番茄Slβ-Hex的分子特征及启动子sgRNA分析

doi:10.6048/j.issn.1001-4330.2025.05.010

新疆农业科学 ›› 2025, Vol. 62 ›› Issue (5): 1131-1138.DOI: 10.6048/j.issn.1001-4330.2025.05.010

• 园艺特产·农产品加工工程 • 上一篇下一篇

番茄Slβ-Hex的分子特征及启动子sgRNA分析

张西英(), 刘江娜, 白云凤, 李荣霞, 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

收稿日期:2024-10-11 出版日期:2025-05-20 发布日期:2025-07-09
通信作者: 张爱萍(1968-),女,新疆人,研究员,研究方向为番茄遗传育种,(E-mail)379409419@qq.com
作者简介:张西英(1984-),女,山东日照人,副研究员,研究方向为作物遗传育种,(E-mail)592354963@qq.com
基金资助:
新疆生产建设兵团科技创新人才计划项目“基于双基因编辑调控番茄果实软化研究”(2023CB019-01);国家自然科学基金项目“基于双基因编辑创制高番茄红素新疆加工番茄新种质”(32160710);新疆生产建设兵团科技创新人才计划项目“加工番茄高可溶性固形物种质创新”(2023CB019-02);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”(2206)

Molecular characteristics and promoter analysis of Slβ-Hex gene in tomato

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

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

Received:2024-10-11 Published:2025-05-20 Online:2025-07-09
Supported by:
S & T Innovation Talent Program Project of XPCC “Study on the Regulation of Tomato Fruit Softening Based on Two-Gene Editing "(2023CB019-01);National Natural Science Foundation of China “New Germplasm of High Lycopene Xinjiang Processed Tomato Based on Double Gene Editing "(32160710);S & T Innovation Talent Program Project of XPCC “Processed Tomato Highly Soluble and Solid Material and Germplasm Innovation "(2023CB019-02);Sixth Division Science and Technology Program Project "Research on Key Technology of Breeding and Physical Enhancement of New Tomato Varieties"(2206)

摘要/Abstract

摘要：

【目的】利用CRISPR-Cas基因编辑系统可对其进行定点突变调控番茄果实软化,为利用CRISPR-Cas系统突变失活番茄细胞壁糖苷水解酶基因β-Hex,抑制N-糖蛋白的降解和游离N-聚糖的产生,降低细胞壁水解软化程度,调控番茄果实过度软化的深入研究奠定基础。【方法】利用生物在线工具,对Slβ-Hex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sgRNAs,促进番茄基因编辑高效表达载体的构建。【结果】番茄Slβ-Hex基因位于番茄第1号染色体的互补链上,由2个外显子、1个内含子组成,编码575个氨基酸。Slβ-Hex主要在番茄的果实中表达,尤其在果皮中表达量较高。该启动子的正链分布7条sgRNA,负链分布20条sgRNA,其中的10条sgRNA含有16个顺式作用元件。【结论】通过选用高特异性sgRNA进行基因编辑,使其所含的顺式元件序列突变,抑制改变Slβ-Hex的表达。

关键词: 番茄; Slβ-Hex分子特征; 启动子; 顺式作用元件; sgRNA

Abstract:

【Objective】 Playing an important role in fruit ripening and softening, In order to inactivate the tomato cell wall glycoside hydrolase gene β-Hex by mutation of CRISPR-Cas system and inhibit the degradation of n-glycoprotein and the production of free n-glycan, to reduce the degree of hydrolytic softening of cell wall and regulate the excessive softening of tomato fruit lay the foundation for further research.CRISPR-Cas gene editing system will be used to regulate tomato fruit softening by site-specific mutation. 【Methods】 The characteristics, conserved domain, genome structure, digital expression profile and promoter cis-acting elements of Slβ-Hex polypeptide were systematically analyzed using biological online tools, and suitable sgRNAs were designed and screened according to CRISPR-Cas9 target design principles to promote the construction of tomato gene editing efficient expression vector.【Results】 Tomato SLβhex gene was located on the complementary chain of tomato chromosome 1, and composed of 2 exons and 1 intron, encoding 575 amino acids. Transcriptome analysis of Slβ-Hex based on RNA-seq showed that Slβ-Hex was mainly expressed in the fruit of tomato, especially in the peel. The positive chain of the promoter distributed 7 Sgrnas, negative chain distributed 20 Sgrnas, of which 10 Sgrnas contained 16 cis-acting elements. 【Conclusion】 High-specific sgRNA is selected for gene editing to mutate the cis-element sequence and inhibit the expression of Slβ-Hex.

Key words: tomato; molecular characteristics of Slβ-Hex; promoter; Cis-acting element; sgRNA

中图分类号:

S641.2
X502

张西英, 刘江娜, 白云凤, 李荣霞, 张爱萍. 番茄Slβ-Hex的分子特征及启动子sgRNA分析[J]. 新疆农业科学, 2025, 62(5): 1131-1138.

ZHANG Xiying, LIU Jiangna, BAI Yunfeng, LI Rongxia, ZHANG Aiping. Molecular characteristics and promoter analysis of Slβ-Hex gene in tomato[J]. Xinjiang Agricultural Sciences, 2025, 62(5): 1131-1138.

