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薛晓菲1,戴云静1,李熙林1,丁艳艳1,王翔1,雷长英1,韩焕勇2,*,贺道华1,*
XUE Xiao-Fei1,DAI Yun-Jing1,LI Xi-Lin1,DING Yan-Yan1,WANG Xiang1,LEI Zhang-Ying1,HAN Huan-Yong2,*,HE Dao-Hua1,*
摘要:
棉酚是储存在棉花色素腺体的萜烯类化合物,其合成与杜松烯合酶(cadinene synthase, CDNs)密切相关。棉籽含有大量棉酚,由于棉酚有毒,限制了人类对棉籽的充分利用,研究棉酚合成相关基因,为创制“棉花植株高棉酚种子低棉酚”材料提供基因资源,对多用途棉花新品种的培育具有十分重要的意义。本研究从陆地棉杜松烯合酶基因家族的转录组数据中筛选出GhCDN10基因,对其进行克隆与序列分析;基于自然群体的基因组重测序数据对该基因进行序列多样性、连锁不平衡(LD)和单核苷酸多态性(SNP)效应及单倍型分析,结合‘棉酚有无’的表型进行关联分析;通过qRT-PCR和转录组RNA-Seq对其进行表达模式分析;通过病毒介导的基因沉默(VIGS)进行功能分析,并进行亚细胞定位。结果表明,GhCDN10由7个外显子组成,上下游的基因间区(IRs)共包含63种类型的顺式作用元件,共533个。重测序数据显示,GhCDN10的SNP频率为7.22 SNPs kb?1,核苷酸多样性π值为0.22595,LD衰减距离为100 kb左右,DNA区段有19个SNP位点,构成9个单倍型;关联分析显示,GhCDN10的19个SNP位点与‘棉酚有无’无显著关联;聚类分析基本能有效地将无酚棉资源和有酚棉资源分开。GhCDN10蛋白含有PF01397和PF03936两个保守结构域,及杜松烯合酶特有的DDTYD、DDVAE等序列模体;亚细胞定位显示该基因产物位于细胞膜和细胞核中;17个物种的同源基因在基因结构、motif组成上存在细微的差异。RNA-Seq数据表明,GhCDN10在有腺体棉中的表达量显著高于无腺体棉,在根中表达量较高,而在花和纤维中表达量很低或不表达。VIGS沉默后GhCDN10的表达量显著降低,仅为对照组的21.3%;通过体视显微镜观察计数叶片中的腺体数量和利用高效液相色谱法(HPLC)测定叶片中棉酚的含量发现,沉默植株的腺体数量和棉酚含量显著低于对照组。本研究结果为进一步剖析棉酚合成途径提供了新见解,也为通过基因工程创制低棉酚材料提供了基因资源。
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