紫花苜蓿MsSQE1的克隆及对皂甙合成的功能分析

doi:10.3864/j.issn.0578-1752.2020.02.003

摘要/Abstract

摘要：

【目的】鲨烯环氧酶（squalene epoxidases,SQE）是苜蓿皂甙合成途径中的一种限速酶,与苜蓿皂甙的合成密切相关。通过对苜蓿鲨烯环氧酶（MsSQE1）基因的克隆及在苜蓿中过表达,探究鲨烯环氧酶对皂甙合成的作用机制。【方法】以模式植物蒺藜苜蓿鲨烯环氧酶基因序列设计引物,同源克隆苜蓿MsSQE1。对MsSQE1进行生物信息学分析;通过基因枪轰击技术,使MsSQE1在洋葱表皮瞬时表达,进行亚细胞定位。利用qRT-PCR方法分析该基因在根、茎、叶中的表达水平,以及在紫外辐射、ABA和GA₃条件下的表达模式。在茉莉酸甲酯（MeJA）的诱导下,分析MsSQE1的转录水平,及对苜蓿皂甙含量的影响。利用根癌农杆菌转化体系,获得过表达MsSQE1的阳性转基因植株,并测定转基因植株的皂甙含量。【结果】克隆了MsSQE1的cDNA序列,开放阅读框1 578 bp,编码525个氨基酸,等电点为8.59。同源性比对分析,其氨基酸序列与蒺藜苜蓿中SQE1氨基酸序列同源性为98.6%,与拟南芥的同源性为80%。亚细胞定位显示,MsSQE1可能定位于细胞膜。组织特异性表达分析显示,MsSQE1在叶中的表达量最高,茎中次之,根中的表达量最低。在紫外辐射、ABA和GA₃的诱导表达显示,紫外辐射诱导24 h,叶中表达量最高; GA₃（50 μmol·L ^-1）和 ABA（100 μmol·L ^-1）处理,均为8 h叶中表达量最高;MeJA处理能诱导MsSQE1表达量上调的同时苜蓿总皂甙含量也随着MsSQE1表达的上调而增加。分析过表达MsSQE1转基因苜蓿和转空载体苜蓿株系发现,MsSQE1的表达水平和总皂甙含量均升高,其中MsSQE1的表达水平是对照的3.11—9.45倍,总皂甙含量比对照提高14.26%—28.05%,暗示MsSQE1是苜蓿皂甙合成途径中的一种关键调节酶。【结论】从豆科牧草紫花苜蓿中克隆了MsSQE1并进行功能分析。在苜蓿中过表达MsSQE1能增加苜蓿总皂甙含量,暗示MsSQE1的表达影响苜蓿皂甙的生物合成,可能对皂甙的合成有重要的调节作用。

关键词: 紫花苜蓿, 鲨烯环氧酶基因（MsSQE1）, 苜蓿皂甙, 实时荧光定量PCR

Abstract:

