番茄SlLCY-B2及其启动子的分子特征和sgRNA分析

doi:10.6048/j.issn.1001-4330.2023.06.019

新疆农业科学 ›› 2023, Vol. 60 ›› Issue (6): 1460-1465.DOI: 10.6048/j.issn.1001-4330.2023.06.019

• 微生物·园艺特产·贮藏保鲜加工 • 上一篇下一篇

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

收稿日期:2022-10-07 出版日期:2023-06-20 发布日期:2023-06-20
通信作者: 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)

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)

摘要/Abstract

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

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

中图分类号:

S641
S188

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.

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.

图/表 5

图1 基于 RNA-seq的SlLCY-B2数字表达谱注:1.果实,子房; 2.未成熟绿果; 3.成熟绿果; 4.破色期果实; 5.成熟红果; 6.花蕾(0~3 mm); 7.花蕾(3~8 mm); 8.开花前花蕾(8 mm); 9.开放的花; 10.发育阶段花的混合物; 11.野生番茄花粉; 12.感染假单胞菌叶片;13.抗假单胞菌叶片;14.混合诱导叶片;15.植株的顶端分生组织(4~6周龄植株); 16.植株顶端分生组织(8周龄植株);17.花前根;18.挂果期根;19.营养匮乏的根;20.完全吸涨5 d后的胚根;21.完全吸涨7 d后的幼苗;22.休眠种子; 23.愈伤组织;24.悬浮培养物;25.冠瘿。PRKM: 每百万reads中来自于特定基因每千碱基长度的reads数

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

图2 SlLCY-B2保守域

Fig.2 Conserved domains on SlLCY-B2

图3 sgRNA在SlLCY-B2的分布注:绿色>示这些sgRNAs位于外显子绿色示sgRNA邻近PAM 12 nt的种子序列在番茄全基因组上是唯一序列;黑色>色示该sgRNA邻近PAM 12 nt 的种子序列在其它染色体的1个位点上有与其匹配的序列;红色<表示这些sgRNAs横跨2个外显子邻接处或含有含连续的TTTT,选用pol III启动子尽力避免选用这些sgRNA序列

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

表1 SlLCY-B2启动子顺式作用元件

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

图4 sgRNA和顺式作用元件在SlLCY-B2启动子的分布注:sgRNA: 绿色>示这些sgRNAs位于外显子绿色示sgRNA邻近PAM 12 nt 的种子序列在番茄全基因组上是唯一序列;黑色>示该sgRNA邻近PAM 12 nt 的种子序列在其它染色体的1个位点上有与其匹配的序列;灰色>表示这些sgRNAs横跨2个外显子邻接处或含有含连续的TTTT,选用pol III启动子尽力避免选用这些sgRNA序列。顺式作用元件:红色下划线示顺式作用元件位于正链,蓝色下划线示元件位于负链

