果蝇F-box基因Ppa促进脂肪储存

doi:10.16288/j.yczz.21-084

遗传 ›› 2021, Vol. 43 ›› Issue (6): 615-622.doi: 10.16288/j.yczz.21-084

• 研究报告 • 上一篇

果蝇F-box基因Ppa促进脂肪储存

杨光武(), 田嫄()

贵州大学生命科学学院,贵阳 550025

收稿日期:2021-03-05 修回日期:2021-04-28 出版日期:2021-06-20 发布日期:2021-05-07
通讯作者: 田嫄 E-mail:ygw1996426@163.com;ytian1@gzu.edu.cn
作者简介:杨光武,在读硕士研究生,专业方向：发育生物学。E-mail: ygw1996426@163.com
基金资助:
国家自然科学基金项目编号(31600962);贵州省科学技术基金资助项目资助编号([2017]1043)

The F-box gene Ppa promotes lipid storage in Drosophila

Guangwu Yang(), Yuan Tian()

College of Life Sciences, Guizhou University, Guiyang 550025, China

Received:2021-03-05 Revised:2021-04-28 Online:2021-06-20 Published:2021-05-07
Contact: Tian Yuan E-mail:ygw1996426@163.com;ytian1@gzu.edu.cn
Supported by:
Supported by the National Natural Science Foundation of China No(31600962);Guizhou Provincial Science and Technology Foundation No([2017]1043)

摘要/Abstract

摘要：

脂质是构成生物体的重要成分之一,脂质代谢的精确调节和稳态维持对人类健康至关重要。泛素化途径通过降解脂质相关蛋白来调控脂质代谢。Ppa (partner of paired)编码一种F-box蛋白,后者是SCF (Skp1-Cullin1-F-box)泛素化复合体成员之一。已有的研究表明Ppa在调控果蝇体节的正常发育和着丝粒组蛋白的正确定位方面发挥重要的作用,但在脂肪代谢方面的功能研究却未见报道。本研究以黑腹果蝇(Drosophila melanogaster)作为研究对象,探究了Ppa在脂肪储存中的功能。通过与绿色荧光蛋白融合表达,检测PPA的亚细胞定位;应用CRISPR/Cas9技术,构建Ppa的缺失突变体;通过BODIPY 493/503或Nile red脂滴染色,对缺失突变体和超量表达Ppa果蝇的脂滴形态改变进行分析。随后,在缺失突变体中超量表达Ppa以验证其功能。结果表明,PPA-GFP融合蛋白定位于唾液腺和脂肪体的细胞核中。与对照组果蝇相比,Ppa缺失突变体表现为脂滴变小;超量表达Ppa显示出脂滴变大。在突变体中超量表达Ppa能够将脂滴回复至正常状态。综上所述,本研究揭示了Ppa在果蝇中具有促进脂肪储存的功能。

关键词: 脂肪储存, 脂滴, CRISPR/Cas9, Ppa

Abstract:

Lipid is one of the important components of living organisms. The precise regulation and homeostasis maintenance of lipid metabolism are essential to human health. The ubiquitination pathway regulates lipid metabolism by degrading lipid-related proteins. Ppa encodes an F-box protein, which is a member of the SCF ubiquitination complex. Previous studies reported that Ppa regulated the body segmentation and the correct localization of centromere histones, while its function in lipid metabolism has not been reported. In this study, Drosophila melanogaster was used to explore the function of Ppa in lipid storage. The subcellular localization of PPA was detected by fusion with green fluorescent protein. The deletion mutant of Ppa was constructed via CRISPR/Cas9 technology. The morphological changes of lipid droplets in deletion mutants and Ppa overexpression flies were analyzed by BODIPY 493/503 or Nile red staining. Further, Ppa was overexpressed in the deletion mutant to verify its function. The results showed that PPA-GFP fusion protein were localized in the nuclei of salivary gland and fat body. Compared with the control flies, the lipid droplets in Ppa deletion mutants became smaller, and overexpression of Ppa exhibited larger lipid droplets. Overexpression of Ppa in the deletion mutant could restore the lipid droplets to normal state. In summary, this study demonstrated that Ppa could promote lipid storage in Drosophila.

Key words: lipid storage, lipid droplets, CRISPR/Cas9, Ppa

杨光武, 田嫄. 果蝇F-box基因Ppa促进脂肪储存[J]. 遗传, 2021, 43(6): 615-622.

