Cloning and Functional Analysis of Squalene Synthase Gene in Citrus

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

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (9): 1641-1652.doi: 10.16420/j.issn.0513-353x.2021-0580

• Research Papers • Next Articles

Cloning and Functional Analysis of Squalene Synthase Gene in Citrus

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

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 E-mail:zhaoxiaochun@cric.cn

Abstract

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

CLC Number:

S666

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.

Figures/Tables 8

References 31

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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

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

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

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

Cloning and Functional Analysis of Squalene Synthase Gene in Citrus

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