino acids. The relative molecular mass of coding protein is 61 574.26 Da and the isoelectric point is 6.61, which is a hydrophilic protein. This protein has a RPE65 conserved domain belonging to carotenoid oxygenase family. The subcellular localization most likely be on the thylakoid within chloroplast stroma.(2) Sequence comparison and phylogenetic tree analysis showed that the amino acid sequence of MlCCD8b had considerable similarity with those of other species, and had the closest evolutionary relationship with the CCD8b protein of Musa acuminata subsp. malaccensis and Ananas comosus. (3) Realtime PCR analysis showed that MlCCD8b exhibited a same tissue expression pattern between the two types of M. lasiocarpa, with the highest expression in rachis, followed by sucker growing point, root tip and leaf, the lowest expression or nonexpression in bract. (4) The expression of MlCCD8b in rachis, sucker growing point, root tip and leaf of RD05 were 4.47, 4.67, 2.09 and 1.10 times than those of YN01. (5) The 5DS content in RD05s root tip was 15.57 times than that in YN01s, which was consistent with the expression pattern of MlCCD8b. The study indicated that the MlCCD8b gene may participate in the synthesis and regulation of strigolactones. These results provided a basis for the further study on regulating the number of suckers in M. lasiocarpa using genetic engineering.

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安 静,马 宏,万友名,等.地涌金莲MlCCD8b基因的克隆与表达分析[J].西北植物学报,2020,40(12):2008-2016

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  • 在线发布日期: 2021-01-26
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