Construction of Yeast Two Hybrid Library and Screening of FvM4K1 Interacting Protein in the Fruit of Fragraria vesca

doi:10.16420/j.issn.0513-353x.2020-0674

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (6): 1067-1078.doi: 10.16420/j.issn.0513-353x.2020-0674

• Research Papers • Previous Articles Next Articles

Construction of Yeast Two Hybrid Library and Screening of FvM4K1 Interacting Protein in the Fruit of Fragraria vesca

GU Si, LIU Lu, LI Anran, ZHANG Weiwei, ZHAO Shuaiqi, XING Yu()

Advanced Innovation Center for Forest Molecular Design and Breeding,College of Plant Science and Technology,Beijing University of Agriculture,Beijing 102206,China

Received:2020-10-19 Revised:2021-03-03 Online:2021-06-25 Published:2021-07-07
Contact: XING Yu E-mail:xingyu@bua.edu.cn

Abstract

Abstract:

cDNA library of strawberry fruit at different stages was constructed and the proteins interacting with strawberry mitogen activated kinase FvM4K1 were screened which revealed the molecular mechanism of FvM4K1 in regulating strawberry fruit development. The total RNA ofFragraria vesca ‘Hawaii 4'was extracted at different fruit development stages related to FvM4K1,and the strawberry cDNA library was established. Specifically,the library capacity was 9.6 × 10 ⁶ CFU,the recombination rate was 96%,and the average length of inserted fragment was more than 1 000 bp. At the same time,the bait vector pGBKT7-FvM4K1 was constructed,and no self-activation activity was detected in yeast. Then 13 proteins interacting with FvM4K1 were screened from the library by co-transformation. The function of these candidate proteins mainly focused on floral development such as pollen development and pollen tube elongation,also affecting flowering time and so on. Meanwhile,the Gene Ontology（GO）pathway annotation of these proteins showed that they mainly participated in cell metabolism process and molecular processes with catalytic activity. Besides,the 13 proteins screened had different expression patterns during fruit development,and four of them were predicted to be closely related to strawberry fruit development.

Key words: strawberry, Fragaria ananassa, cDNA library, MAPK

CLC Number:

S668.4

GU Si, LIU Lu, LI Anran, ZHANG Weiwei, ZHAO Shuaiqi, XING Yu. Construction of Yeast Two Hybrid Library and Screening of FvM4K1 Interacting Protein in the Fruit of Fragraria vesca[J]. Acta Horticulturae Sinica, 2021, 48(6): 1067-1078.

Figures/Tables 12

References 44

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蛋白 Protein number	基因 Gene number	正向序列（5′-3′） Sequence of forward primer	反向序列（5′-3′） Sequence of reverse primer
XP_004302175	LOC101293013	TGGAACGACTTCATCAATG	GCACCAATACAGTCAACA
XP_004303711	LOC101305492	TCCATACCAAGTGAACCA	CATAAGCCTCTCCTCCTT
XP_004299172	LOC101296956	CGTGCCTGCTAATAATGA	GAGTGTTGTTCCGTATCC
XP_004299687	LOC101307208	CTCGGAGCAACAATAACA	TGACATTGAGGTGAAGGA
XP_011466791	LOC101304014	CTGTGGTGTTCTTGTCTG	CAGCCTTGACATTCTCTT
XP_011470680	LOC101297868	AGGTCCTGATGAAGAGTT	CATAGCACTGTCTAAGCATA
XP_004306495	LOC101308794	AGAAGACAACGAAGACAAG	CAAGTGGAGGAAGGAGTA
XP_004299659	LOC101299003	ATGTCCACGACCAAGAAG	GATTGGGAGGCATTTAGC
XP_004303975	LOC101290785	GATGGGATGTTTCTGCTAT	TTCCTCCTTACACCTATCAA
XP_004297088	LOC101313011	AGATCATCATCGACACCTA	CACCAATGGCATAAGACA
XP_004307305	LOC101294984	CTAAGTGCTTCTTCTGACAT	ACAGGATGAGGTTGATGA
XP_011463919	LOC101291534	CTCAAGAAGCAGCCTAAG	GATGGTCTGTAATCAGCAG
XP_004291272	LOC101305235	GATTGACAACCTGGACAG	CATCTTATCATTCACGACCA

