Genome-wide Identification of Non-specific Phospholipase C Gene Family in Hordeum vulgare L. and Stress Expression Analysis at Seedling Stage

doi:10.13560/j.cnki.biotech.bull.1985.2024-0201

Abstract

Abstract:

【Objective】Non-specific phospholipase C（NPC）is a type of phospholipase C, which plays an important role in plant growth and development and response to abiotic stresses. Identifying the NPC gene family members from the whole barley genome, and analyzing the expression characteristics of the related genes would provide a theoretical basis for the study of barley NPC gene function and genetic improvement.【Method】Based on the barley genome-wide data included in the EnsemblPlants database, bioinformatics methods were used to identify NPC gene family in barley and analyze their physicochemical properties, subcellular localization, phylogenetic relationship, gene structure, protein structural domains, and promoter cis-acting elements. Transcriptome data and RT- qPCR were used to analyse the expression characteristics of HvNPCs in different tissues as well as at seedling stage under low temperature, drought and salt stress.【Result】Five NPC members were identified in the barley genome, and the encoded proteins were structurally similar to NPC genes from other plants, all of which had a phosphoesterase structural domain ; the sequence lengths of the HvNPC proteins ranged from 477 to 553 aa, the isoelectric points ranged from 5.22 to 8.83, and the molecular weights ranged from 53.12 to 61.33 kD. Subcellular localization showed that the HvNPC gene was localized in chloroplasts, membrane-intrinsic proteins, and cytoplasm. The HvNPC promoter region contained a large number of cis-acting elements associated with adversity stresses, and real-time PCR analysis confirmed that the HvNPC gene was induced to express by drought, high salt and low temperature. Among them, HvNPC2, HvNPC3 and HvNPC5 were induced to be expressed by low-temperature stress and drought stress; and HvNPC3 and HvNPC5 were induced to be expressed by salt stress.【Conclusion】A total of five HvNPC genes were identified in the barley genome, and members of the HvNPC genes were organ-specofc expression and different genes responded differently to abiotic stresses.

Key words: Hordeum vulgare L., non-specific phospholipase C, genome-wide identification, abiotic stress, expression analysis

CUI Yuan-yuan, WANG Zhao-yi, BAI Shuang-yu, REN Yu-zhao, DOU Fei-fei, LIU Cai-xia, LIU Feng-lou, WANG Zhang-jun, LI Qing-feng. Genome-wide Identification of Non-specific Phospholipase C Gene Family in Hordeum vulgare L. and Stress Expression Analysis at Seedling Stage[J]. Biotechnology Bulletin, 2024, 40(8): 74-82.

Figures/Tables 7

Table 1 Primers used for RT-qPCR

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	退火温度Annealing temperature/℃
HvNPC1-F	AACAAGCCCCCATCACCG	58.9
HvNPC1-R	CCACCTTGAAGGCGTCGT	58.6
HvNPC2-F	ACTACCAGTACCCGCCGT	58.9
HvNPC2-R	ACCACGTAGTTGGGCAGC	58.6
HvNPC3-F	GACGTGCTGGACCAGGTC	59.4
HvNPC3-R	CGGAGGAGAGCAGGGAGT	59.8
HvNPC4-F	GATGCCGACGAGGATCCG	59.2
HvNPC4-R	ATCCCCGTTCAGCACTGC	58.8
HvNPC5-F	CGTCTTCGACCGCTGGTT	58.4
HvNPC5-R	CGTTGAAGGTGAGCCCGT	58.6
HvActin-F	CAGGTATCGCTGACCGTATGAG	57.5
HvActin-R	TGGAAAGTGCTGAGTGAGGCT	58.9

Table 2 Basic physico-chemical properties of NPC family in barley（Hordeum vulgare L）

基因名称 Gene name	基因ID Transcript ID	物理位置 Physical position	相对分子量 Molecular weight/kD	等电点 pI	亲水性总平均值 Grand average of hydrophilicity	蛋白长度 Protein length/aa	亚细胞定位 Subcellular location
HvNPC1	HORVU.MOREX.r3.7HG0752530.1	7H:631001620-631003350	53.12	5.22	-0.317	477	细胞质
HvNPC2	HORVU.MOREX.r3.1HG0085590.1	1H:496122549-496124474	61.33	6.53	-0.364	553	叶绿体
HvNPC3	HORVU.MOREX.r3.3HG0232030.1	3H:28367740-28369602	58.77	8.83	-0.204	532	叶绿体
HvNPC4	HORVU.MOREX.r3.3HG0322080.1	3H:600541735-600546962	59.07	5.98	-0.424	533	液泡膜
HvNPC5	HORVU.MOREX.r3.5HG0527960.1	5H:566994044-566997667	60.76	6.80	-0.367	545	叶绿体

Fig. 1 Phylogenetic tree of NPC gene family in different species

Fig. 2 Structural features of NPC gene family in barely A: Purple bars indicate the exon, lines indicate the intron while grey color bars indicate the un-translated region（UTR）. B: The yellow bar indicates the signature domain phosphoesterase（PF04185）

Fig. 3 Cis-acting elements in the promoters of HvNPC gene family members ABRE: Abscisic acid responsive element. CGTCA-motif, TGACG-motif: MeJA responsive element. GARE-motif, P-box, TATC-box: Gibberellin responsive element. TGA-element: Auxin responsive element. TCA-element: Salicylic acid responsive element. MBS, MYC, MYB: Drought responsive element. TC-rich: Defense and stress responsive element. LTR: Low-temperature responsive element. ARE: Essential for the anaerobic responsive element. O2-site: Zein metabolism regulatory element. RY-element: Seed-specific regulatory element. Sp1, TCT-motif, AE-box, BoxII, I-box: Light responsive element

Fig. 4 Expression profile of NPC gene in 14 tissues and organs of barley A: Embryo（4 d after germination）. B: Roots from the seedlings（shoot length 10 cm）. C: Shoots（10 cm）. D: Etiolated seedling （10 d old at dark）. E: Developing tillers（six leaf stge, 3rd internode）. F: Root（4 weeks）. G: Epidermis（4 weeks）. H: Florescences（5 mm）. I: Florescences（1-1.5 cm）. J: Rachis（5 weeks）. K: Lemma（6 weeks）. L: Lodicule（6 weeks）. M: Palea（6 weeks）. N: Senescing leaf（2 weeks）

Fig. 5 Relative expression pattern of HvNPCs genes under low temperature, NaCl, and drought treatments A: Gene expression profiles of HvNPCs under 4℃ treatment. B: Gene expression profiles of HvNPCs under NaCl treatment. C: Gene expression profiles of HvNPCs under PEG treatment. Different lower letters indicate significant differences（P<0.05）

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