Construction of Yeast Two-Hybrid Three-Frame cDNA Library and Screening of Interacting Proteins in FWL1 Membrane System of Pear Young Fruit

doi:10.6048/j.issn.1001-4330.2022.08.008

Abstract

Abstract:

【Objective】 Screening of proteins that interact with FWL1 provides a basis of in-depth exploration of the mechanism of FWL1 gene regulation of fruit size. 【Methods】 Using ‘Duli’ pear, Korla fragrant pear, and Yali pear and Zaomeixiang were used as materials, to extract the mixed RNA of pulp tissue in the critical period of fruit cell division of four pear varieties was extracted. And then a yeast two-hybrid triple-frame cDNA library of pear young fruit FWL1 membrane system was constructed, and the quality of the library was identified. After that, The the bait vector was constructed and transformed into yeast, and the proteins that interacted with FWL1 were screened. The positive yeast clones that were initially screened were sequenced and Blast alignment was performed in NCBI to determine the candidate interacting proteins. 【Results】 The library capacity is was about 3×10⁷ CFU, greater than 1×10⁷ CFU, the average insert fragment is was greater than 1,000 bp, and the positive rate is was ≧98%. After testing the bait vector had no self-activating function, and 272 proteins interacting with FWL1 were screened out by co-transformation method. 【Conclusion】 After testing, the yeast two-hybrid three-frame cDNA library of the pear young fruit membrane system meets the basic conditions of the yeast two-hybrid library and is suitable for later screening of interacting proteins. The screened 272 interacting proteins were are expressed differently during fruit development, among which collagen and calcium-binding EGF domain-containing protein 1 and metallothionein-like protein may be related to pear fruit size development.

Key words: pear; fruit size; fruit weight 2.2; construction of three-frame cDNA library; screening of interacting proteins

摘要：

【目的】筛选与FWL1互作的蛋白,为研究FWL1基因调节果实大小机理提供依据。【方法】以杜梨、库尔勒香梨、鸭梨、早美香为材料,提取4个梨品种果实细胞分裂关键时期的果肉组织混样RNA。构建梨幼果FWL1膜系统酵母双杂交三框cDNA文库,并鉴定文库质量。构建诱饵载体并转化酵母菌,筛选与FWL1互作的蛋白,对初步筛选出的阳性酵母克隆进行测序并在NCBI中进行Blast比对,确定候选互作蛋白。【结果】文库库容约为3×10⁷ CFU,大于1×10⁷ CFU,平均插入片段大于1 000 bp,阳性率为≥98%。诱饵载体无自激活功能,利用共转化方法,筛选出272个与FWL1互作的蛋白质。【结论】梨幼果膜系统酵母双杂交三框cDNA文库符合酵母双杂文库的基本条件,适用于后期筛选相互作用的蛋白。筛选出的272个互作蛋白在果实发育过程中表达不同,其中collagen and calcium-binding EGF domain-containing protein 1和metallothionein-like protein可能与梨果实大小发育有关。

关键词: 梨, 果实大小, fw2.2, 三框cDNA文库, 互作蛋白筛选

CLC Number:

S661.2

SAI Jingyi, WEN Yue, HAO Zhichao, TIAN Jia. Construction of Yeast Two-Hybrid Three-Frame cDNA Library and Screening of Interacting Proteins in FWL1 Membrane System of Pear Young Fruit[J]. Xinjiang Agricultural Sciences, 2022, 59(8): 1877-1888.

赛静忆, 温玥, 郝志超, 田嘉. 梨幼果FWL1膜系统酵母双杂交三框cDNA文库构建及互作蛋白的筛选[J]. 新疆农业科学, 2022, 59(8): 1877-1888.

