Molecular characteristics and promoter analysis of SlLCY-B2 gene in tomato

doi:10.6048/j.issn.1001-4330.2023.06.019

Xinjiang Agricultural Sciences ›› 2023, Vol. 60 ›› Issue (6): 1460-1465.DOI: 10.6048/j.issn.1001-4330.2023.06.019

• Microbes·Horticultural Special Local Products·Storage and Preservation Processing • Previous Articles Next Articles

Molecular characteristics and promoter analysis of SlLCY-B2 gene in tomato

LIU Jiangna(), ZHANG Xiying, LI Rongxia, ZHANG Xiaowei, BAI Yunfeng, ZHANG Aiping()

Institute of Agriculture Sciences, Sixth Division, Xinjiang Production and Construction Corps, Wujiaqu Xinjiang 831300, China

Received:2022-10-07 Online:2023-06-20 Published:2023-06-20
Correspondence author: ZHANG Aiping(1968-), female, bachelor, researcher, mainly engaged in plant molecular breeding, (E-mail) 592354963@qq.com
Supported by:
National Natural Science Foundation of China "Improvement of Storage Quality of the Processing Tomato in Xinjiang via the CRISPR-Cas9 System"(32160710);Creation of Lycopene-Rich Processing Tomato Germplasm in Xinjiang Based on Double-Gene Editing"(31760571);Scientific and Technological Talents Innovation Program of XPCC "Improvement of lycopene Content in Tomato Fruits via CRISPR-Cas9 Bidirectional Regulation(2021CB037);Sixth Division Technology Program "Study on Key Technologies of Breeding and Improving Quality and Efficiency of New Processing Tomato Varieties(1903)

番茄SlLCY-B2及其启动子的分子特征和sgRNA分析

刘江娜(), 张西英, 李荣霞, 张小伟, 白云凤, 张爱萍()

新疆生产建设兵团第六师农业科学研究所,新疆五家渠 831300

通讯作者: 张爱萍(1968-),女,安徽人,研究员,研究方向为植物分子育种,(E-mail) 592354963@qq.com
作者简介:刘江娜(1985-),女,河北人,硕士,副研究员,研究方向为植物分子育种,(E-mail)2849847373@qq.com
基金资助:
国家自然科学基金项目“基于CRISPR-Cas9定点突变改良新疆加工番茄耐贮性研究”(32160710);基于双基因编辑创制高番茄红素新疆加工番茄新种质(31760571);兵团科技人才创新计划项目“基于 CRISPR-Cas9 双向调控提高番茄果实中番茄红素含量的研究”(2021CB037);第六师科技计划项目“加工番茄新品种引育及体质增效关键技术研究”(1903)

Abstract

Abstract:

【Objective】 Lycopene synthesis can enter the metabolic pathway catalyzed by lycopene-β-cyclase sllcy-B, and inhibition of the expression of SlLCY-B2 can increase the accumulation of lycopene. The molecular characteristics and sgRNA of lycopene metabolism regulation gene SlLCY-B1 were systematically analyzed in order to utilize CRISPR/ Cas9 Site-directed mutation or fragment deletion of SlLCY-B2 and its upstream promoter sequences was performed to regulate the expression of SlLCY-B2 and reduce metabolic pathways to improve the content of lycopene in tomato fruits.【Methods】 The characteristics, conserved domain, genome structure, digital expression profile and promoter cis-acting elements of SlLCY-B2 polypeptide were systematically analyzed by online biological tools. According to the design principle of CRISPR-Cas9 target, suitable sgRNA was designed and screened out.【Results】 SlLCY-B2 was located on chromosome 6 of tomato and encodes 498 amino acids. The gDNA did not contain introns and was specifically expressed in fruit. SlLCY-B2 distributed 145 Sgrnas, of which 21 were highly specific. There were 15 cis-acting elements and 112 Sgrnas in the 1 500 bp promoter sequence upstream of SlLCY-B2.【Conclusion】 l Gene editing of SlLCY-B2 and its promoter region can regulate the expression of SlLCY-B2 and increase lycopene content.

Key words: tomato; SlLCY-B2; molecular characteristics; promoter; cis-acting element; sgRNA

摘要：

【目的】番茄红素合成后会在β-番茄红素环化酶SlLCY-B(lycopene-β-cyclase)催化下进入代谢途径,抑制SlLCY-B2的表达可增加番茄红素的积累。对番茄红素代谢调控基因SlLCY-B1的分子特征及sgRNA进行系统分析,为利用CRISPR/ Cas9 技术对SlLCY-B2及其上游启动子序列进行定点突变或片段删除、调控SlLCY-B2的表达以减少代谢的途径,提高番茄果实的番茄红素含量。【方法】利用生物在线工具,对SlLCY-B2多肽特性及保守结构域、基因组结构、数字表达谱、启动子顺式作用元件进行系统分析,根据CRISPR-Cas9靶点设计原则,设计筛选出适宜的sgRNA。【结果】SlLCY-B2位于番茄6号染色体,编码498 个氨基酸,gDNA不含内含子,呈果实特异性表达。SlLCY-B2分布145条sgRNA,其中21条有高特异性。SlLCY-B2上游-1 500 bp 启动子序列分布15个顺式作用元件和112条sgRNA。【结论】对SlLCY-B2和其启动子区进行基因编辑,可调控SlLCY-B2的表达,将提高番茄红素含量。

