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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (11): 2949-3295.doi: 10.3724/SP.J.1006.2023.24224

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

Identification and relative expression pattern of PLA1 gene family in flax

ZHAO Li-Rong(), LI Wen, WANG Li-Min, QI Yan-Ni, LI Wen-Juan, XIE Ya-Ping, DANG Zhao, ZHAO Wei, ZHANG Jian-Ping()   

  1. Institute of Crops, Gansu Academy of Agricultural Sciences, Lanzhou 730070, Gansu, China
  • Received:2022-10-09 Accepted:2023-04-17 Online:2023-11-12 Published:2023-05-23
  • Supported by:
    Modern Biology Breeding Project of Gansu Academy of Agricultural Sciences(2020GAAS08);Modern Biology Breeding Project of Gansu Academy of Agricultural Sciences(2022GAAS04);China Agriculture Research System (Oil) of MOF and MARA, the Science and Technology Project of Gansu Province(21JR7RA722);China Agriculture Research System (Oil) of MOF and MARA, the Science and Technology Project of Gansu Province(21JR1RA354);Major Science and Technology Project of Gansu Province(21ZD4NA022-02);Science and Technology Planing Project of Anning District(2022-JB-10)

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Abstract:

Phospholipase A1 (PLA1) plays an important role in plant growth and development and stress responses. However, there is no function study of PLA1 in flax. In this study, we identified the PLA1 gene family in seven species including flax by bioinformatics, and analyzed the sequence characteristics, phylogenetic evolution, cis-acting elements, collinearity, and replication events of PLA1 (LuPLA1) genes in flax. Transcriptome data were used to analyze the relative expression patterns in different genetic backgrounds and organs, and qRT-PCR was used to analyze the relative expression patterns in different tissues and developmental stages, and under different stress treatments. The results showed that there were 14, 21, 15, 20, 41, 18, and 35 PLA1 members identified in flax, Arabidopsis, maize, rice, soybean, castor, and cassava, respectively. LuPLA1 were distributed on 8 chromosomes. Sequence analysis showed that except for LuPLA1-7 and LuPLA1-10, all the other members had introns and most of them had one intron, with protein length of 388-1759 aa, isoelectric point of 5.99-9.19, and molecular weight of 41.55-192.61 kD. All the LuPLA1 proteins were hydrophilic, and most of them localized in vacuoles and contained 4-25 motifs. Phylogenetic analysis revealed that the PLA1 proteins were divided into four clades, and clade IV had the largest number of PLA1 members. Collinearity analysis indicated that LuPLA1 had homologous genes in Arabidopsis, maize, rice, cassava, and soybean, and had the most homologous genes with soybean and cassava. There were two tandem duplicated gene pairs and seven segment duplicated genes pairs of LuPLA1 family members, and all the duplicated genes underwent purification selection. Transcriptome analysis showed that most LuPLA1 members exhibited organ-specific expression patterns. The promoter regions of LuPLA1 contained a large number of hormone and stress response elements. The qRT-PCR further confirmed that the relative expression of LuPLA1 genes were induced by hormone, drought, high salt, low temperature, and high temperature. LuPLA1-1 was induced by IAA. LuPLA1-1 and LuPLA1-6 were induced by NAA. LuPLA1-1, LuPLA1-5, and LuPLA1-6 were induced by GA3. LuPLA1-12/14 was induced by NaCl and PEG. Except for LuPLA1-2/4, the other genes were induced by high temperature. All LuPLA1 members were induced by low temperature, and the response to low temperature was the most obvious. This study laid a foundation for further analysis of the function of LuPLA1 gene family.

Key words: flax, LuPLA1, gene family, the relative expression pattern

Table S1

Primers for qRT-PCR"

基因名称
Gene name		 上游引物
Forward primer (5′-3′)		 下游引物
Reverse primer (5′-3′)
GAPDH CTTTACCCTCAGCAAATCCG AGGTTCTTCCCGCTCTCAAT
PLA1-1 TCTTCCCTCAGAACAGAGGTGTATTT AGTCTTGTTGGACCCTGTCCCTAG
PLA1-2/4 CCTCTTCTCGTCCCTTGCTTTGA GGAGAAGAGCAGGGAACGAAACT
PLA1-3/11 ATTTCCGTTTATGGGAGGTTTGCC TAAAGGCAAATACCCTCCAAACGG
PLA1-5 CTTGATGGGGAGAATGTGAGGA GTTGGAGCTGCGGAAGTTGATA
PLA1-6 GCTTGATGGGAAGGATGTGAGGAT AGAGGGCGTTTGTTGTCATTTGGT
PLA1-7/10 CGAATTGACCCTAAAGGACACG GCTTAACTGGGATTTCCTGTGC
PLA1-8/9 GAACAGCGACGAATAATCCAACG CTTGTCGCTGCTTATTAGGTTGC
PLA1-12/14 ATGGGAGGTTTGCCGGGCTAC TACCCTCCAAACGGCCCGATG

Table S2

Accession numbers of transcriptome data"

名称
Name		 登录号
Accession number		 名称
Name		 登录号
Accession number
Longya-10-fruit-1 SRR8281918 Heiya-14-fruit-1 SRR8281912
Longya-10-fruit-2 SRR8281919 Heiya-14-fruit-2 SRR8281913
Longya-10-stem-1 SRR8281920 Heiya-14-stem-1 SRR8281916
Longya-10-stemt-2 SRR8281921 Heiya-14-stem-2 SRR8281917

Fig. 1

Chromosome location of LuPLA1 genes The orange line indicates segmental duplication, and the red rectangle indicates tandem duplication."