Guangwu Yang, Yuan Tian. The F-box gene Ppa promotes lipid storage in Drosophila[J]. Hereditas(Beijing), 2021, 43(6): 615-622.

图/表 7

表1

表2

图1

图2

图3

图4

图5

参考文献 27

[1]	Fahy E, Cotter D, Sud M, Subramaniam S. Lipid classification, structures and tools. Biochim Biophys Acta, 2011,1118(11):637-647.
[2]	Welte MA, Gould AP. Lipid droplet functions beyond energy storage. Biochim Biophys Acta Mol Cell Biol Lipids, 2017,1862(10 Pt B):1260-1272.
[3]	Welte MA. Expanding roles for lipid droplets. Curr Biol, 2015,25(11):R470-R481.
[4]	Filipe A, Mclauchlan J. Hepatitis C virus and lipid droplets: finding a niche. Trends Mol Med, 2015,21(1):34-42.
[5]	Inloes JM, Hsu KL, Dix MM, Viader A, Masuda K, Takei T, Wood MR, Cravatt BF. The hereditary spastic paraplegia-related enzyme DDHD2 is a principal brain triglyceride lipase. Proc Natl Acad Sci USA, 2014,111(41):14924-14929.
[6]	Xu X, Park JG, So JS, Lee AH. Transcriptional activation of Fsp27 by the liver-enriched transcription factor CREBH promotes lipid droplet growth and hepatic steatosis. Hepatology, 2015,61(3):857-869.
[7]	Wilfling F, Wang HJ, Haas JT, Krahmer N, Gould TJ, Uchida A, Cheng JX, Graham M, Christiano R, Fröhlich F, Liu XR, Buhman KK, Coleman RA, Bewersdorf J, Farese RV, Walther TC. Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets. Dev Cell, 2013,24(4):384-399.
[8]	Krahmer N, Guo Y, Wilfling F, Hilger M, Lingrell S, Heger K, Newman HW, Schmidt-supprian M, Vance DE, Mann M, Farese RV, Walther TC. Phosphatidylcholine synthesis for lipid droplet expansion is mediated by localized activation of CTP:phosphocholine cytidylyltransferase. Cell Metab, 2011,14(4):504-515.
[9]	Wang XY, Sun LP, Zhang JF, Li H, Lv WQ, Zhang QQ. F-box proteins and their functions. Chin Bull Life Sci, 2008,20(5):807-811.
	王秀燕, 孙莉萍, 张建锋, 李辉, 吕文清, 张其清. F-box蛋白家族及其功能. 生命科学, 2008,20(5):807-811.
[10]	Kipreos ET, Pagano M. The F-box protein family. Genome Biol, 2000, 1(5): REVIEWS3002.
[11]	Chen K, Cheng HH, Zhou RJ. Molecular mechanisms and functions of autophagy and the ubiq-uitin-proteasome pathway. Hereditas(Beijing), 2012,34(1):5-18.
	陈科, 程汉华, 周荣家. 自噬与泛素化蛋白降解途径的分子机制及其功能. 遗传, 2012,34(1):5-18.
[12]	Xie CM, Wei WY, Sun Y. Role of SKP1-CUL1-F-box- protein (SCF) E3 ubiquitin ligases in skin cancer. J Genet Genomics, 2013,40(3):97-106.
[13]	Sohn JH, Lee YK, Han JS, Jeon YG, Kim JI, Choe SS, Kim SJ, Yoo HJ, Kim JB. Perilipin 1 (Plin1) deficiency promotes inflammatory responses in lean adipose tissue through lipid dysregulation. J Biol Chem, 2018,293(36):13974-13988.
[14]	Ju LP, Han JF, Zhang XY, Deng YJ, Yan H, Wang CR, Li XH, Chen SQ, Alimujiang M, Li X, Fang QC, Yang Y, Jia WP. Obesity-associated inflammation triggers an autophagy- lysosomal response in adipocytes and causes degradation of perilipin 1. Cell Death Dis, 2019,10(2):121.