蛋白 Protein number	基因 Gene number	正向序列（5′-3′） Sequence of forward primer	反向序列（5′-3′） Sequence of reverse primer
XP_004302175	LOC101293013	TGGAACGACTTCATCAATG	GCACCAATACAGTCAACA
XP_004303711	LOC101305492	TCCATACCAAGTGAACCA	CATAAGCCTCTCCTCCTT
XP_004299172	LOC101296956	CGTGCCTGCTAATAATGA	GAGTGTTGTTCCGTATCC
XP_004299687	LOC101307208	CTCGGAGCAACAATAACA	TGACATTGAGGTGAAGGA
XP_011466791	LOC101304014	CTGTGGTGTTCTTGTCTG	CAGCCTTGACATTCTCTT
XP_011470680	LOC101297868	AGGTCCTGATGAAGAGTT	CATAGCACTGTCTAAGCATA
XP_004306495	LOC101308794	AGAAGACAACGAAGACAAG	CAAGTGGAGGAAGGAGTA
XP_004299659	LOC101299003	ATGTCCACGACCAAGAAG	GATTGGGAGGCATTTAGC
XP_004303975	LOC101290785	GATGGGATGTTTCTGCTAT	TTCCTCCTTACACCTATCAA
XP_004297088	LOC101313011	AGATCATCATCGACACCTA	CACCAATGGCATAAGACA
XP_004307305	LOC101294984	CTAAGTGCTTCTTCTGACAT	ACAGGATGAGGTTGATGA
XP_011463919	LOC101291534	CTCAAGAAGCAGCCTAAG	GATGGTCTGTAATCAGCAG
XP_004291272	LOC101305235	GATTGACAACCTGGACAG	CATCTTATCATTCACGACCA

蛋白号 Protein number	基因号 Gene number	蛋白名称 Protein name	相关蛋白功能预测 Speculative function
XP_004302175	LOC101293013	似E3泛素蛋白连接酶ARI8 Probable E3 ubiquitin-protein ligase ARI8	花结构,胚胎发育,延迟开花等 Flower structure,embryonic development,delayed flowering
XP_004303711	LOC101305492	似TIFY 11A蛋白 Protein TIFY 11A-like	昼夜节律,气孔开关,开花时间 Circadian rhythm,stomata switch,flowering time
XP_004299172	LOC101296956	豇豆球蛋白 Vignain	细胞循环,初生根生长,花粉发育 Cell cycle,primary root growth,pollen development
XP_004299659	LOC101299003	磷酸-2-脱氢-3-脱氧庚酸醛缩酶2,类叶绿体 Phospho-2-dehydro-3-deoxyheptonate aldolase 2,chloroplastic-like	提高幼苗光响应能力,花粉萌发,花粉管伸长,细胞壁结构,向重力性 Improve the light response ability of seedlings,pollen germination,pollen tube elongation,cell wall structure,gravity
XP_004303975	LOC101290785	亚硫酸氧化酶 Sulfite oxidase	抗氧反应,花粉萌发,花粉管伸长 Antioxidant response,pollen germination,pollen tube elongation
XP_004299687	LOC101307208	Transcription factor bHLH96	DNA结合蛋白
XP_011466791	LOC101304014	果糖二磷酸醛缩酶,胞质同工酶样亚型X2 Fructose-bisphosphate aldolase, cytoplas-mic isozyme-like isoform X2	花粉萌发,花粉管伸长,细胞壁结构,向重力性 Pollen germination,pollen tube elongation,cell wall structure,gravity
XP_011470680	LOC101297868	未表征的甲基转移酶At2g41040,似氯塑料的同工型X3 Uncharacterized Methyltransferase At2g41040, chloroplastic-like isoform X3	提高幼苗光响应能力 Improve the light response ability of seedlings
XP_004306495	LOC101308794	营养细胞壁蛋白gp1 Vegetative cell wall protein gp1	未知Unknown
XP_004297088	LOC101313011	S-腺苷甲硫氨酸合酶2 S-adenosylmethionine synthase 2	响应水杨酸、油菜素内酯,种子萌发、花粉萌发、花粉管伸长,细胞壁 Response to salicylic acid,brassinolide,seed germination,pollen germination,pollen tube elongation,cell wall
XP_004307305	LOC101294984	尿酸降解双功能蛋白TTL亚型X1 Uric acid degradation bifunctional protein TTL isoform X1	未知Unknown
XP_011463919	LOC101291534	40S核糖体蛋白S10 40S ribosomal protein S10	核糖体蛋白Ribosomal protein
XP_004291272	LOC101305235	E3泛素蛋白连接酶AIP2 E3 ubiquitin-protein ligase AIP2	脱落酸信号通路中负调控因子 Negative regulator of abscisic acid signaling pathway