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

参考文献 20

[1]	吕慧芳, 刘四运. 植物番茄红素生物合成及分子调控机理研究进展[J]. 池州学院学报, 2016, 30(3):43-47.
	LV Huifang, LIU Siyun. Biosynthesis of lycopene from plants And advances in molecular regulation mechanism[J]. Journal of Chizhou University, 2016, 30(3):43-47.
[2]	Ronen G, Carmel-Goren L, Zamir D, et al. An alternative pathway to beta-carotene formation in plant chromoplasts discovered by map-based cloning of beta and old-gold color mutations in tomato[J], Proc. Natl. Acad. Sci., USA (PNAS). 2000, 97(20): 11102-11107. DOI URL
[3]	Rosati C, Aquilani R, Dharmapuri S et al. Metabolic engineering of beta-carotene and lycopene content in tomato fruit[J]. Plant J, 2000, 24:413-419. DOI PMID
[4]	马超, 马兵钢, 郝青南. 番茄红素β环化酶基因(Lyc-β)RNAi载体构建及表达鉴定[J]. 农业生物技术学报, 2010, 18(1): 10-17.
	MA Chao, MA Binggang, HAO Qinnan. Construction of RNA Interference Vector for Lycopene Cyclase β Gene (Lyc-β) and its Expression Identification in Lycopersicun esculentum[J]. Journal of Agricultural Biotechnology, 2010, 18(1):10-17.
[5]	万群, 张兴国, 宋明. 果实特异性 RNAi 介导的 Lcy 基因沉默来增加番茄中番茄红素的含量[J]. 生物工程学报, 2007, 23(3): 429-433. PMID
	WAN Qun, ZHANG Xinguo, SONG Min. Fruit-specific RNAi-mediated Lcy Gene Silencing Enhances Content of Lycopene in Tomatoes[J]. Chinese Journal of Biotechnology. 2007, 23(3): 429-433 DOI PMID
[6]	Voytas D F. Plant genome engineering with sequence-specific nucleases[J]. Annu. Rev. Plant. Biol, 2013, 64(1):327-350. DOI URL
[7]	Li X, Wang Y, Chen S, et al. Lycopene is enriched in tomato fruit by CRISPR/Cas9-mediated multiplex genome editing[J]. Frontiers in Plant Science, 2018, 9:559. DOI PMID
[8]	Farboud B, Meyer B J. Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design[J]. Genetics, 2015, 199: 959-971. DOI PMID
[9]	Lescot M, Déhais P, Moreau Y, et al. PlantCARE: a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J]. Nucleic Acids Res, 2002, 30(1): 325-327. DOI URL
[10]	Arumuganathan K, Earle ED. Nuclear DNA content of some important plant species[J]. Plant Molecular Biology Reports, 1991, 9: 208-218. DOI URL
[11]	Fu Y, Foden J, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR/Cas nucleases in human cells[J]. Nature Biotechnology, 2013, 31(9): 822-826. DOI
[12]	Hsu p, Scott D, Weinstein J, et al. DNA targeting specificity of RNA-guided Cas9 nucleases[J]. Nature Biotechnology, 2013, 31(9): 827-832. DOI PMID
[13]	Friedland, A. E., Y. B. Tzur, et al. Church et al. Heritable genome editing in C. elegans via a CRISPR-Cas9 system[J]. Nat. Methods, 2013, 10: 741-743. DOI PMID
[14]	Doudna J A, Charpentier E,. Genome editing. The new frontier of genome engineering with CRISPR-Cas9[J]. Science, 2014, 346 (6213): 1258096. DOI URL
[15]	Doench J G, Hartenian E, Graham D B, et al. Rational design of highly active sgRNAs for CRISPR/Cas9-mediated gene inactivation[J]. Natura Biotechnology, 2014, 32(12):1262-127. DOI
[16]	Zhang S J, Zhang R Z, Song G Q, et al. Construction of promoter region CRISPR/Cas9 genome editing vector of pinb gene in wheat[J]. Shandong Agricultural Sciences, 2020, 52(1):1-9.
[17]	张淑娟, 张荣志, 宋国琦, 等. 小麦 Pinb 基因启动子区 CRISPR / Cas9 基因编辑载体的构建[J]. 山东农业科学, 2020, 52(1):1-9.
	ZHANG Sunjuan, ZHANG Rongzhi, SONG Guoqi, et al. Construction of promoter region CRISPR/Cas9 genome editing vector of pinb gene in wheat.Shandong Agricultural Sciences[J]. Shandong Agricultural Sciences, 2020, 52(1):1-9
[18]	Rodríguez-Leal D, Lemmon Z H, Man J, et al. Engineering quantitative trait variation for crop improvement by genome editing[J]. Cell, 2017, 171(2): 470-480. DOI PMID
[19]	Huang L, Li Q, Zhang C, et al. Creating novel Wx alleles with fine‐tuned amylose levels and improved grain quality in rice by promoter editing using CRISPR/Cas9 system[J]. Plant Biotechnology Journal, 2020a, https://doi.org/10.1111/pbi.13391.
[20]	Huang L, Sreenivasulu N, Liu Q. Waxy editing: Old meets new[J]. Trends in Plant Science,2020b https://doi.org/10.1016/j.tplants.2020.07.009.

