Gene Cloning and Interaction Proteins Screening of DcPMK in Dioscorea composite

doi:10.7525/j.issn.1673-5102.2022.05.017

Abstract

Abstract:

To explore the synthesis path of terpenoid in Dioscorea composite and to provide theoretical reference for studying， D. composite was used as material， DcPMK gene was cloned by RT-PCR and the bioinformatic analysis， tissue-specific expression analysis and expression analysis after salicylic acid （SA） treatment were carried out respectively， and the bait vector of DcPMK was constructed to screen interacting proteins from Arabidopsis yeast library. The results showed that the open reading frame （ORF） of DcPMK was 1 536 bp （GenBank accession No. MZ171241）， which encoded 511 amino acids. By multiple sequence alignment， the results showed that similarity between DcPMK and PMK amino acid sequence of other plants was as high as 88.85%， DcPMK obtained 1 ATP binding site Gly-X-Gly-XX-Ala. The result of phylogenetic tree showed that DcPMK was closely related to monocotyledon such as Phonenix dactylifera．HPLC analysis showed that the content of diosgenin was the highest in rhizome， qRT-PCR analysis indicated that DcPMK was constitutively expressed among flower， leaf， stem， rhizome and root， with the highest levels in old stem and the lowest in rhizome. After SA treatment， the changes of diosgenin content were in parallel with the expression levels of DcPMK， the diosgenin content was improved as DcPMK up-regulated expression in leaf. Yeast two-hybrid results showed that DcPMK was no autoactivation and cell toxicity， then 27 significant interactional genes were screened， which were growth and development-related genes AtKCR1（AT1G67730）， AtRPS9M（AT3G49080）， AtASY4（AT2G33793）， abiotic and biotic stress-related genes AtVDAC2（AT5G67500）， AtVDAC3（AT5G15090）， AtRH8（AT4G00660）， anthocyanin accumulation-related gene AtPHR2（AT2G47590）. The results illustrated that DcPMK could participate in the synthesis of terpenoids in D. composite， and was widely involved ingrowth and stress response pathways through protein-protein interaction.

Key words: Dioscorea composite, PMK, bioinformation, expression pattern, interaction proteins

CLC Number:

S567.9

Hongpeng WANG, Yidan LI, Yao WANG, Xiaoyu TAN, Chengbin CHEN, Lipeng ZHANG. Gene Cloning and Interaction Proteins Screening of DcPMK in Dioscorea composite[J]. Bulletin of Botanical Research, 2022, 42(5): 855-865.

