Expression Pattern Analysis and Functional Identification of Potato Expansion Protein Gene StEXLB1a

doi:10.7668/hbnxb.20195691

Abstract

Abstract:

To explore the expression pattern of potato gene StEXLB1a and provide a theoretical basis for the resistance to potato disease,based on the previous research of the Laboratory of Plant Resources and Molecular Biology at Northeast Agricultural University,the study using the Atlantic variety of potato as material, cloned the StEXLB1a gene, and preliminarily analyzed its bioinformatics, expression pattern, and disease resistance. The results showed that the full-length cDNA sequence of StEXLB1a gene was 768 bp,encoding 255 amino acids.The annotation indicated that it belonged to the extend protein family genes.Subcellular localization showed that the protein was localized to the cell wall.Among different tissues of potato,StEXLB1a gene expression was highest in leaves,followed by roots and stems,and lowest in flowers and tubers.The expression level of StEXLB1a gene changed significantly under various stress treatments,such as hormone(indoleacetic acid,gibberellin,abscisic acid),stress(high temperature,low temperature,salt,drought),fungal disease dry rot(Fusarium avenaceum),bacterial disease soft rot(Erwinia carotovora subsp,Ecc)and bacterial wilt(Ralstonia solanacearum,RS).The overexpressed transgenic potato plants were inoculated with the dry rot pathogen Fusarium avenaceum,and the transgenic plants were more seriously infected than the wild-type plants.The enzyme activities of active oxygen scavenging system in overexpressed plants were significantly changed,POD and SOD activities were inhibited,MDA content was higher than that of wild-type plants,and the damage degree of plants was aggravated.The results showed that transgenic potato plants with overexpression of StEXLB1a had lower resistance to dry rot than those with wild type.

Key words: Potato, Expansin, Gene cloning, Expression pattern analysis, Functional identification

CLC Number:

DU Chong, HE Fumeng, SUI Jia, ZHAO Xiaocan, CHE Yunzhu, ZHANG Zengli, LIU Dan, WANG Xue, LI Fenglan. Expression Pattern Analysis and Functional Identification of Potato Expansion Protein Gene StEXLB1a[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(5): 161-170. doi: 10.7668/hbnxb.20195691.

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Figures/Tables 12

Tab.1 Primer sequence of StEXLB1

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')	功能 Function
StEXLB1a-F	ATGGCTCCTCTTCAAGTTCT	克隆基因
StEXLB1a-R	TTATGCATCTTCTTGTATTC
Q-StEXLB1a-F	ACTGTAGTTGTAACAGACCAAGG	表达特异性分析
Q-StEXLB1a-R	GTGGCAATATAGAAACAGCAGCA
Q-β-actin-F	GCTTCCCGATGGTCAAGTCA
Q-β-actin-R	GGATTCCAGCTGCTTCCATTC
LB1a-PstⅠ-F	AAAA CTGCAGATGGCTCCTCTTCAAGTTCTCTCTG	亚细胞定位载体构建
LB1a-SalⅠ-R	ACGC GTCGACTGCATCTTCTTGTATTCCTGAGTCG
LB1a-XbaⅠ-F	GC TCTAGAATGGCTCCTCTTCAAGTTCT	过表达载体的构建
LB1a-BamHⅠ-R	CG GGATCCTTATGCATCTTCTTGTATTC
GUS-F	GTCGCGCAAGACTGTAACCA	过表达植株鉴定
GUS-R	CGGCGAAATTCCATACCTG

Fig.1 Electrophoresis results of PCR amplification products M.DL2000 Marker;1,2.Target fragment.

Fig.2 Bioinformatics analysis of StEXLB1a protein A.Conserved domain analysis of StEXLB1a protein;B.StEXLB1a protein signal peptide prediction;C.Prediction of hydrophilic and hydrophobic properties of StEXLB1a protein;D.StEXLB1a protein secondary structure prediction;E.Prediction of the tertiary structure of StEXLB1a protein.

Fig.3 Phylogenetic tree of StEXLB1a protein

Fig.4 Relative expression of StEXLB1a in different potato tissues Different letter indicate significant differences(P<0.05). The same as Fig.5—7,9,11.

Fig.5 Effects of exogenous hormones(IAA,GA₃,ABA)on the relative expression of StEXLB1a in potato leaves at different time

Fig.6 Effects of abiotic stress(high temperature,low temperature,NaCl,PEG) on the relative expression of StEXLB1a in potato leaves at different time

Fig.7 Analysis of resistance specificity of StEXLB1a gene

Fig.8 Subcellular localization of StEXLB1a protein

Fig.9 Genetic transformation,positive identification and qRT-PCR results of overexpressed potato in the potato test tube A—D.Microscopic potato slices sprouting,differentiating,rooting and growing;E:M.DNA molecular weight standard 2000,+.Positive control(PCR product with recombinant plasmid as template),-.Negative control(DNA from wild-type plants as template),1—6.DNA from transgenic StEXLB1a plants as template;F.qRT-PCR detection results of overexpression plants.

Fig.10 Changes of leaf lesions after pathogen treatment

Fig.11 Determination of physiological indexes of disease resistance in transgenic plant leaves treated with pathogenic bacteria

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