番茄SlLCY-B2及其启动子的分子特征和sgRNA分析

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

在线阅读

PDF下载

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 20

相关文章 15

编辑推荐

Metrics

[1]	王丹丹, 李燕, 张庆银, 李世东, 庞永超, 马琨芝, 马龙, 牛瑞生, 钟增明, 齐连芬, 师建华. 不同微生物菌处理对番茄土壤微生物多样性的影响[J]. 新疆农业科学, 2023, 60(9): 2248-2257.
[2]	蒲敏, 阮向阳, 肖乐乐, 索常凯, 陈国永, 冶军, 高波. 枸溶性钙镁肥对加工番茄钙、镁吸收及品质的影响[J]. 新疆农业科学, 2023, 60(8): 1987-1995.
[3]	肖林刚, 马艳, 宋兵伟, 焦锐斌, 邢剑飞. 温室膜下微喷灌溉技术研究进展[J]. 新疆农业科学, 2023, 60(7): 1731-1740.
[4]	杜红艳, 庞胜群, 马海翔, 吉雪花. 加工番茄早熟突变体农艺性状相关性及通径分析[J]. 新疆农业科学, 2023, 60(4): 943-950.
[5]	朱普生, 刘慧英, 曹泽, 刘凯歌, 李雪珍. 外源GSNO对NaCl胁迫下番茄幼苗生长及光合特性的影响[J]. 新疆农业科学, 2023, 60(2): 351-358.
[6]	曾军, 武磊, 高雁, 张卓, 林青, 刘建伟, 杨红梅, 霍向东, 史应武. 光合细菌叶面肥喷施对设施有机番茄产量和品质的影响[J]. 新疆农业科学, 2023, 60(12): 3018-3024.
[7]	刘升学, 李思琪, 王晓东, 杨德松. 番茄早疫病菌dsRNA多样性分析[J]. 新疆农业科学, 2023, 60(12): 3057-3064.
[8]	芦大伟, 哈丽哈什·依巴提, 李青军. 磷肥用量对滴灌加工番茄产量、品质及磷肥利用率的影响[J]. 新疆农业科学, 2023, 60(1): 185-191.
[9]	李岳雁, 李玉姗, 王帆, 郭雅文, 王飞燕, 高杰, 宋羽. 不同品种番茄果实性状遗传多样性及聚类分析[J]. 新疆农业科学, 2022, 59(9): 2147-2157.
[10]	李春雨, 谭占明, 程云霞, 束胜, 何涛, 靳钰婕, 马新超, 杜佳庚, 张婧. 水肥耦合对沙培番茄生长发育及品质的影响[J]. 新疆农业科学, 2022, 59(9): 2158-2169.
[11]	马雪红, 刘婷, 李传天, 朱金芳. HPLC法测定番茄红素胶束中番茄红素Z/E异构体含量[J]. 新疆农业科学, 2022, 59(9): 2209-2216.
[12]	古宁宁, 张蒲, 谢彦如, 唐丹, 赵志信, 董瑞芳, 崔拥民, 秦勇. 基质中化肥施入量对番茄幼苗生长的影响[J]. 新疆农业科学, 2022, 59(8): 1929-1934.
[13]	许俊锋, 杨蓉, 詹发强, 侯敏, 包慧芳, 王宁, 龙宣杞, 张志东, 崔卫东. 有机-无机液体复合肥功能菌筛选及对设施番茄品质、生理活性的影响[J]. 新疆农业科学, 2022, 59(7): 1758-1766.
[14]	王强, 刘会芳, 韩宏伟, 庄红梅, 王柏柯, 王娟, 杨涛, 王浩, 秦勇. 基于 TMT和PRM 技术筛选番茄响应盐胁迫差异表达蛋白[J]. 新疆农业科学, 2022, 59(6): 1418-1428.
[15]	林青, 史应武, 王娜, 华兰兰, 杨红梅, 楚敏, 曾军, 高雁, 霍向东. 加工番茄系统抗性诱导促生菌的筛选鉴定及其促生防病效果[J]. 新疆农业科学, 2022, 59(6): 1466-1474.