[15]	Dui W, Lu W, Ma J, Jiao RJ. A systematic phenotypic screen of F-box genes through a tissue-specific RNAi- based approach in Drosophila. J Genet Genomics, 2012,39(8):397-413.
[16]	Clark E, Akam M. Odd-paired controls frequency doubling in Drosophila segmentation by altering the pair-rule gene regulatory network. eLife, 2016,5: e18215.
[17]	Raj L, Vivekanand P, Das TK, Badam E, Fernandes M, Finley RL, Brent R, Appel LF, Hanes SD, Weir M. Targeted localized degradation of Paired protein in Drosophila development. Curr Biol, 2000,10(20):1265-1272.
[18]	Heun P, Erhardt S, Blower MD, Weiss S, Skora AD, Karpen GH. Mislocalization of the Drosophila centromere- specific histone CID promotes formation of functional ectopic kinetochores. Dev Cell, 2006,10(3):303-315.
[19]	Moreno-moreno O, Medina-giró S, Torras-llort M, Azorín F. The F box protein partner of paired regulates stability of Drosophila centromeric histone H3, CenH3(CID). Curr Biol, 2011,21(17):1488-1493.
[20]	Zhao JJ, Xiong XL, Li Y, Liu X, Wang T, Zhang H, Jiao Y, Jiang JJ, Zhang HJ, Tang QQ, Gao X, Li XJ, Lu Y, Liu B, Hu C, Li XY. Hepatic F-box protein FBXW7 maintains glucose homeostasis through degradation of fetuin-A. Diabetes, 2018,67(5):818-830.
[21]	Zhang C, Chen F, Feng L, Shan Q, Zheng GH, Wang YJ, Lu J, Fan SH, Sun CH, Wu DM, Li MQ, Hu B, Wang QQ, Zhang ZF, Zheng YL. FBXW7 suppresses HMGB1- mediated innate immune signaling to attenuate hepatic inflammation and insulin resistance in a mouse model of nonalcoholic fatty liver disease. Mol Med, 2019,25(1):29.
[22]	Cooke PS, Holsberger DR, Cimafranca MA, Meling DD, Beals CM, Nakayama K, Nakayama KI, Kiyokawa H. The F box protein S phase kinase-associated protein 2 regulates adipose mass and adipocyte number in vivo. Obesity (Silver Spring), 2007,15(6):1400-1408.
[23]	Scott NA, Sharpe LJ, Capell-Hattam IM, Gullo SJ, Luu W, Brown AJ. The E3 ubiquitin ligase MARCHF6 as a metabolic integrator in cholesterol synthesis and beyond. Biochim Biophys Acta Mol Cell Biol Lipids, 2021,1866(1):158837.
[24]	Scott NA, Sharpe LJ, Capell-hattam IM, Gullo SJ, Luu W, Brown AJ. The cholesterol synthesis enzyme lanosterol 14α-demethylase is post-translationally regulated by the E3 ubiquitin ligase MARCH6. Biochem J, 2020,477(2):541-555.
[25]	Ghosh M, Niyogi S, Bhattacharyya M, Adak M, Nayak DK, Chakrabarti S, Chakrabarti P. Ubiquitin ligase COP1 controls hepatic fat metabolism by targeting ATGL for degradation. Diabetes, 2016,65(12):3561-3572.
[26]	Liu LL, Guo AW, Li QQ, Wu PF, Yang YJ, Chen FF, Li SH, Guo PJ, Zhang Q. The regulation of ubiquitination in milk fat synthesis in bovine. Hereditas(Beijing), 2020,42(6):548-555.
	刘莉莉, 郭爱伟, 李青青, 吴培福, 杨亚晋, 陈粉粉, 李素华, 郭盘江, 张勤. 泛素化途径在奶牛乳脂生成过程中的调控作用. 遗传, 2020,42(6):548-555.
[27]	Leber V, Nans A, Singleton MR. Structural basis for assembly of the CBF3 kinetochore complex. EMBO J, 2018,37(2):269-281.