Table 1

Duplication analysis of LuPLA1 genes in flax"

基因对
Gene pairs		 复制事件
Duplication events		 同义替换率
Ks		 非同义替换率
Ka		 Ka/Ks 选择类型
Selection type		 分歧时间
Divergence time (Mya)
LuPLA1-7/LuPLA1-8 串联复制
Tandem duplication		 0.5411 0.1419 0.2622 纯化选择
Purifying selection		 44.3525
LuPLA1-9/LuPLA1-10 串联复制
Tandem duplication		 0.5093 0.1448 0.2843 纯化选择
Purifying selection		 41.7459
LuPLA1-2/LuPLA1-4 片段复制
Segmental duplication		 0.1189 0.0213 0.1791 纯化选择
Purifying selection		 9.7459
LuPLA1-3/LuPLA1-11 片段复制
Segmental duplication		 0.1217 0.0061 0.0501 纯化选择
Purifying selection		 9.9754
LuPLA1-3/LuPLA1-12 片段复制
Segmental duplication		 1.0065 0.0692 0.0687 纯化选择
Purifying selection		 82.5000
LuPLA1-7/LuPLA1-9 片段复制
Segmental duplication		 0.5776 0.1400 0.2424 纯化选择
Purifying selection		 47.3443
LuPLA1-11/LuPLA1-12 片段复制
Segmental duplication		 0.9443 0.0758 0.0803 纯化选择
Purifying selection		 77.4016
LuPLA1-11/LuPLA1-14 片段复制
Segmental duplication		 0.9403 0.0770 0.0819 纯化选择
Purifying selection		 77.0738
LuPLA1-12/LuPLA1-14 片段复制
Segmental duplication		 0.0845 0.0135 0.1598 纯化选择
Purifying selection		 6.9262

Fig. 2

Collinearity of LuPLA1 genes in flax and its collinearity with other species"

Table 2

Basic characteristics of LuPLA1 family members"

基因名称
Gene name		 基因号
Gene ID		 染色体位置
Chromosome
location		 外显子数量
No. of
exons		 蛋白序列长度
Protein
length (aa)		 分子量
Molecular
weight (kD)		 等电点
Isoelectric point		 亚细胞定位
Subcellular
location
LuPLA1-1 L.us.o.m.scaffold73.66 Chr1:21780419-
21783081(-)		 7 459 50.96 5.99 液泡
Vacuole
LuPLA1-2 L.us.o.m.scaffold17.61 Chr5:5454111-
5456030(-)		 2 388 41.55 8.08 液泡
Vacuole
LuPLA1-3 L.us.o.m.scaffold255.68 Chr5:17092681-
17095829(-)		 2 463 50.92 8.00 液泡
Vacuole
LuPLA1-4 L.us.o.m.scaffold11.49 Chr6:4217828-
4219482(+)		 2 390 41.81 7.52 液泡
Vacuole
LuPLA1-5 L.us.o.m.scaffold100.2 Chr7:7572800-
7576425(+)		 7 400 44.50 6.97 液泡
Vacuole
LuPLA1-6 L.us.o.m.scaffold284.7 Chr7:7630026-
7633875(+)		 7 391 43.23 6.92 液泡
Vacuole
LuPLA1-7 L.us.o.m.scaffold219.29 Chr8:17228540-
17230314(+)		 1 483 52.59 9.19 细胞膜, 液泡
Cell membrane,
vacuole
LuPLA1-8 L.us.o.m.scaffold219.27 Chr8:17242561-
17243859(+)		 2 421 45.31 8.99 液泡
Vacuole
LuPLA1-9 L.us.o.m.scaffold253.44 Chr11:1866126-
1867424(-)		 2 421 45.30 8.99 液泡
Vacuole
LuPLA1-10 L.us.o.m.scaffold253.45 Chr11:1884093-
1885373(-)		 1 426 45.90 9.15 叶绿体, 液泡
Chloroplast,
vacuole
LuPLA1-11 L.us.o.m.scaffold144.142 Chr11:13906282-
13908918(-)		 2 463 50.94 7.55 叶绿体, 液泡
Chloroplast,
vacuole
LuPLA1-12 L.us.o.m.scaffold263.24 Chr12:5589259-
5592930(+)		 2 453 49.49 7.01 液泡
Vacuole
LuPLA1-13 L.us.o.m.scaffold220.83 Chr15:8920678-
8929774(-)		 28 1759 192.61 6.80 液泡
Vacuole
LuPLA1-14 L.us.o.m.scaffold0.620 Chr15:11287238-
11289213(-)		 2 452 49.43 6.97 叶绿体, 液泡
Chloroplast,
vacuole

Fig. S1

Prediction of hydrophilicity and hydrophobicity of PLA1 gene family in flax"

Fig. S2

Secondary structure of LuPLA1 encoding proteins"

Fig. 3

Phylogenetic tree of PLA1 family in different species"

Fig. 4

Phylogenetic tree, conserved motif, and gene structure of LuPLA1 gene family"

Fig. 5

Cis-acting elements in promoters of LuPLA1 family members"

Fig. 6

Heat map of the relative expression pattern of LuPLA1 genes in different varieties and organs"

Fig. S3

Relative expression pattern of LuPLA1 genes in seeds and anther at different developmental stages of Longya 15 (L15)"

Fig. 7

Relative expression pattern of LuPLA1 genes under IAA (A) and NAA (B) treatments Different lowercase letters mean significant differences at the 0.05 probability level."

Fig. 8

Relative expression pattern of LuPLA1 genes under GA3 (A) and NaCl (B) treatments Different lowercase letters mean significant differences at the 0.05 probability level."

Fig. 9

Relative expression pattern of LuPLA1 genes under PEG (A) and different temperature (B) treatments Different lowercase letters mean significant differences at the 0.05 probability level."

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