关键词: 番茄, SlLCY-B2, 分子特征, 启动子, 顺式元件, sgRNA

CLC Number:

S641
S188

LIU Jiangna, ZHANG Xiying, LI Rongxia, ZHANG Xiaowei, BAI Yunfeng, ZHANG Aiping. Molecular characteristics and promoter analysis of SlLCY-B2 gene in tomato[J]. Xinjiang Agricultural Sciences, 2023, 60(6): 1460-1465.

刘江娜, 张西英, 李荣霞, 张小伟, 白云凤, 张爱萍. 番茄SlLCY-B2及其启动子的分子特征和sgRNA分析[J]. 新疆农业科学, 2023, 60(6): 1460-1465.

Figures/Tables 5

Fig.1 The digital expression profile of SlLCY-B2 baded on RNA-sequence Note: 1.TA496 fruit, ovary; 2.TA496 fruit,developing/immature green; 3.TA496 fruit, mature green; 4.TA496 fruit, breaker 5.TA496 fruit, red ripe; 6.TA496 flower, 0-3 mm buds; 7.TA496 flower, 3-8 mm buds; 8.TA496 flower, 8 mm-preanthesis buds; 9.TA496 open flower; 10.TA496 flower, a mixture of developmental stages (mixed flower); 11.TA56 wild tomato pollen; 12.R11-13 (Rio Grande x Money Maker) leaf, Pseudomonas susceptible; 13.R11-12 (35S::Pto in Rio Grande x Money Maker) leaf, Pseudomonas resistant; 14.Rio Grande PtoR leaf, mixed elicitor; 15. TA496 shoot/meristem, 4-6 week old plants; 16.TA496 shoot meristem, 8 week old plants; 17.TA496 root, plant at pre-anthesis; 18.TA496 root, plant at fruit set; 19.TA496 root, nutrient deficient; 20.TA496 radicle, seedlings 5 days post-imbibition; 21.TA496 whole seedling,7 days post imbibtion; 22.TA496 quiescent seed; 23.TA496 callus; 24.TA496, E6203 Suspension culture; 25.TA496 crown gall. PRKM: Reads Per Kilo bases per Million reads

Fig.2 Conserved domains on SlLCY-B2

Fig.3 The position of sgRNAs in SlLCY-B2 Note:Green shows these sgRNAs located in exon green seed sequence on the whole tomato genome; blue shows the seed sequence of the sgRNA adjacent PAM 12 nt has a matching sequence at one site of other chromosomes; grey indicates these sgRNAs across two exon neighbors or containing continuous TTTT, using pol III promoter to avoid these sgRNA sequences

Tab.1 Predicted cis-regulatory elements in SlLCY-B2 promoter

元件 Elements	序列及位置 Sequences and position	数量 Number	功能 Function
A-box	CCGTCC: -598	1	顺式调控元件
GATA-motif	AAGGATAAGG: -1048	1	部分光响应元件
GT1-motif	GGTTAAT: - 27,	1	光响应元件
GT1-motif	GGTTAA: - 27,	1	光响应元件
I-box	atGATAAGGTC: -1046	1	部分光响应元件
LTR	CCGAAA : -378,-707	2	低温响应顺式调控元件
MBS	CAACTG:- 439	1	与干旱诱导相关的 MYB 结合位点
P-box	CCTTTTG:-914,-1346	2	赤霉素响应元件
P-box	CAACAAACCCCTT: -1290	1	赤霉素响应元件和部分光响应元件
TC-rich repeats	ATTCTCTAAC: -1402	1	防御和胁迫响应顺式调控元件
TGA-element	AACGAC:-990	1	生长素响应元件
chs-CMA2a	TCACTTGA: -759	1	部分光响应元件
Circadian	CAAAGATATC: -289	1	昼夜节律控制顺式调控元件

Fig.4 The position of sgRNAs and cis-element in SlLCY-B2 promoter Note: sgRNA: Green shows these sgRNAs located in exon green seed sequence on the whole tomato genome; blue shows the seed sequence of the sgRNA adjacent PAM 12 nt has a matching sequence at one site of other chromosomes; grey indicates these sgRNAs across two exon neighbors or containing continuous TTTT, using pol III promoter to avoid these sgRNA sequences. Cis action element: red underscore element is located in the positive chain and blue underscore element in the negative chain

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Molecular characteristics and promoter analysis of SlLCY-B2 gene in tomato

番茄SlLCY-B2及其启动子的分子特征和sgRNA分析

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