Figures/Tables 12

References 39

[1]	BREUNINGER H, LENHARD M. Control of Tissue and Organ Growth in Plants[J]. Current Topics in Developmental Biology, 2010, 91:185-220. DOI PMID
[2]	GONZALEZ N, VANHAEREN H, INZE D. Leaf size control: complex coordination of cell division and expansion: Leaf size control: complex coordination of cell division and expansion[J]. Trends in Plant Science, 2012, 17(6): 332-340. DOI PMID
[3]	LIM SD, YIM WC, LIU D, et al. A Vitis vinifera basic helix-loop-helix transcription factor enhances plant cell size, vegetative biomass, and reproductive yield[J]. Plant Biotechnology Journal, 2018, 16(9):1595-1615. DOI URL
[4]	郁网庆, 宋烨, 王达, 等. 库尔勒香梨采后生理及品质调控综合技术研究进展[J]. 中国果菜, 2018, 38(11):6-9,20.
	YU Wangqing, SONG Ye, WANG Da, et al. Research on Postharvest Physiology and Comprehensive Technologiesfor Quality Control of Korla Fragrant Pear[J]. Chinese fruits vegetables, 2018, 38(11):6-9,20.
[5]	CONG B, TANKSLEY S D. FW2.2 and cell cycle control in developing tomato fruit: a possible example of gene co-option in the evolution of a novel organ[J]. Plant Molecular Biology, 2006, 62(6):867-880. PMID
[6]	熊文涛. 水稻OsFWL家族部分基因的生物学功能研究[D]. 湖北: 华中农业大学, 2018.
	XIONG Wentao. Functional analysis of part of genes in OsFWL gene family in rice[D]. Hubei: Huazhong Agricultural University, 2018.
[7]	李志超. 酸浆属fw2.2同源基因调控浆果大小及其自然变异的机理研究[J]. 北京: 中国科学院大学, 2013.
	LI Zhichao. Role of FW2.2-like genes in controlling berry size and itsnatural variation within Physalis[J]. Beijing: University of Chinese Academy of Sciences, 2013.
[8]	XU J, XIONG W, CAO B, et al. Molecular characterization and functional analysis of "fruit-weight2.2-like" gene family in rice[J]. Planta, 2013, 238(4): 643-655. DOI URL
[9]	GUO M, RUPE M A, DIETER J A, et al. Cell number regulator1 affects Plant and organ size in maize: Implications for Crop Yield Enhancement and Heterosis[J]. Plant Cell, 2010, 22(4): 1057-1073. DOI URL
[10]	DAHAN Y, ROSENFELD R, ZADIRANOV V, et al. A proposed conserved role for an avocado fw2.2-like gene as a negative regulator of fruit cell division[J]. Planta, 2010, 232(3): 663-676. DOI PMID
[11]	LIBAULT M, ZHANG X C, GOVINDARAJULU M, et al. A member of the highly conserved FWL(tomato FW2.2-like) gene family is essential for soybean nodule organogenesis[J]. Plant Journal for Cell & Molecular Biology, 2010, 62(5):852-864.
[12]	雷海英, 白凤麟, 段永红, 等. 玉米酵母双杂交cDNA文库的构建及ZmCEN互作蛋白的筛选[J]. 西北植物学报, 2018, 38(4):598-606.
	LEI Haiying, BAI Feng, DUAN Yonghong. et al. Construction of yeasttwo-hybrid cDNA library and screening of interaction proteins of ZmCEN in Maize[J]. Acta Botanica Boreali-Occidentalia Sinica, 2018, 38(4): 598-606.
[13]	王洋, 张政, 王莹莹, 等. 利用番茄cDNA酵母双杂交文库筛选Pti4互作蛋白[J]. 安徽农业科学, 2019, 47(18):111-114.
	WANG Yang, ZHANG Zheng, WANG Yingying, et al. Screening of Pti4 interaction proteins by using yeast two-hybrid cDNA library of tomato[J]. Journal Of Anhui Agricultural Sciences, 2019, 47(18):11
[15]	TIAN J, ZENG B, LUO SP, et al. Cloning, localization and expression analysis of two fw2.2-like-genes in small- and large-fruited pear species[J]. Journal of Integrative Agriculture, 2016, 15(2): 282-294. DOI
