园艺学报 ›› 2021, Vol. 48 ›› Issue (9): 1641-1652.doi: 10.16420/j.issn.0513-353x.2021-0580

• 研究论文 •    下一篇


王福生, 刘晓纳, 徐媛媛, 刘小丰, 朱世平, 赵晓春*()   

  1. 西南大学/中国农业科学院柑桔研究所,国家柑桔工程技术研究中心,重庆400712
  • 收稿日期:2021-06-16 修回日期:2021-08-27 出版日期:2021-09-25 发布日期:2021-09-30
  • 通讯作者: 赵晓春
  • 基金资助:

Cloning and Functional Analysis of Squalene Synthase Gene in Citrus

WANG Fusheng, LIU Xiaona, XU Yuanyuan, LIU Xiaofeng, ZHU Shiping, ZHAO Xiaochun*()   

  1. Citrus Research Institute of Southwest University/Chinese Academy of Agricultural Sciences,National Citrus Engineering Research Center,Chongqing 400712,China
  • Received:2021-06-16 Revised:2021-08-27 Online:2021-09-25 Published:2021-09-30
  • Contact: ZHAO Xiaochun


从18个柑橘品种中克隆角鲨烯合成酶(squalene synthase,SQS)基因,分析其序列和表达特性,通过遗传转化研究该基因在柑橘类柠檬苦素生物合成过程的作用。结果表明,除了‘融安金柑’和‘小果罗浮’外,SQS基因的开放阅读框(open reading frame,ORF)为1 242 bp,编码413个氨基酸。序列比对发现,18个品种的氨基酸序列保守性为97.1% ~ 100%,‘小果罗浮’‘融安金柑’和‘4号宜昌橙’等品种在CDS区第14位碱基发生G/C非同义突变。系统进化树显示,‘小果罗浮’‘融安金柑’和‘4号宜昌橙’的SQS具有较近的遗传距离。qRT-PCR结果表明,柑橘SQS基因在花中的表达水平最高,成熟茎中其次,幼嫩茎、根和叶片中再次,幼果中最低。‘琯溪蜜柚’不同发育时期种子中SQS基因表达水平与类柠檬苦素含量呈极显著正相关。4株SQS基因干扰的‘锦橙’株系(SiN-1 ~ SiN-4)叶片中的SQS基因表达水平为对照的61.00% ~ 79.00%;柠檬苦素含量为对照的35.83% ~ 81.56%;SiN-1和SiN-2叶片中的诺米林含量分别为对照的80.11%和94.94%,而SiN-3和SiN-4中的诺米林含量分别比对照提高52.76%和35.30%。SQS基因干扰植株中控制三萜类和甾醇类生物合成的氧化鲨烯环化酶(oxidosqualene cyclase,OSC)基因出现差异性调控,大多数OSC基因的表达水平降低。以上结果表明,SQS是控制类柠檬苦素、三萜类和植物甾醇类物质生物合成的关键基因,能显著影响类柠檬苦素化合物的生物合成。

关键词: 柑橘, 角鲨烯合成酶, 类柠檬苦素, 生物合成, RNA干扰, SNP


A squalene synthase(SQS)gene was isolated from 18 citrus accessions,and its sequence and expression characteristics were analyzed. The roles in the biosynthesis of limonoids were identified by genetic transformation. The results showed that the full length of SQS ORF(open reading frame)in citrus is 1 242 bp,coding 413 amino acids except in the accessions of Rongan Kumquat(Fortunella classifalia)and Xiaoguo Kumquat(F. margarita). Clustal analysis showed that the deduced amino acids of SQS shared from 97.1% to 100% homology among the 18 citrus accessions. SQS in Xiaoguo kumquat,Rongan kumquat,and No.4 Ichang Papeda(Citrus ichangensis)shared a G/C nonsynonymous mutation at the fourteenth nucleotide of CDS region. Phylogenetic analysis result of SQS proteins showed that the SQS of those accessions clustered together. qRT-PCR analysis showed that the highest expression of SQS was observed in flower,followed by that in stem,root,leaf,and young fruit. The expression level of SQS gene is positively correlated with the limonoids contents in seeds of Guanxi Mi You(C. grandis)variety at different development stages. Four positive SQS interference transgenic citrus plants(SiN-1-SiN-4)were obtained via citrus genetic transformation. The expression levels of SQS gene in four transgenic lines were 61.00% to 79.00% of the control plants. Obvious deduction on limonin content was observed in all the four transgenic lines(about 35.83% to 81.56% of the control plants). The contents of nomilin in SiN-1 and SiN-2 lines were decreased to 80.11% and 94.94% of the control,respectively. However,the nomilin content increased by 52.76% and 35.30% in SiN-3 and SiN-4 lines,respectively. The expression levels of genes belonging to oxidosqualene cyclase(OSC)superfamily involved in triterpenoids and sterol biosynthesis were down-regulated in SQS interference transgenic citrus plants. This may due to the reduced supply of precursor for triterpenoids and sterol biosynthesis. This study indicated that SQS gene contributed greatly to the production of limonoids in citrus.

Key words: citrus, squalene synthase, limonoids, biosynthesis, RNA interference, SNP