利用酵母双杂交系统筛选花生AhSAP1的互作蛋白

doi:10.3864/j.issn.0578-1752.2024.21.016

Abstract

Abstract:

【Objective】Seed size directly affects the yield of peanut. The key transcription factor AhSAP1, which regulates peanut seed size, was obtained by QTL mapping in the early stage, but the molecular mechanism of AHSAP1 regulating peanut seed size remains unclear. In this paper, a peanut embryo yeast two-hybrid cDNA library was constructed and AhSAP1 was used as bait to screen interacting proteins, and the spatial and temporal expression characteristics of candidate interacting protein genes were analyzed. It laid the foundation for further study on the molecular mechanism of AhSAP1 regulating peanut kernel development. 【Method】The peanut embryo Escherichia coli cDNA library was constructed and identified by SMART (switching mechanism at 5′ end of the RNA transcript) method. The decoy vector pGBKT7-AhSAP1 was constructed and its toxicity and self-activation to yeast cells were evaluated. The peanut embryo cDNA library plasmid and the bait plasmid pGBKT7-AhSAP1 were co-transformed into Y2H Gold yeast strains, and the positive colonies with good growth and blue color were screened and sequenced to obtain the candidate interacting protein gene sequences and predict the biological functions. The expression characteristics of candidate interacting protein genes in different tissues and organs of peanut, induced by exogenous plant hormones and induced by low calcium stress were determined by RNA-seq. According to the functional annotation results, the candidate factors that may be involved in plant seed development were selected, their CDS full-length sequences were amplified, and the target vector pGADT7 was constructed, and point-to-point yeast two-hybrid interaction was verified with pGBKT7-AhSAP1 co-transformed yeast cells. 【Result】The titer of peanut embryo Escherichia coli secondary cDNA library was 1.05×108 cfu/mL, the recombination rate was 98%, the average insert fragment size was more than 1 000 bp, and the library quality was high. The yeast two-hybrid decoy vector pGBKT7-AhSAP1 was successfully constructed, which had no self-activation in yeast cells and no toxicity to yeast. Sixty-eight yeast-positive clones were screened, and 60 candidate interacting proteins were obtained by sequence similarity comparison and removal of duplicating, which were mainly involved in energy production and metabolism, translation, ribosome structure and biological development, transcription, signal transduction mechanism, post-translational modification, inorganic ion transport and metabolism, chromatin structure, etc. Twelve candidate interacting proteins were selected for one-to-one yeast two-hybrid verification with AhSAP1, and 8 candidate interacting proteins were found to interact with AhSAP1. 【Conclusion】The mixed cDNA library of peanut embryo development at different stages was successfully constructed, and 60 candidate interacting proteins with AhSAP1 were screened. The candidate interacting proteins were mainly involved in energy production and metabolism, translation, ribosome structure and biogenesis, transcription, signal transduction mechanism, post-translational modification, inorganic ion transport and metabolism, chromatin structure, etc. These candidates interacting protein genes were expressed in root, stem, leaf, inflorescence, fruit needle, pericarp, seed coat and embryo, and the interaction between 8 candidate interacting proteins and AhSAP1 was confirmed.

Key words: peanuts, seed size, AhSAP1, yeast two-hybrid, interacting protein

ZHU YanTing, DANG Hao, NIU SiJie, LIN JingYi, YANG Hua, YANG Qiang, ZHANG Chong, CAI TieCheng, ZHUANG WeiJian, CHEN Hua. Screening of Interaction Proteins with AhSAP1 in Peanut Using the Yeast Two-Hybrid System[J].Scientia Agricultura Sinica, 2024, 57(21): 4376-4390.

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URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2024.21.016

https://www.chinaagrisci.com/EN/Y2024/V57/I21/4376

Figures/Tables 7

Fig. 1

Fig. 2

Fig. 3

Table 1

Sequence analysis of partial interacting candidate proteins with AhSAP1 by yeast two-hybrid system"