[16]	张艳, 田嘉, 张峰, 等. 梨FWL5基因克隆及表达分析[J/OL]. 分子植物育种, 1-15(2022-2-18).
	ZHANG Yan, TIAN Jia, ZHANG Feng, et al. Cloning and expression analysis of pear FWL5 gene[J/OL]. Molecular Plant Breeding, 1-15(2022-2-18).
[17]	THOMMA B, ESSE H, CROUS P W, et al. Cladosporiumfulvum(syn. Passalorafulva), a highly specialized plant pathogen as a model for functional studies on plant pathogenic Mycosphaerellaceae[J]. Molecular Plant Pathology, 2010, 6(4):379-393. DOI URL
[18]	AUERBACH D, THAMINY S, HOTTIGER M O, et al. The post-genomic era of interactive proteomics: facts and perspectives: Facts and perspectives[J]. Proteomics. 2015, 2(6): 611-623. DOI URL
[19]	王磊, 李冉辉, 邓湘赢, 等. 利用酵母双杂交技术筛选与生殖支原体黏附蛋白(MgPa)相互作用的宿主蛋白[J]. 微生物学杂志, 2019, 39(2): 38-43.
	WANG Lei, LI Ranhui, DENG Xiangying, et al. Screening of host proteins interacting with Mycoplasma genitaliumadhesion protein of using yeast double-hybrid system[J]. Journal of Microbiology, 2019, 39(2): 38-43.
[20]	ZHANG Z X, ZHANG F D, TANG W H, et al. Construction and characterization of normalized cDNA library of maize inbred MO17 from multiple tissues and developmental stages[J]. Molecular Biology, 2005. 39(2): 178-184.
[21]	王淑红, 邹志华, 张子平, 等. 副溶血弧菌感染的九孔鲍血细胞均一化全长cDNA文库的构建[J]. 台湾海峡, 2008, 27(3):278-285.
	WANG Shuhong, ZOU Zhihua, ZHANG Ziping, et al. Construction a normalized full-length cDNAlibrary of haemocytesfrom Haliotisdiversicolorsupertexta infectedwith Vibrio parahaemolvticus[J]. Journal of TaiwanStrait, 2008, 27(3): 278-285.
[22]	DU W, XIIA J, ZHANG Y, et al. Expression of recombinant myostatinpropeptide pPIC9K-Msp plasmid in Pichia pastoris[J]. Geneticsand Molecular Research, 2015, 14(4): 18414-18420.
[23]	朱慧慧, 许学年, 高春花, 等. 细粒棘球蚴cDNA文库的构建和免疫筛选[J]. 国际医学寄生虫病杂志, 2012, 39(5): 272-275.
	ZHU Huihui, XU Xuenian, GAO Chunhua, et al. Construction and immunoscreening of cDNA library of Echinococcusgranulosus[J]. International Journal of Medical Parasitic Diseases. 2012, 39(5): 272-275.
[24]	OSAMU O, GARY T. Directional cDNA library construction assisted by the in vitro recombination reaction[J]. Nucleic Acids Research, 2001(4): E22. DOI PMID
[25]	雷龑, 谢倩, 陈婷, 等. 炭疽菌侵染后刺葡萄果皮酵母双杂交cDNA文库构建[J]. 福建农业学报, 2020, 35(12):1330-1335.
	LEI Yan, XIE Qian, CHEN Ting, et al. Yeast two-hybrid cDNA library constructed from Vitisdavidiifexpericarpsinfected by grape ripe rot pathogen, Colletotrichumviniferum[J]. Fujian Journal of Agricultural Sciences, 2020, 35(12):1330-1335.
[26]	韦春艳, 秦腾飞, 董娜, 等. 陆地棉酵母双杂交cDNA文库及VdSCP7诱饵载体的构建[J]. 核农学报, 2021, 35(3):549-555. DOI
	WEI Chunyan, QIN Tengfei, DONG Na, et al. Construction of a yeast two-hybrid cDNA library of cotton(Gossypiumhirsutum L.) and VdSCP7 Bait Vector[J]. Journal of Nuclear Agricultural Sciences, 2021, 35(3):549-555.
[27]	韩晓蕾, 高仕祺, 张帆, 等. 酵母双杂交筛选与小麦黄花叶病毒P2互作的寄主因子[J]. 浙江农业学报, 2021, 33(3):497-505. DOI
	HAN Xiaolei, GAO Shiqi, ZHANG Fan, et al. Screening of host factors interacting with wheat yellow mosaic virus P2 by yeast two-hybrid system[J]. Acta Agriculturae Zhejiangensis, 2021, 33(3):497-505. DOI