Figures/Tables 9

Table 1

Fig.1

Fig.2

Fig.3

Fig.4

Fig.5

Fig.6

Table 2

The interaction genes function information of DcPMK

阳性菌编号 No.	拟南芥基因号 Gene ID	基因大小Length /bp	基因名称 Name	基因注释信息 Description
4	ATCG00740	990	RPOA	RNA聚合酶α亚基 RNA polymerase alpha subunit.
5，31	AT5G06340	1 035	ATNUDX27	nudix水解酶27 Nudix hydrolase homolog 27
6	AT3G51130	1 233	unknown	跨膜蛋白 Transmembrane protein.
7	AT1G67730	957	ATKCR1	编码β-酮酰基还原酶（KCR），该酶催化VLCFA延伸过程中的第一次还原反应 Beta-ketoacyl reductase（KCR），which catalyzes the first reduction during VLCFA elongation
8，44	AT2G47590	1 344	PHR2	光解酶/蓝光光感受器PHR2 mRNA photolyase/blue light photoreceptor PHR2 mRNA
9，40	AT3G10910	546	DAFL1	RING/U-box超家族蛋白 RING/U-box superfamily protein
11，19，29，41	AT3G60770	456	unknown	核糖体蛋白S13/S15 Ribosomal protein S13/S15
14	AT4G16555	525	unknown	类HSP20伴侣 HSP20-like chaperone
16	AT2G05325	536	unknown	新的转录区；在根，心皮等中检测到 Novel transcribed region；detected in root，carpel，and so on
17	AT2G43235	1 314	unknown	磷酸核糖甲酰甘氨酸脒合酶 phosphoribosylformylglycinamidine synthase
22	AT3G60570	795	EXPB5	β-扩张蛋白成员 member of BETA-EXPANSINS
23	AT3G55330	693	PPL1	促进光系统Ⅱ超级复合物的组装，并在波动光下优化植物的垂体 Facilitates the assembly of the photosystem Ⅱ supercomplexes and optimizes plant pitness under fluctuating light
24	AT3G49080	1 293	RPS9M	功能丧失导致配子致死 Loss of function results in gametophyte lethality
26	AT3G47080	1 548	unknown	类超家族蛋白-TPR TPR-like superfamily protein
27，28	AT4G00660	1 518	RH8	类RNA解旋酶-8 RNA helicase-like 8
30，34，39	AT2G43130	645	ARA4	其涉及细胞内囊泡运输 Which are implicated in intracellular vesicular traffic
32	AT1G78170	666	unknown	泛素连接酶E3 E3 ubiquitin-protein ligase
33	AT5G20290	669	unknown	核糖体蛋白S8e家族蛋白 Ribosomal protein S8e family protein
35	AT2G18770	783	unknown	含有P环的核苷三磷酸水解酶超家族蛋白 P-loop containing nucleoside triphosphate hydrolases superfamily protein
36	AT1G55120	1 785	FRUCT5	植物的生长发育中起作用 Plays a role in the growth and development of plants
38	AT5G15090	825	VDAC3	编码依赖电压的阴离子通道。它们参与细胞器和胞质溶胶之间的代谢物交换 Encodes a voltage-dependent anion channel. They are involved in metabolite exchange between the organelle and the cytosol
42	AT5G16710	1 088	DHAR3	调节细胞质中抗坏血酸-谷胱甘肽循环的氧化还原状态 Modulate redox states of ascorbate-glutathione cycle in the cytosol
43	AT5G67500	912	VDAC2	它们参与细胞器和胞质溶胶之间的代谢物交换 They are involved in metabolite exchange between the organelle and the cytosol
45	AT5G47190	1 009	PRPL19	核糖体蛋白L19家族蛋白 Ribosomal protein L19 family protein
46	AT5G41910	561	MED10A	介体复合物亚基10 Mediator complex，subunit Med10
47	AT2G33793	639	ASY4	编码突触前染色体轴的蛋白质成分。突变体在减数分裂重组中显示出定量缺陷 Encodes a protein component of the pre-synaptic chromosome axis；Mutants show quantitative defects in meiotic recombination
48	AT2G33800	912	SCA1	等位基因sca1-rps5在质体16S rRNA加工中表现出缺陷，并导致光合积累的减少 The sca1-rps5 allele exhibits defects in plastid 16S rRNA processing and a resulting decrease in accumulation of photosynthetic

Table 2

Fig.7

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引物 Primer	引物序列 Primer sequence（5′—3′）	用途 Strategy
DcPMK-F	ATGGCGGTTGTGGCTTCAGC	基因克隆 gene cloning
DcPMK-R	CTAGTTGATATTGATGGAAGAAATAGACG	基因克隆 gene cloning
DcPMK-BDCE-F	tatggccatggaggccgaattcATGGCGGTTGTGGCTTCAGC	酵母双杂交引物 Yeast two-hybrid primers
DcPMK-BDCE-R	gccgctgcaggtcgacggatccCCTAGTTGATATTGATGGAAGAA	酵母双杂交引物 Yeast two-hybrid primers
DcTUB7-qF	AGACAACATCAACCCTGGACT	内参基因引物 Internal reference primers
DcTUB7-qR	GAGGCTGAGAGCAACATGAAT	内参基因引物 Internal reference primers
DcPMK-qF	TGTCTTCTGCTCAGGCATTGGGTG	荧光定量PCR引物 Primers for qRT-PCR
DcPMK-qR	TCACAAAGCCAACCATTGAAGGAGTA	荧光定量PCR引物 Primers for qRT-PCR

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