【Objective】Squalene epoxidase (SQE) is a rate-limiting enzyme in sterols and triterpenoids biosynthesis pathway, which is closely related to the synthesis of saponins. This study focused on the regulatory function of MsSQE1 on saponin biosynthesis in alfalfa,【Method】MsSQE1 was isolated from alfalfa based on SQE sequence of model plant Medicago truncatula. MEGA software was used for bioinformatic analysis of MsSQE1. Subcellular localization of MsSQE1 protein was examined by micro projectile bombardment. qRT-PCR was used to examine tissue-specific expression and the expression patterns of MsSQE1 under UV radiation, MeJA, ABA and GA₃ treatments. The MsSQE1 overexpressing transgenic alfalfa was obtained by Agrobacterium-mediated transformation, and the saponins content of the transgenic plants was determined by spectrophotometer【Result】The cDNA sequence of MsSQE1 was cloned, which contained an ORF of 1 578 bp encoding a protein of 525 amino acids with an isoelectric point of 8.59. Bioinformatic analysis showed that the deduced of MsSQE1 shared high sequence homology with SQE1 from M. truncatula (98.6%) and Arabidopsis thaliana (80%). Subcellular localization indicated that MsSQE1 may be located in the cell membrane of onion epidermis. The expression level of MsSQE1 was highest in leaves compared with stems and roots. MsSQE1 was enhanced by the simulation of UV radiation, ABA and GA₃. The expression level of MsSQE1 in leaves was highest in 24 h treated by UV, and in 8 h treated of GA₃ (50 μmol·L ^-1) and ABA(100 μmol·L ^-1). Moreover, MsSQE1 was upregulated by MeJA, and the enhanced expression of MsSQE1 resulted in the increase of total saponins. Overexpression of MsSQE1 in alfalfa led to the accumulation of total saponins in the transgenic plants and MsSQE1 expression level was correlated with the contents of saponins. The expression level of MsSQE1 in transgenic alfalfa was 3.11-9.45 times of control, and the content of saponins was 14.26%-28.05% higher than control. These findings suggested that the novel identified MsSQE1 encodes epoxidase and it contributes to the synthesis of saponins in alfalfa.【Conclusion】This study reported the cloning and functional characterization of squalene epoxidase (SQE) encoding gene from legume forage alfalfa (MsSQE). Overexpression MsSQE1 in alfalfa improved the content of total saponins suggesting that MsSQE affects the saponin biosynthesis.

Key words: Medicago sativa L.(alfalfa), squalene epoxidase(SQE), saponins, qRT-PCR

康俊梅,张俏燕,蒋旭,王珍,张铁军,龙瑞才,崔会婷,杨青川. 紫花苜蓿MsSQE1的克隆及对皂甙合成的功能分析[J]. 中国农业科学, 2020, 53(2): 247-260.

KANG JunMei,ZHANG QiaoYan,JIANG Xu,WANG Zhen,ZHANG TieJun,LONG RuiCai,CUI HuiTing,YANG QingChuan. Cloning MsSQE1 from Alfalfa and Functional Analysis in Saponin Synthesis[J]. Scientia Agricultura Sinica, 2020, 53(2): 247-260.

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引物名称 Primer name	引物序列 Sequence of primer (5′-3′)	用途 Function
P1	GTTCTTTGAGATGGATTATCAGTAT	ORF分离 ORF isolation
P2	AAATGACATGAATACTACAGTGACA	ORF分离 ORF isolation
SQE1-PE-SPt-f	GAAAAGAAGAAAACAGGTTCAAGTT	原核表达 Prokaryotic expression
SQE1-PE-f	ATGGATTATCAGTATATTCTTGGAGGG
SQE1-PE-r	ATGGACAGGAGGCATTCTGTAATAT
SQE1-GFP-f	CCctcgagATGGATTATCAGTATATTCTTGGAGGG	亚细胞定位 Subcellular localization
SQE1-GFP-r	TGgtcgacATGGACAGGAGGCATTCTGTAATAT	亚细胞定位 Subcellular localization
SQE1-RTf-1	GCAAAGGCTCCTCTCACCATAG	实时荧光定量 PCR/qRT-PCR
SQE1-RTr-1	GCACATCAACCAAACAGCGAAT	实时荧光定量 PCR/qRT-PCR
Actin-s	CAAAAGATGGCAGATGCTGAGGAT	内参基因 House-keeping gene
Actin-a	CATGACACCAGTATGACGAGGTCG	内参基因 House-keeping gene
SQE1-P1	tctagaATGGATTATCAGTATATTCTTGGAGGG	扩增目的基因 Amplification of target genes
SQE1-P2	ggatccTTAATGGACAGGAGGCATTCTGTAATATGC	扩增目的基因 Amplification of target genes
35S-f	ACTATCCTTCGCAAGACCCTTCCTC	转基因株系的鉴定 Identification of transgenic lines
SQE1-r	TTAATGGACAGGAGGCATTCTGTAAT	转基因株系的鉴定 Identification of transgenic lines