[28]	谷思, 刘璐, 李安然, 等. 草莓果实酵母双杂交文库的构建及FvM4K1互作蛋白的筛选[J]. 园艺学报, 2021, 48(6):1067-1078.
	GU Si, LIU Lu, LI Anran, et al. Construction of yeast two-hybrid library and screening of FvM4K1interacting protein in the fruit of Fragrariavesca[J]. Acta Horticulturae Sinica, 2021, 48(6):1067-1078.
[29]	杨子平, 孙艺桓, 杨倩, 等. 剑麻酵母双杂交cDNA表达文库的构建及与AhKNOX2相互作用蛋白的筛选[J]. 热带作物学报, 2020, 41(9): 1748-1755.
	YANG ZiPing, SUN Yihuan, YANG Qian, et al. Construction of yeast two-hybrid library of agave hybrid No.11648 and screening of proteins interacting with AhKNOX2[J]. Chinese Journal of Tropical Crops, 2020, 41(9):1748-1755.
[30]	许向阳, 裴童, 吴泰茹, 等. Cf-19介导的抗番茄叶霉病(Cladosporiumfulvum)免疫应答酵母双杂交cDNA文库构建和鉴定[J]. 东北农业大学学报, 2020, 51(5):10-16.
	XUXiangyang, PEI Tong, WU Tairu, et al. Construction and identification of yeast two-hybrid cDNA library forCf-19 mediated resistance to Cladosporiumfulvum infection in tomato[J]. Journal of Northeast Agricultural University, 2020, 51(5):10-16.
[31]	COHEN S. Purification of a nerve-growth promoting protein from the mouse salivary gland and its neuro-cytotoxic antiserum[J]. Proceedings of the National Academy of Sciences, 1960, 46(3): 302-311. DOI URL
[32]	SAVAGE C R, INAGAMI T, COHEN S. The primary structure of epidermal growth factor[J]. Journal of Biological Chemistry, 1972, 247(23):7612-7621. PMID
[33]	MARQUARDT H, HUNKAPILLER M W, HOOD L E, et al. Transforming growth factors produced by retrovirus-transformed rodent fibroblasts and human melanoma cells: amino acid sequence homology with epidermal growth factor[J]. Proc Natl Acad, U S A, 1983, 80(15): 4684-4688. DOI URL
[34]	MARQUARDT H, TODARO G J. Human transforming growth factor. Production by a melanoma cell line, purification, and initial characterization.[J]. Journal of Biological Chemistry, 1982, 257(9): 5220. PMID
[35]	庞实锋, 姜潮, 李文荣, 等. 大豆油体与EGF融合基因的克隆及其在红花种子中的表达[J]. 中国生物工程杂志, 2014, 34(4):71-77.
	PANG Shifeng, JIANG Chao, LI Wenrong, et al. Cloning of soybean oleosin and EGF fusion gene andexpression in safflower seeds[J]. China Biotechnology, 2014, 34(4):71-77.
[36]	芮志佩. 金属硫蛋白(MT)基因植物表达载体的构建及其在水生美人蕉中表达的初步研究[D]. 江苏: 扬州大学, 2009.
	RUI Zhipei. Construction of plant expression vector of metallothionein(MT) gene and preliminary study on its expression in Aquatic canna[D]. Jiangsu: Yangzhou University, 2009.
[37]	YUAN J, CHEN D, REN YJ, et al. Characteristic and expression analysis of a metallothioneingene, OsMT2b, down-regulated by cytokinin suggests functions in root development and seed embryo germination ofrice[J]. Plant Physiology, 2008, 146(4): 1637-1650. DOI URL
[38]	WU C S, CHEN D Y, CHANG C F, et al. The promoter and the 5'-untranslatedregion of rice metallothionein OsMT2b gene are capable of directing high-level gene expression in germinated rice embryos[J]. Plant Cell Reports, 2014, 33(5): 793-806. DOI URL
[39]	金淑梅. 水稻类金属硫蛋白(rgMT)基因功能解析及对苜蓿的遗传转化研究[D]. 哈尔滨: 东北林业大学, 2006.
	JIN Shumei. Functional analysis of rgMT from rice and its genetic transformation in alfalfa[D]. Harbin: Northeast Forestry University, 2006.