蛋白号 Protein number	基因号 Gene number	蛋白名称 Protein name	相关蛋白功能预测 Speculative function
XP_004302175	LOC101293013	似E3泛素蛋白连接酶ARI8 Probable E3 ubiquitin-protein ligase ARI8	花结构,胚胎发育,延迟开花等 Flower structure,embryonic development,delayed flowering
XP_004303711	LOC101305492	似TIFY 11A蛋白 Protein TIFY 11A-like	昼夜节律,气孔开关,开花时间 Circadian rhythm,stomata switch,flowering time
XP_004299172	LOC101296956	豇豆球蛋白 Vignain	细胞循环,初生根生长,花粉发育 Cell cycle,primary root growth,pollen development
XP_004299659	LOC101299003	磷酸-2-脱氢-3-脱氧庚酸醛缩酶2,类叶绿体 Phospho-2-dehydro-3-deoxyheptonate aldolase 2,chloroplastic-like	提高幼苗光响应能力,花粉萌发,花粉管伸长,细胞壁结构,向重力性 Improve the light response ability of seedlings,pollen germination,pollen tube elongation,cell wall structure,gravity
XP_004303975	LOC101290785	亚硫酸氧化酶 Sulfite oxidase	抗氧反应,花粉萌发,花粉管伸长 Antioxidant response,pollen germination,pollen tube elongation
XP_004299687	LOC101307208	Transcription factor bHLH96	DNA结合蛋白
XP_011466791	LOC101304014	果糖二磷酸醛缩酶,胞质同工酶样亚型X2 Fructose-bisphosphate aldolase, cytoplas-mic isozyme-like isoform X2	花粉萌发,花粉管伸长,细胞壁结构,向重力性 Pollen germination,pollen tube elongation,cell wall structure,gravity
XP_011470680	LOC101297868	未表征的甲基转移酶At2g41040,似氯塑料的同工型X3 Uncharacterized Methyltransferase At2g41040, chloroplastic-like isoform X3	提高幼苗光响应能力 Improve the light response ability of seedlings
XP_004306495	LOC101308794	营养细胞壁蛋白gp1 Vegetative cell wall protein gp1	未知Unknown
XP_004297088	LOC101313011	S-腺苷甲硫氨酸合酶2 S-adenosylmethionine synthase 2	响应水杨酸、油菜素内酯,种子萌发、花粉萌发、花粉管伸长,细胞壁 Response to salicylic acid,brassinolide,seed germination,pollen germination,pollen tube elongation,cell wall
XP_004307305	LOC101294984	尿酸降解双功能蛋白TTL亚型X1 Uric acid degradation bifunctional protein TTL isoform X1	未知Unknown
XP_011463919	LOC101291534	40S核糖体蛋白S10 40S ribosomal protein S10	核糖体蛋白Ribosomal protein
XP_004291272	LOC101305235	E3泛素蛋白连接酶AIP2 E3 ubiquitin-protein ligase AIP2	脱落酸信号通路中负调控因子 Negative regulator of abscisic acid signaling pathway

Construction of Yeast Two Hybrid Library and Screening of FvM4K1 Interacting Protein in the Fruit of Fragraria vesca

