柑橘SQS基因的克隆及功能分析

doi:10.16420/j.issn.0513-353x.2021-0580

摘要/Abstract

摘要：

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

Abstract:

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

中图分类号:

S666

王福生, 刘晓纳, 徐媛媛, 刘小丰, 朱世平, 赵晓春. 柑橘SQS基因的克隆及功能分析[J]. 园艺学报, 2021, 48(9): 1641-1652.

WANG Fusheng, LIU Xiaona, XU Yuanyuan, LIU Xiaofeng, ZHU Shiping, ZHAO Xiaochun. Cloning and Functional Analysis of Squalene Synthase Gene in Citrus[J]. Acta Horticulturae Sinica, 2021, 48(9): 1641-1652.

图/表 8

表1 目的片段扩增和基因qRT-PCR分析所用引物

Table 1 Primers for gene amplification and qRT-PCR analysis

引物名称 Primer	序列（5′-3′） Sequence
SQS-GSP	F：CATCATGTTGCAACTGCTTTTCTG;R：GATTACGCCAAGCTTCCACATGCATCAGAGCATTGGTGAC
gene-specific 5′UTR	TCTCTCCTCAAGTCCAATTCAGTCAGTAT
3′ adapter-UAP	GGCCACGCGTCGACTAGTAC
SQSRNAi	F：TCTAGAGCTCAGCAGGACTTGTAGGATTAGGT;R：GGATCCGAGCTCTGAGGGATAGCACAGAATCG
Nos+SQS-sense	F ：GATCATGAGCGGAGAATTAA;R：CCTCTTTCTACCTTCCCACA
pGBiantisense	F：ACTTACTTACACTTGCCTTGGAGT;R：AAGGATCTGAGCTACACATGCTC
N + P	F：CCTGATGCTCTTCGTCCAGA;R：GGATTGATGTGATAACATGGTGG
SQS-qPCR	F：TCCAGAAGTTTCGCTCTCGTTA;R：TGGTGTCATCCTCAACAGTGTC
123βAS	F：CAGCTGCGACTACAGCATTGAG;R：CTTGCGATGCTCAGACTCAGCT
330βAS	F：ACTGCAATGAAGAGGGCTGCT;R：TCTCTGCGATGCTCAACTGTG
490CAMS1	F：ACTTCTAAAGGAGGGTGGACAT;R：TTCTGGTGGCAGCATTGAG
195LUP2	F：GGATGGGGATTACACGTTGAG;R：TCGAGCTCTTGCACAAGCGT
632βAS	F：ATTACACGTTGAGGGTCACAGC;R：AGATGGTATGCTGGTTGCTCC
789LUP2	F：CGTTGAAGAGCTAGCTGAGGT;R：CTGAATTTGCCAAAGGAGGTC
602CAS1	F：TGCTTTGCTGTTGGGTGGAA;R：CGGCAAAAGCAGTGTCCCAT
GAPDH	F：GGAAGGTCAAGATCGGAATCAA;R：CGTCCCTCTGCAAGATGACTCT

图1 18个柑橘材料中SQS基因CDS第14位核苷酸的G/C非同义突变 G：下划线的G为CDS第14位核苷酸。

Fig. 1 G/C nonsynonymous mutation at14th nucleotide acids of SQS CDS in eighteen citrus accessions G：G with underline is the 14th nucleotide acids in CDS.

图2 柑橘SQS氨基酸序列的进化树进化树节点上的数字代表置信度。

Fig. 2 Phylogenetic tree of deduced citrus SQS amino acids Numbers at node represent bootstrap support values.

图3 ‘黄花尤力克’柠檬中SQS基因的组织特异性表达

Fig. 3 Tissue special expression of SQS gene in Citrus limonia

图4 柚不同品种成熟种子和各发育时期种子中SQS基因表达水平（A、B）和类柠檬苦素含量（C、D） MLZY：勐仑早柚;GXMY：琯溪蜜柚;PSY：坪山柚;GXSTY：广西沙田柚;JLY：金兰柚;MWY：梅塆柚;BP：缅甸柚。

Fig. 4 SQS expression level（A,B）and limonoids contents（C,D）in mature seeds and seeds at different developmental stages of pummelos MLZY：Citrus grandis‘Menglun Zao You’;GXMY：C. grandis‘Guanxi Mi You’;PSY：C. grandis‘Pingshan You’;GXSTY：C. grandis‘Guangxi Shatian You’;JLY：C. grandis‘Jinlan You’;MWY：C. grandis‘Meiwan You’;BP：C. grandis‘Burma Pummelo’.

图5 SQS基因干扰锦橙植株及其PCR验证 C-：非转基因植株对照;SiN-1 ~ SiN-4：SQS基因干扰株系。

Fig. 5 PCR detection and SQS RNAi transgenic plants C-：Nontransgenic plants;SiN-1-SiN-4：SQS RNAi transgenic plants.

图6 SQS基因干扰植株叶片中OSC类型基因的表达 C-：非转基因柑橘;SiN-1 ~ SiN-4：SQS基因干扰株系。

Fig. 6 qRT-PCR results of relative expression level of OSC genes in leaf of SQS RNAi transgenic plants C-：Nontransgenic plants;SiN-1-SiN-4：SQS RNAi transgenic plants.

表2 SQS基因干扰植株SiN-1 ~ SiN-4叶片中的类柠檬苦素含量

Table 2 Limonoids contents in leaf of transgenic plants SiN-1-SiN-4 mg · kg-1 FW

植株Line	柠檬苦素Limonin	诺米林Nomilin	类柠檬苦素Limonoids
SiN-1	120.99 ± 1.92 c	235.93 ± 3.95 e	356.92 ± 5.87 d
SiN-2	80.18 ± 3.18 d	279.61 ± 9.56 d	359.79 ± 12.74 d
SiN-3	162.78 ± 5.80 b	449.87 ± 2.98 a	611.78 ± 8.78 a
SiN-4	71.51 ± 0.88 d	398.45 ± 2.81 b	469.96 ± 3.69 c
C-（非转基因植株Nontransgenic plants）	199.57 ± 10.25 a	294.50 ± 13.72 c	494.07 ± 1.00 b

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