Bioinformatics of Huipizhi Black soybean GmPUB24 and expression under Heterodera glycines infection

doi:10.3969/j.issn.1004-1524.2022.06.03

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (6): 1124-1132.DOI: 10.3969/j.issn.1004-1524.2022.06.03

• Crop Science • Previous Articles Next Articles

Bioinformatics of Huipizhi Black soybean GmPUB24 and expression under Heterodera glycines infection

LI Wenchen(), LIU Xin, QI Zezheng, YU Lu, WANG Fang()

College of Life Sciences and Agroforestry, Qiqihar University, Qiqihar 161006, Heilongjiang, China

Received:2021-08-21 Online:2022-06-25 Published:2022-06-30
Contact: WANG Fang

Abstract

Abstract:

In order to clarify the mechanism of soybean U-box E3 ubiquitin ligase mediating plant disease resistance, coding sequence (CDS) of GmPUB24 gene from resistant cultivar Huipizhi black bean was cloned by RT-PCR. Bioinformatics and expression analysis of this gene after inoculation with soybean cyst nematode were carried out. The results showed that the length of CDS was 1 254 bp, encoded a total of 417 amino acids with a molecular weight of 46.78 ku. Protein secondary structure analysis showed that the protein encoded by GmPUB24 contained alpha helix, random coil, extended chain and beta turn, and the alpha helix accounted for the highest proportion of 58.9%. Protein encoded by GmPUB24 gene was a hydrophilic protein without transmembrane domain and signal peptide. Protein phylogenetic tree displayed that it had the highest affinity with wild soybean. Subcellular location predicted that it localized in the cytoplasm. Promoter analysis of 1 500 bp upstream of GmPUB24 gene showed that it contained CGTCA-motif, TGACG-motif and other elements that respond to disease resistance pathways, as well as ABRE, WUN-motif, TATA-box and other corresponding abiotic stress elements. Quantitative real-time PCR (qRT-RCR) results showed that the expression of GmPUB24 was induced by soybean cyst nematode, and the highest expression level in roots was 6.14 times compared with the uninoculated sample on the third day, indicating that GmPUB24 gene was induced by soybean cyst nematode and might be involved in the process of soybean defense against cyst nematode. These results provided a basis for clarifying the regulatory mechanism of soybean U-box family gene in resistance to soybean cyst nematode disease.

Key words: soybean, cloning, promoter, bioinformatics, expression

CLC Number:

LI Wenchen, LIU Xin, QI Zezheng, YU Lu, WANG Fang. Bioinformatics of Huipizhi Black soybean GmPUB24 and expression under Heterodera glycines infection[J]. Acta Agriculturae Zhejiangensis, 2022, 34(6): 1124-1132.

Figures/Tables 9

Fig.1 Agarose gel electrophoresis results of bacterial liquid PCR of GmPUB24 geneM, DL2000 marker; 1-3, 3 replicates of bacterial fluid samples.

Fig. 2 U-box domain of protein encoded by GmPUB24 gene

Fig. 3 Secondary structure of protein encoded by GmPUB24 Red, Extended strand; Blue, Alpha helix; Green, Beta turn; Purple, Random coil.

Fig. 4 Hydrophilicity and hydrophobicity of GmPUB24 protein Taking 0 as the limit, positive values represented hydrophobic amino acids and negative values represented hydrophilic amino acids.

Fig. 5 Homologous alignment of U-box domains of Huipizhi black bean GmPUB24 with other plant associated proteins Black shading meant amino acids with 100% identity, pink meant more than 75% identity, blue meant 50%-75% identity, and white means less than 50% identity. KAG4974237, Glycine soja; XP_003539150, Glycine max; XP_027911296, Vigna unguiculata; XP_014523587, Vigna radiata var. Radiata; XP_027348689, Abrus precatorius; KYP39063, Cajanus cajan; XP_004506267, Cicer arietinum; OAP04810, Arabidopsis thaliana; XP_004229574, Solanum lycopersicum; XP_015635733, Oryza sativa; PWZ26684, Zea mays. The same as below.

Fig. 6 Phylogenetic tree of GmPUB24 protein from Huipizhi black bean and other plant homologous proteinsNeighbor Joining method with 1 000 bootstrap replicates. The scale bar represented the genetic distance.

Table 1 Analysis of cis element of GmPUB24 promoter

名称 Name	数量 Number	序列 Sequence	功能 Function
ABRE	3	ACGTG	参与脱落酸反应 Involved in the abscisic acid reaction
Box 4	7	ATTAAT	参与光反应有关的保守DNA Conserved DNA elements involved in photo reaction
Box II	2	CCACGTGGC	参与光响应Components involved in light response
CAAT-box	20	CCAAT	启动子和增强子区常见的顺式作用元件 Involved in promoter and enhancer regions
CAT-box	1	GCCACT	参与分生组织相关表达 Involved in the expression of meristems
CGTCA-motif	3	CGTCA	参与茉莉酸反应 Involved in the jasmonic acid reaction
TATA-box	35	ATATAT	参与核心启动子30位点转录 Transcription element involved in the 30 position of the core promoter
TGACG-motif	3	TGACG	参与茉莉酸反应 Involved in the jasmonic acid reaction
Unnamed-1	4	GAATTTAATTAA	60s蛋白质活性位点 Involved in 60s protein active site
WUN-motif	1	AAATTTCCT	参与植物伤口反应 Elements involved in plant wound response
ARE	1	AAACCA	无氧诱导 Necessary for anaerobic induction
AT-rich element	1	ATAGAAATCAA	结合蛋白（ATBP-1）的结合位点 Binding site element of binding protein (ATBP-1)
G-box	3	GCCACGTGGA	参与光响应Involved in light response
O2-site	1	GATGATGTGG	参与玉米醇溶蛋白代谢调节 Involved in the regulation of zein metabolism

Fig. 7 Electrophoresis detection of GmPUB24 and TUA4 PCR products M, DL2000 marker; 1, TUA4; 2, GmPUB24.

Fig. 8 Expression of soybean GmPUB24 gene at different time of soybean cyst nematode infection ** represented P ≤ 0.01, *** represented P ≤ 0.001, ns represented the difference was not significant.

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Bioinformatics of Huipizhi Black soybean GmPUB24 and expression under Heterodera glycines infection

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