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

基因型	来源	用途
w¹¹¹⁸	中国科学院遗传与发育生物学研究所黄勋研究员实验室	对照组
w; ln(2LR)noc^4LSco^rv9R, b¹/CyO, P {ActGFP}JMR1*	中国科学院遗传与发育生物学研究所黄勋研究员实验室	平衡致死系
da-Gal4	清华大学果蝇中心	全身表达的Gal4
y¹M{nos-Cas9.P}ZH-2A w*	Bloomington	表达Cas9蛋白
w;L/CyO	本实验室	平衡致死系
Ppa-sgRNA	本实验构建	表达两段sgRNA
UAS-Ppa-RA/RC	本实验构建	超量表达PPA
UAS-Ppa-RA/RC-eGFP	本实验构建	PPA的亚细胞定位

引物名称	引物序列(5′→3′)
Ppa-sgRNA1-F	gtcgCGGATCCGGGAGCAACGGAG
Ppa-sgRNA1-R	aaacCTCCGTTGCTCCCGGATCCG
Ppa-sgRNA2-F	gtcgTCAGGTTTGCACCGCATGGC
Ppa-sgRNA2-R	aaacGCCATGCGGTGCAAACCTGA
Ppa-chk-F	CCCTTCCATCATCAAACA
Ppa-chk-R	GAACGCCCAGGACCAAAT

果蝇F-box基因Ppa促进脂肪储存

The F-box gene Ppa promotes lipid storage in Drosophila

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 27

相关文章 15

编辑推荐

Metrics

[1]	王秉政, 张超, 张佳丽, 孙锦. 利用单转录本表达Cas9和sgRNA条件性编辑果蝇基因组[J]. 遗传, 2023, 45(7): 593-601.
[2]	刘梅珍, 王立人, 李咏梅, 马雪云, 韩红辉, 李大力. 利用CRISPR/Cas9技术构建基因编辑大鼠模型[J]. 遗传, 2023, 45(1): 78-87.
[3]	张潇筠, 徐坤, 沈俊岑, 穆璐, 钱泓润, 崔婕妤, 马宝霞, 陈知龙, 张智英, 魏泽辉. 一种新型提高HDR效率的CRISPR/Cas9-Gal4BD供体适配基因编辑系统[J]. 遗传, 2022, 44(8): 708-719.
[4]	张充, 魏子璇, 王敏, 陈瑶生, 何祖勇. 利用CRISPR/Cas9在人类黑色素瘤细胞中编辑MC1R与功能分析[J]. 遗传, 2022, 44(7): 581-590.
[5]	刘尧, 周先辉, 黄舒泓, 王小龙. 引导编辑：突破碱基编辑类型的新技术[J]. 遗传, 2022, 44(11): 993-1008.
[6]	韩玉婷, 许博文, 李羽童, 卢心怡, 董习之, 邱雨浩, 车沁耘, 朱芮葆, 郑丽, 李孝宸, 司绪, 倪建泉. 模式动物果蝇的基因调控前沿技术[J]. 遗传, 2022, 44(1): 3-14.
[7]	彭定威, 李瑞强, 曾武, 王敏, 石翾, 曾检华, 刘小红, 陈瑶生, 何祖勇. 编辑MSTN半胱氨酸节基元促进两广小花猪肌肉生长[J]. 遗传, 2021, 43(3): 261-270.
[8]	王娜, 甲芝莲, 吴强. RFX5调控原钙粘蛋白α基因簇的表达[J]. 遗传, 2020, 42(8): 760-774.
[9]	李国玲, 杨善欣, 吴珍芳, 张献伟. 提高CRISPR/Cas9介导的动物基因组精确插入效率研究进展[J]. 遗传, 2020, 42(7): 641-656.
[10]	陈赢男, 陆静. CRISPR/Cas9系统在林木基因编辑中的应用[J]. 遗传, 2020, 42(7): 657-668.
[11]	刘思远, 易国强, 唐中林, 陈斌. 基于CRISPR/Cas9系统在全基因组范围内筛选功能基因及调控元件研究进展[J]. 遗传, 2020, 42(5): 435-443.
[12]	鲍莉雯, 周一叶, 曾凡一. 基于CRISPR/Cas9技术的β-地中海贫血和血友病基因治疗研究进展[J]. 遗传, 2020, 42(10): 949-964.
[13]	林珉婷, 赖璐璐, 赵淼, 林必玮, 姚香平. 利用CRISPR/Cas9 AAV系统构建纹状体Slc20a2基因敲除小鼠模型[J]. 遗传, 2020, 42(10): 1017-1027.
[14]	刘沛峰, 吴强. CRISPR/Cas9基因编辑在三维基因组研究中的应用[J]. 遗传, 2020, 42(1): 18-31.
[15]	郑晓飞,黄海燕,吴强. 染色质架构蛋白CTCF调控UGT1基因簇的表达[J]. 遗传, 2019, 41(6): 509-523.