克隆编号 ID	基因编号 Gene ID	功能描述 Function description	代谢途径 KEGG	G<BOLD>O</BOLD>注释 GO annotation
114	AH17G19530	原卟啉原氧化酶1，叶绿体Protoporphyrinogen oxidase 1, chloroplastic	K00231:PPOX	GO:0016491, GO:0055114
96	AH14G06790	胸苷激酶Thymidine kinase	K00857:tdk	GO:0004797, GO:0005524
103	AH05G16760	2-C-甲基-D-赤藓糖醇2,4-环二磷酸合酶，叶绿体型2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, chloroplastic	K01770:ispF	GO:0008685, GO:0016114
120	AH11G18540	ATP合成酶δ链，叶绿体型ATP synthase delta chain, chloroplastic	K02113:ATPF1D	GO:0015986, GO:0046933
107	AH09G34040	ATP合成酶亚基d，线粒体型ATP synthase subunit d, mitochondrial	K02138:ATPeF0D	GO:0000276, GO:0015078, GO:0015986
148,155	AH05G02620	SKP1同源蛋白1B SKP1-like protein 1B	K03094:SKP1	GO:0006511
28	AH19G33100	延长因子1-α Elongation factor 1-alpha	K03231:EEF1A	GO:0003924, GO:0005525
87	AH02G05110	翻译起始因子eIF-2B亚基α Translation initiation factor eIF-2B subunit alpha	K03239:EIF2B1	GO:0044237
54	AH17G22760	过氧化氢酶同工酶1 Catalase isozyme 1	K03781:katE	GO:0004096, GO:0006979, GO:0020037, GO:0055114
30,31	AH13G07880	NADH脱氢酶[泛醌]1β亚复合体亚基9 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 9	K03965:NDUFB9	GO:0005747, GO:0006120
56,135	AH03G06830	NADH脱氢酶[泛醌]1β亚复合体亚基9 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 9	K03965:NDUFB9	GO:0005747, GO:0006120
321	AH13G07880	NADH脱氢酶[泛醌]1β亚复合体亚基9 NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 9	K03965:NDUFB9	GO:0005747, GO:0006120
104	AH18G19590	核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基结合蛋白β亚基，叶绿体型 RuBisCO large subunit-binding protein subunit beta, chloroplastic	K04077:groEL	GO:0005524, GO:0005737, GO:0042026
123	AH14G35300	SNF1相关蛋白激酶催化亚基α KIN10 SNF1-related protein kinase catalytic subunit alpha KIN10	K07198:PRKAA	GO:0004672, GO:0005524, GO:0006468
153	AH01G21220	核转录因子Y亚基A-3 Nuclear transcription factor Y subunit A-3	K08064:NFYA	GO:0003700, GO:0006355
78	AH11G01650	类似AGAMOUS的MADS-box蛋白AGL5 Agamous-like MADS-box protein AGL5	K09264:K09264	GO:0003700, GO:0005634, GO:0006355, GO:0003677, GO:0046983, GO:0000977, GO:0045944
55	AH19G40840	F-box蛋白SKIP22 F-box protein SKIP22	K10293:FBXO7	GO:0005515
166	AH01G21740	含有WD-40重复序列的MSI4蛋白 WD-40 repeat-containing protein MSI4	K10752:RBBP4	GO:0005515
85	AH03G20510	Mec-8和Unc-52蛋白抑制子同源体2 Suppressor of mec-8 and unc-52 protein homolog 2	K13109:IK	GO:0005634
13	AH18G00610	假组氨酸磷酸转移蛋白6 Pseudo histidine-containing phosphotransfer protein 6	K14490:AHP	GO:0000160, GO:0004871
69	AH03G34280	核内前mRNA域包含蛋白1B的调控 Regulation of nuclear pre-mRNA domain-containing protein 1B	K15559:RTT103	NA
82	AH15G31710	葡糖醛酸激酶1 Glucuronokinase 1	K16190:GLCAK	GO:0005524
65	AH03G22980	E3泛素-蛋白质连接酶RGLG2 E3 ubiquitin-protein ligase RGLG2	K16280:RGLG	NA
24	AH13G04580	线粒体导入内膜转位酶亚基Tim9 Mitochondrial import inner membrane translocase subunit Tim9	K17777:TIM9	NA
102	AH03G33130	NAD(P)H依赖型6′-脱氧查尔酮合成酶 NAD(P)H-dependent 6′-deoxychalcone synthase	K22374:DMAS1	GO:0016491, GO:0055114
184	AH05G02930	叶绿体DNAJ-11伴侣蛋白 Chaperone protein dnaJ 11, chloroplastic	NA	NA
28	AH03G16330	含染色质结构域蛋白LHP1 Chromo domain-containing protein LHP1	NA	GO:0005634
27	AH09G24080	多梳蛋白增强子同源物2 Enhancer of polycomb homolog 2	NA	GO:0006357, GO:0032777, GO:0035267
131	AH16G06440	光依赖同源盒蛋白LUMINIDEPENDENS Homeobox protein LUMINIDEPENDENS	NA	GO:0003677
35	AH13G51890	MADS-box转录因子1 MADS-box transcription factor 1	NA	GO:0003700, GO:0005634, GO:0006355, GO:0003677, GO:0046983, GO:0000977, GO:0045944
48	AH07G01050	OBERON类似蛋白（片段） OBERON-like protein (Fragment)	NA	NA
21	AH15G07140	含五肽重复序列的蛋白质At3g18110，叶绿体 Pentatricopeptide repeat-containing protein At3g18110, chloroplastic	NA	NA
22	AH13G25080	WRKY转录因子11 Probable WRKY transcription factor 11	NA	GO:0003700, GO:0006355, GO:0043565
93	AH08G23910	质体转录活性蛋白7 Protein PLASTID TRANSCRIPTIONALLY ACTIVE 7	NA	NA
37	AH17G03830	SCAF11蛋白Protein SCAF11	NA	NA
77	AH08G16900	转录因子bHLH115 Transcription factor bHLH115	NA	GO:0046983
187	AH08G22230	转录因子bHLH130 Transcription factor bHLH130	NA	GO:0046983
70	AH13G36200	转录因子TGA5 Transcription factor TGA5	NA	GO:0006351, GO:0043565
75	AH07G05790	尿苷二磷酸糖基转移酶92A1 UDP-glycosyltransferase 92A1	NA	GO:0008152, GO:0016758
108	AH03G40650	多聚半乳糖醛酸酶-1非催化亚基β Polygalacturonase-1 non-catalytic subunit beta	NA	NA
89	AH15G05570	VAN3结合蛋白VAN3-binding protein	NA	NA

Table 1

Fig. 4

Fig. 5

Fig. 6

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Screening of Interaction Proteins with AhSAP1 in Peanut Using the Yeast Two-Hybrid System

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