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[3]	ZHAO Xia, LI Gang, LIU Lifeng, SONG Yanhong, and ZHOU Houcheng. A New Strawberry Cultivar‘Huashuo 1’ [J]. Acta Horticulturae Sinica, 2022, 49(S2): 81-82.
[4]	DONG Jing, CHANG Linlin, WANG Guixia, ZHONG Chuanfei, WEI Yongqing, SUN Jian, SUN Rui, ZHANG Hongli, GAO Yongshun, XU Liping, TAO Pang, LUO Zhiwei, and ZHANG Yuntao, . A New Ever-bearing Strawberry Cultivar‘Jinghong’ [J]. Acta Horticulturae Sinica, 2022, 49(S1): 61-62.
[5]	YANG Liyuan, WANG Qian, WANG Xuhui, XU Tongda, MA Jun. The Important Biological Function of T111,an Evolutional Conserved Amino Acid in FveTAA1,a Key Enzyme in Strawberry Auxin Biosynthesis [J]. Acta Horticulturae Sinica, 2021, 48(9): 1695-1705.
[6]	HONG Yanhong, YE Qinghua, LI Zekun, WANG Wei, XIE Qian, CHEN Qingxi, CHEN Jianqing. Accumulation of Anthocyanins in Red-flowered Strawberry‘Meihong’Petals and Expression Analysis of MYB Gene [J]. Acta Horticulturae Sinica, 2021, 48(8): 1470-1484.
[7]	ZHANG Zhiqiang, LU Shixiong, MA Zonghuan, LI Yanbiao, GAO Caixia, CHEN Baihong, MAO Juan. Bioinformatic Identification and Expression Analysis of Candidate Genes of LIM Protein Family in Strawberry Under Abiotic Stresses [J]. Acta Horticulturae Sinica, 2021, 48(8): 1485-1503.
[8]	HUANG Qianru, NIU Yonghao, WU Kuan, LIU Zhanjie, CAO Mengji, ZHAO Lei, WU Yunfeng. LncRNA Sequencing Identification of Viruses Infecting Strawberry in Shaanxi [J]. Acta Horticulturae Sinica, 2021, 48(8): 1589-1594.
[9]	HUANG Weijian, LI Meng. Status and Prospects Whole Genome Sequencing in Fruit Trees [J]. Acta Horticulturae Sinica, 2021, 48(4): 733-748.
[10]	QI Yongzhi, SUN Yaru, WANG Bing, GUO Hanfei, MA Ke, ZHEN Wenchao. Research Progress on Allelopathy of Root Exudates and Decompositions of Strawberry [J]. Acta Horticulturae Sinica, 2021, 48(4): 778-790.
[11]	LIU Lu, GU Si, ZHANG Weiwei, ZHAO Shuaiqi, LI Anran, XING Yu. A Primary Study on FvMOB1 Interact with FvM4K1 and Response to Auxin Regulation in Fragraria vesca [J]. Acta Horticulturae Sinica, 2021, 48(11): 2185-2196.
[12]	CHEN Dao, ZHANG Jie, WU Zujian, DING Xinlun. Genome Sequence of Strawberry Pallidosis-associated Virus Isolated in China [J]. Acta Horticulturae Sinica, 2021, 48(1): 146-152.
[13]	ZHENG Qiaoling, SHEN Wei, YAO Wenkong, and XU Weirong, . Construction of Yeast Two-hybrid Three-frame cDNA Library of Vitis amurensis and Screening of VaCIPK18 Interaction Protein [J]. Acta Horticulturae Sinica, 2020, 47(12): 2301-2316.
[14]	LUO He, LI Weijia, LI He, and ZHANG Zhihong. FaRGA1 Gene Silencing Changes the Characteristics of Flowering and Runner Producing in Strawberry [J]. Acta Horticulturae Sinica, 2020, 47(12): 2331-2339.
[15]	LONG Qin, XIE Yu, LEI Tiangang, HE Yongrui, XU Lanzhen, ZOU Xiuping, and CHEN Shanchun. Bioinformatics Analysis of MAPK Genes in Citrus and Their Expression in Response to Canker Disease [J]. Acta Horticulturae Sinica, 2020, 47(11): 2095-2106.