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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (1): 24-35.doi: 10.3724/SP.J.1006.2023.14233

• CROP GENETICS & BREEDING ·GERMPLASM RESOURCES ·MOLECULAR GENETICS • Previous Articles     Next Articles

Function of GmPIN2 family gene in regulating root development in soybean

LIANG Zheng1,3(), KE Mei-Yu1,3, CHEN Zhi-Wei2, CHEN Xu3,*(), GAO Zhen3,*()   

  1. 1College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    3Haixia Institute of Science and Technology, Horticultural Plant Biology and Metabolomics Center, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
  • Received:2021-12-08 Accepted:2022-05-05 Online:2023-01-12 Published:2022-05-13
  • Contact: CHEN Xu,GAO Zhen E-mail:1879949813@qq.com;chenxu@inbox.com;gaozhen0695@fafu.edu.cn
  • Supported by:
    Distinguished Young Scholar Program of Fujian Agriculture and Forestry University(XJQ201921)

Abstract:

Auxin is one of the important plant hormones. Auxin transport is involved in the regulation of plant tissue and organ development. In Arabidopsis thaliana, auxin transport is mainly regulated by PIN family proteins, among which AtPIN2 controls auxin basipetal transport and regulates root gravitation in Arabidopsis. However, GmPIN2 family proteins and their functions are still largely unknown in soybean. In this study, we identified two AtPIN2 homologous genes of GmPIN2a and GmPIN2b through phylogenetic and protein domain analysis. The relative expression level showed that GmPIN2a and GmPIN2b were highly expressed in root and nodule. Both genes were highly expressed in root epidermis and cortex, and the vascular bundles of the root nodule. Only GmPIN2a expressed in the epidermis and outer cortex at the tip of the root nodule. Furthermore, Gmpin2ab mutants were generated by CRISPR/Cas9 (Clustered Regularly Interspersed Short Palindromic Repeats/CRISPR Associated 9) gene editing technology. Gmpin2ab mutants had a significant loss of root gravitropism. Moreover, the loss of Gmpin2 resulted in a significant decrease of root area and lateral root length, while the lateral root angle of Gmpin2ab significantly increased. Overexpression of GmPIN2b also resulted in a decrease in root area and lateral root length, but the lateral root angle unchanged. In conclusion, GmPIN2a and GmPIN2b play important roles in regulation of soybean root development via mediating auxin basipetal transport. This study laid a foundation for further analysis of the function and mechanism of GmPIN proteins in soybean root morphogenesis.

Key words: Glycine max (soybean), auxin, PIN protein, root

Table 1

Primer sequences"

引物用途
Purpose of primers
引物名称
Primer name
引物序列
Primer sequence (5°-3°)
启动子序列扩增引物
Promoter amplification primers
pGmPIN2a-F AGATGGTTAGAGAGGCATGAGCCACAATGGAAC
pGmPIN2a-R AGCTCTTATACTCGACATGGTTCATGCGGG
pGmPIN2b-F AGATGGTTAGAGAGGTGATGTTCTTGGCCACTG
pGmPIN2b-R AGCTCTTATACTCGAGATCATGGTTAATGCAGGG
定量PCR引物序列
RT-PCR primers
GmPIN2a-F CCACACGAAACTGTTGCCTC
GmPIN2a-R AAGCTCCCTCTGATCTCCGT
GmPIN2b-F GGGAATTTGAGGCATGCAGT
GmPIN2b-R CAGGCTCCCTCTCTCCACTC
GmActin-11-F GAGCTATGAATTGCCTGATGG
GmActin-11-R CGTTTCATGAATTCCAGTAGC
sgRNA序列 sgRNA sequence sgRNA-GmPIN2ab TGGAACACCTTCACAAAACATGG
验证编辑载体的引物序列
Primer sequence for verifying the gene editing vectors
pCas9-F GCCAAGAGCGAGCAGGAAAT
pCas9-R GGTGGATGATATTCTCGGCCT

Fig. 1

Phylogenetic tree of GmPIN and AtPIN proteins The red box represents soybean GmPIN2 and Arabidopsis AtPIN2."

