Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (12): 2321-2330.doi: 10.3864/j.issn.0578-1752.2020.12.001

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

Genome-Wide Identification and Expression Analysis of PIN Genes Family in Wheat

LIU PeiXun,WAN HongShen,ZHENG JianMin,LUO JiangTao,PU ZongJun()   

  1. Crop Research Institute, Sichuan Academy of Agricultural Sciences/Key Laboratory of Wheat Biology and Genetic Improvement on Southwestern China, Ministry of Agriculture and Rural Areas, Chengdu 610066
  • Received:2019-09-04 Online:2020-06-16 Published:2020-06-25
  • Contact: ZongJun PU E-mail:pzjun68@163.com

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Abstract:

【Objective】 The puroindoline (PIN) gene is a family specific to plants and plays an important role in controlling the grain hardness in wheat. In silico identification and expression analysis of PIN family genes in Triticum aestivum on whole genome level lay the foundation for elucidation the biological function of wheat PIN family. 【Method】 The known sequences of wheat PIN proteins and barley hordoindoline (HIN) proteins were used to query the newly released wheat peptides dataset of Chinese spring variety with HMM and BLASTP profiles. UniProt, URGI, PFAM, CDD, expVIP and other databases were used for bioinformatics analysis with Clustal X, MEGA 7.0, ExPASy, MEME, GSDS, TBtools, GraphPad Prism5 and other programs. The expression profiles of TaPIN genes in different wheat harness seeds were validated with quantitative real-time polymerase chain reaction (qRT-PCR). 【Result】 A total of 19 TaPINs were identified in wheat genome, which were clustered in homologous groups of chromosome 1, 5 and 7. These TaPIN proteins contained 148 to 327 amino acids, as their relative molecular weight varied from 16.39 to 37.19 kD and the isoelectric points ranged from 6.35 to 9.34. Phylogenetic and conserved domain analyses showed that the 19 TaPIN proteins were divided into A and B categories, respectively. Most TaPIN genes harbored only one exon, and there were many cis-acting regulatory elements involved in stress responsiveness and seed-specific regulation. RNA-Seq showed that this gene family expressed largely in wheat grain and hardly expressed in other tissues. The qRT-PCR results indicated that the relative expression level among TaPIN genes were significantly changed. TaPIN9 and TaPIN10 were highly expressed. The expression levels of TaPIN9 and TaPIN10, as well as its expression ratio, were up-regulated with the decrease of wheat grain hardness, while TaPIN16 and TaPIN6 showed an opposite trend. 【Conclusion】 Pina and Pinb genes were the main factors regulating grain hardness in wheat. It is speculated that other members of this gene family have similar functions, but have little influence on grain hardness because of low expression level. According to the evolutionary relationship of this gene, Aegilops tauschii is most closely related to wheat, followed by oats, rye and barley.

Key words: Triticum aestivum, grain hardness, PIN gene, gene expression

Table 1

Primer sequences used in quantitative real-time PCR analysis of TaPINs"

基因名称
Gene name		 正向引物序列
Forward primer sequence (5′-3′)		 反向引物序列
Revers primer sequence (5′-3′)
TaPIN6 TATGCCGCTCTCTTGGGT GATCGCCTTGGATTGATG
TaPIN7 AGCTATGCAAGCTCCCAC CACAACTTCTCTTCCCCC
TaPIN8 TATGCCGCTCTCTTGGTT GATCGCCTTGGATTGATG
TaPIN9 AGCTCCTTGGGGAGTGTT CAGGTTCTTGGCTTCTTG
TaPIN14 GGCGGTGAAGGGTTTTTC GCTATCGGGCGTAGTTGC
TaPIN15 AAGGATTATGTGATGGAG GCTGGTAACACTGGTCTA
TaPIN16 AAAGAAGTGCCGATGTGAGG GCTGAAAGCCAAAGACGC
TaPIN19 TGTGAACAAGAAGCCCTA TGCTGAAAACCAAAGATG
TaPIN10 TGAGCATGAGGTTCGGGA TTGCACTTTGAGGGGAGG
β-Actin ATGTACCGTGGTGATGTT CCTGGTGGCTGGTAGTTG

Fig. 1

Phylogenetic tree of TaPINs using the complete protein sequences PINA and PINB were puroindoline proteins reported in Triticum aestivum, AINA and AINB were homologous proteins in Avena sativa, SINA and SINB in Secale cereale, HINA and HINB in Hordeum vulgare, EMT21351 and EMT21353 in Aegilops tauschii, respectively"

Fig. 2

The conserved motifs and gene structure analysis of 19 TaPINs The sectional intron of TaPIN15 gene was deleted manually, as it was too long (17881 bp), and only 1200 bp was retained for showing the gene structure"

Fig. 3

Cis-acting element analysis of 19 TaPINs ABRE: cis-acting element involved in the abscisic acid responsiveness; CGTCA-motif: cis-acting regulatory element involved in the MeJA-responsiveness; TGACG-motif: cis-acting regulatory element involved in the MeJA-responsiveness; GCN4_motif: cis-regulatory element involved in endosperm expression; P-box: Gibberellin-responsive element; MBS: MYB binding site involved in drought-inducibility; LTR: cis-acting element involved in low-temperature responsiveness; O2-site: cis-acting regulatory element involved in zein metabolism regulation; TC-rich repeats: cis-acting element involved in defense and stress responsiveness; TCA-element: cis-acting element involved in salicylic acid responsiveness; GARE-motif: Gibberellin-responsive element; RY-element: cis-acting regulatory element involved in seed-specific regulation"

Fig. 4

Expression profiles of TaPINs in different growth stages and different tissues of Chinese Spring A: Root_3 leaf stage; B: Spike_emergence; C: Spike_anthesis; D: Stem_anthesis; E: Grain_2dpa; F: Shoot_5 leaf stage; G: Leaf_7 leaf stage; H: Grain_14dp; I: Aleurone layer_20dpa; J: Endosperm_10dpa; K: Endosperm_20dpa; L: Endosperm_30dpa"

Fig. 5

Relative expression of TaPINs belong to class A The value in the figure is the average value of three replicates. SM969MN: SM969, Medium nitrogen application; SM969LN: SM969, Low nitrogen treatment; CM601LN: CM601, Low nitrogen treatment"

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