试剂 Reagent	单位 Unit
First-Strand cDNA	2
Deionized H₂O	80
10× Advantage 2 PCR Buffer	10
50× dNTP Mix	2
M1 PCR Primer	4
50× Advantage 2 Polymerase Mix	2

试剂 Reagent	单位 Unit
First-Strand cDNA	2
Deionized H₂O	80
10× Advantage 2 PCR Buffer	10
50× dNTP Mix	2
M1 PCR Primer	4
50× Advantage 2 Polymerase Mix	2

试剂 Reagent	第1次扩增（μL） First amplification/μL	第2次扩增（μL） Second amplification/μL
cDNA(DSN处理稀释后)
cDNA (after DSN treatment and dilution)	1	0
cDNA(稀释后) cDNA (after dilution)	0	2
纯化水purified water	40.5	80
10× Advantage 2 PCR Buffer	5	10
50× dNTP Mix (10mM of each)	1	2
Evrogen PCR primer M1	1.5	0
Evrogen PCR primer M2	0	4
50× Advantage 2 Polymerase Mix	1	2

试剂 Reagent	第1次扩增（μL） First amplification/μL	第2次扩增（μL） Second amplification/μL
cDNA(DSN处理稀释后)
cDNA (after DSN treatment and dilution)	1	0
cDNA(稀释后) cDNA (after dilution)	0	2
纯化水purified water	40.5	80
10× Advantage 2 PCR Buffer	5	10
50× dNTP Mix (10mM of each)	1	2
Evrogen PCR primer M1	1.5	0
Evrogen PCR primer M2	0	4
50× Advantage 2 Polymerase Mix	1	2

引物名称 Primers	引物序列（5’→3’） Primers sequence
FWL1-F（pBT3-N）	GAATTCCTGCAGGGCCATTACGGCCATGTACTCGTCACACCCAAGGG
FWL1-R（pBT3-N）	TACTTACCATGGGGCCGAGGCGGCTTTATCTCGACTCATGCCTTCCTC
FWL1-F（pBT3-C）	CACTAATCTAGACGGCCATTACGGCCATGTACTCGTCACACCCAAGGG
FWL1-R（pBT3-C）	ATGGAGGCCTTTGGCCGAGGCGGCTTTATCTCGACTCATGCCTTCCTC
FWL1-F（pBT3-SUC）	AATATCTGCAATGGCCATTACGGCCATGTACTCGTCACACCCAAGGG
FWL1-R（pBT3-SUC）	ATTCCTGCAGATGGCCGAGGCGGCTTTATCTCGACTCATGCCTTCCTC
FWL1-F（pBT3-STE）	TATTTTATGTAATGGCCATTACGGCCATGTACTCGTCACACCCAAGGG
FWL1-R（pBT3-STE）	ATTCCTGCAGATGGCCGAGGCGGCTTTATCTCGACTCATGCCTTCCTC