Fig. 2

Bioinformatics analysis of PIN2 protein families in Arabidopsis thaliana, Glycine max, Medicago truncatula, Lotus japonicus, Oryza sativa, and Zea mays A: gene structure of the PIN2 protein family in Arabidopsis thaliana, Glycine max, Medicago truncatula, Lotus japonicus, Oryza sativa, and Zea mays. B: analysis of gene promoter regions of the PIN2 protein family in Arabidopsis thaliana, Glycine max, Medicago truncatula, Lotus japonicus, Oryza sativa, and Zea mays. C: protein sequence alignment of the PIN2 protein family in Arabidopsis thaliana, Glycine max, Medicago truncatula, Lotus japonicus, Oryza sativa, and Zea mays. AT5G57090: AtPIN2; Glyma.13G101900: GmPIN2a; Glyma.17G057300: GmPIN2b; LOC_Os06g44970: OsPIN2; Medtr4g127100: MtPIN2a; Medtr4g127090: MtPIN2b; Lj4g3v2139970: LjPIN2; Zm00001d046893_ P001: ZmPIN2."

Table 2

Homology comparison of PIN2 protein families in Arabidopsis thaliana, Glycine max, Medicago truncatula, Lotus japonicus, Oryza sativa, and Zea mays (%)"

Seq-> AtPIN2 GmPIN2a GmPIN2b OsPIN2 MtPIN2a MtPIN2b LjPIN2 ZmPIN2
AtPIN2 100.0 76.5 77.2 61.7 75.0 63.7 76.5 61.0
GmPIN2a 76.5 100.0 96.8 63.9 85.1 68.3 90.7 61.9
GmPIN2b 77.2 96.8 100.0 63.5 85.5 68.8 90.8 61.9
OsPIN2 61.7 63.9 63.5 100.0 63.8 53.8 63.4 87.3
MtPIN2a 75.0 85.1 85.5 63.8 100.0 75.4 85.0 63.6
MtPIN2b 63.7 68.3 68.8 53.8 75.4 100.0 68.8 53.4
LjPIN2 76.5 90.7 90.8 63.4 85.0 68.8 100.0 61.7
ZmPIN2 61.0 61.9 61.9 87.3 63.6 53.4 61.7 100.0

Fig. 3

Tissue expression pattern analysis and histochemical localization of GmPIN2a and GmPIN2b genes A: the relative expression pattern of GmPIN2a and GmPIN2b genes; B: histochemical localization of GmPIN2a and GmPIN2b in root and nodule. Bars: 100 μm."

Fig. 4

Construction of homozygous mutants of Gmpin2ab-#1 and Gmpin2ab-#2 A: the gene structure of GmPIN2a and GmPIN2b and target sites of designed sgRNAs. The black arrow represents the sgRNA position, black boxes represent exons, red boxes represent 5′UTR and 3′UTR and black lines represent introns. B: the validation of gene editing vectors in Gmpin2ab-L1 and Gmpin2ab-L2. White numbers from left to right indicate the wild type, 35S::GmPIN2b, Gmpin2ab-L1, and Gmpin2ab-L2. C: the relative expression level of GmPIN2 family in wild type, 35S::GmPIN2b, Gmpin2ab-L1, and Gmpin2ab-L2. P-values are determined by a two-tailed Student’s t-test assuming equal variances, ** indicates that Gmpin2ab-L1, Gmpin2ab-L2 significantly different from wild type at P < 0.01; *** indicates that 35S::GmPIN2b significantly different from wild type at P < 0.001; **** indicates that Gmpin2ab-L1, Gmpin2ab-L2 significantly different from wild type at P < 0.0001. ns: not significant. D: the sequence alignment of Gmpin2ab-#1 and Gmpin2ab-#2. Green letters represent sgRNA targeting sequences; red letters and red dash lines represent added or missing codons. E: the protein sequence of WT, Gmpin2ab-#1 and Gmpin2ab-#2 homogenous mutants. AA represents amino acid. The black arrow represents the positions of frameshift mutations. The purple box represents the frame-shifting mutant protein sequence. The number represents the number of frameshift amino acids."

Fig. 5

Plant phenotype of wild type, Gmpin2ab-L1, Gmpin2ab-L2, and 35S::GmPIN2b A: Gmpin2ab-L1 and Gmpin2ab-L2 are defective in root gravitropism. The red box indicates that the mutant is growing toward the ground. B: the scanning pattern of seven-day wild-type, Gmpin2ab-L1, Gmpin2ab-L2, and 35S::GmPIN2b, Bars: 1 cm. P-values are determined by a two-tailed Student’s t-test assuming equal variances, **** indicates P < 0.0001. ns: not significant. n = 7."

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