图/表 9

图1 Slβ-Hex的基因组结构和染色体位置

Fig.1 Slβ-Hex genomic constructure and its location on the chromosome of tomato

图2 基于 RNA-seq的番茄栽培品种及野生近缘种醋栗番茄不同组织的Slβ-Hex的转录组注:1.花蕾;2.开放的花;3. 1 cm大的果实;4. 2 cm大的果实;5. 3 cm大的果实;6.青熟果实;7.转色果实; 8.转色后期果实; 9.根; 10.叶; 11. 醋栗番茄未熟绿果; 12. 醋栗番茄转色果实;13. 醋栗番茄转色后期果实; 14. 醋栗番茄叶片

Fig.2 Transcriptomic analysis of Slβ-Hex in different tissues of tomato cultivars and wild relatives gooseberry tomato based on RNA-seq Notes:1. unopened flower buds;2. fully opened flowers; 3. 1 cm fruits; 4. 2 cm fruits;5.3 cm fruits;6. mature green fruits;7. breaker fruits;8.breaker+10 fruits;9.roots;10.leaves;11. pimpinellifolium immature green fruits;12.pimpinellifolium breaker fruits; 13. pimpinellifolium breaker+5 fruits;14. pimpinellifolium leaves.

图3 基于RNA-sequence 的番茄Slβ-Hex在果实不同部位的转录组注:1. SA3花后4 d的表皮和外果皮;2. SA3花后4 d的隔膜和种子;3. SA3花后7 d的表皮;4. SA3花后7 d的隔膜;5. SA3花后7 d的种子;6. SA3花后10 d的表皮;7. SA3花后10 d的隔膜; 8. SA3花后10 d的种子; 9. SA4花后4 d的表皮和外果皮; 10. SA4花后4 d的隔膜和种子; 11. SA4花后7 d的表皮; 12. SA4花后7 d的隔膜;13. SA3花后7 d的种子;14. SA3花后10 d的表皮;15. SA3花后10 d的隔膜;16. SA4花后10 d的种子。

Fig.3 The Slβ-Hex transcriptome profiling of three fruit tissuesd baded on RNA-sequence Notes:1. SA3 Pericarp and exocarp, 4 Days Post Anthesis; 2. SA3 Septum and seeds, 4 Days Post Anthesis; 3. SA3 Pericarp, 7 Days Post Anthesis; 4. SA3 Septum, 7 Days Post Anthesis; 5. SA3 Seeds, 7 Days Post Anthesis ; 6. SA3 Pericarp, 10 Days Post Anthesis ;7. SA3 Septum, 10 Days Post Anthesis; 8. SA3 Seeds, 10 Days Post Anthesis; 9. SA4 Pericarp and exocarp, 4 Days Post Anthesis; 10. SA4 Septum and seeds, 4 Days Post Anthesis;11. SA4 Pericarp, 7 Days Post Anthesis; 12. SA4 Septum, 7 Days Post Anthesis; 13. SA4 Seeds, 7 Days Post Anthesis; 14. SA4 Pericarp, 10 Days Post Anthesis; 15. SA4 Septum, 10 Days Post Anthesis; 16. SA4 Seeds, 10 Days Post Anthesis

表1 Slb-Hex启动子顺式作用元件

Tab.1 Predicted cis-regulatory elements in Slb-Hex promoter

元件 Elements	序列及位置 Sequences and position	数量 Number	功能 Function
ABRE	ACGTG, 424(-)	1	脱落酸响应顺式作用元件
AE-box	AGAAACAA, 187(+)	1	部分光响应元件
ARE	AAACCA, 306(-),628(+)	2	厌氧诱导反应顺式调控元件
Box4	ATTAAT, 371(+), 495(-)	2	光响应顺式调控元件
G-box	CACGTT, 424(+)	1	光响应顺式调控元件
GCN4_motif	TGAGTCA,234(-)	1	参与胚乳表达的顺式作用元件
GT1-motif	GGTTAA, 556(-)		光应答因子
HD-Zip 3	GTAAT(G/C)ATTAC,527(-)		蛋白质结合位点
TCT-motif	TCTTAC,133(+)		部分光应答因子
TGA-motif	AACGAC,293(-), 570(+)	2	生长素应答因子
chs-CMA2a	TCACTTGA,261(+)	1	部分光应答因子

图4 Slβ-Hex启动子包含的诱导型顺式作用元件和sgRNAs

Fig. 4 The position of cis-acting elements and sgRNAs in the promoter of Slβ-Hex

表2 基因克隆所用引物

Tab.2 Primersused in thisstudy

引物名称 Primername	引物序列(5'→ 3') Primer sequence(5'→ 3')
Slβ-Hex-F	ctatgagaggagagaaaacattctcc
Slβ-Hex-R	catcagctagtaaatgaatgaactg

图5 Hex电泳CDL 2000 Marker:2000、1000、750、500、250和100 bp

Fig.5 Hex electroph-ores is 2000、1000、750、500、250 and 100 bp

表3 包含β-Hex sgRNA的引物序列

Tab.3 Primer sequence containingβ-Hex sgRNA

图6 双sgRNA 靶位点在Slβ-Hex 中的位置

Fig.6 The position of the dual sgRNA targeting sife in Slβ-Hex

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番茄Slβ-Hex的分子特征及启动子sgRNA分析

Molecular characteristics and promoter analysis of Slβ-Hex gene in tomato

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