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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (6): 1070-1081.doi: 10.3724/SP.J.1006.2021.04133

• SPECIAL SECTION: GENOMICS AND GENETIC IMPROVEMENT IN MAIN BAST FIBER CROPS • Previous Articles     Next Articles

Transcriptome profiling of flax (Linum usttatissimum L.) response to low potassium stress

HUANG Wen-Gong1(), JIANG Wei-Dong1, YAO Yu-Bo1, SONG Xi-Xia1, LIU Yan1, CHEN Si1, ZHAO Dong-Sheng1, WU Guang-Wen1, YUAN Hong-Mei1, REN Chuan-Ying2, SUN Zhong-Yi3, WU Jian-Zhong4, KANG Qing-Hua1,*()   

  1. 1Institute of Industrial Crops, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang, China
    2Food Processing Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang, China
    3Institute of Animal Husbandry Research, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang, China
    4Institute of Forage and Grassland Science, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, Heilongjiang, China
  • Received:2020-06-22 Accepted:2020-11-13 Online:2021-06-12 Published:2020-12-15
  • Contact: KANG Qing-Hua E-mail:huangwengong1736@163.com;qinghuakang111@163.com
  • Supported by:
    The National Key Research and Development Program of China(2018YFD0201100);The Heilongjiang Province Modern Agricultural Industry Technology Collaborative Innovation System-Hemp (medicinal) Resources Genetic Improvement and Innovative Utilization Collaborative Innovation Post(YYM19SQ-24);The China Agriculture Research System(CARS-16-E04);The Science and Technology Innovation Project of Heilongjiang Academy of Agricultural Sciences(2019JCQN003);The Science and Technology Innovation Project of Heilongjiang Academy of Agricultural Sciences(HNK2019CX08-05)

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Abstract:

Potassium (K) is an essential element for the growth and development in flax. Transcriptome sequencing and qRT-PCR were used to investigate the regulation of differential gene expression after 12 h and 96 h of low-K + treatment. The results showed that the leaf edge of flax treated with low-K + for 7 days turned yellow, and the plants were dwarfed compared with the control. LusKC1 (Lus K channel 1), LusSKOR (Lus STELAR K + outward rectifier) and LusHAK5 (Lus high affinity K + transporter 5) were detected to respond to low-K + with response peak time of 12 h and 96 h. Compared with the control, 1154 differentially expressed genes (DEGs) (508 up-regulated and 646 down-regulated genes) were identified in low-K + treatment for 12 h. GO enrichment showed that DEGs were mainly concentrated on five categories: metabolic process, cellular process, single biological process, catalytic activity and binding function. KEGG pathway enrichment showed that DEGs involved in energy metabolism, carbohydrate metabolism, carbon metabolism, amino acid metabolism, terpenoid metabolism and plant hormone signal transduction pathways. Furthermore, 7 genes directly related to K (4K transporters, 2K channel proteins and 1 sodium-potassium-calcium exchanger protein), 13 genes related to hormone and 6 genes related to cellulose synthesis were screened. Among the 7 genes directly related to K, the relative expression of 2 genes were up-regulated by 1.75 and 2.64 times and 5 genes down-regulated by 1.21-9.57 times. In summary, DEGs preliminarily revealed the transcriptional regulation pathway involved in low-K + in flax, which laid a foundation for cloning and functional verification of flax low-K + tolerance related genes.

Key words: flax, low potassium, transcriptome profiling, differentially expressed genes

Table 1

Primers used for qRT-PCR in this study"

引物名称
Primers		 正向引物
Forward primers (5'-3')		 反向引物
Reverse primers (5'-3')
LusKC1-1 CATTGTCCTCACTTTCTTTGTCGC CGAACCATGGCTTAGTGAGATACC
LusKC1-2 CATTGTCCTCACTTTCTTTGTCGC TAGATGAACTGAAACGGTAGGGTG
LusKC1-3 CGACTTGGCGGTGGATGCTT TAGATGAACTGAAACGGTAGGGTG
LusSKOR-1 GTCTCGTCATTCATCCCGACAACA CCAACAATGTCCAAAATGAAGAGAT
LusSKOR-2 GTCTCGTCATTCATCCCGACAACA TAATCCCCTGAAGAATCCAAACTCC
LusSKOR-3 TTCCATCACGTCTCCACCGGT TAATCCCCTGAAGAATCCAAACTCC
LusAKT2-1 TTCATAAACGGAGGTCATCTCTTCT GACAATCTCAGCAGCATAAAGCCTC
LusAKT2-2 TTCATAAACGGAGGTCATCTCTTCT TCAGGCTTAAGATCTCGATGCACAA
LusAKT2-3 GTCATCCATCCTTTCATCGTCCAGC GACAATCTCAGCAGCATAAAGCCTC
LusHAK5-1 GTTTCCAAGACAACACAAGGCG GATGTTCCGATGTCGCCGTA
LusHAK5-2 GTTTCCAAGACAACACAAGGCG ATGTCGCCGTAGACCACTCCA
LusHAK5-3 CCAAGACAACACAAGGCGGC GATGTTCCGATGTCGCCGTA
LusKUP2-1 TGTTCAAGTATGTGTTCATTGTTCT GGACAATGATTCATCTGAGACCTGC
LusKUP2-2 TGTTCAAGTATGTGTTCATTGTTCT TGATTCATCTGAGACCTGC
LusKUP2-3 GCTGATGACAATGGAGAGGGT GCAAACAAGGCTACCAGTATGAT
LusKUP3-1 CACCCCTGCCCTTTCTGTTT TAGATGAACTGAAACGGTAGGGTG
LusKUP3-2 CACCCCTGCCCTTTCTGTTT TATGATGCATGTAGCTGGGAGT
LusKUP3-3 CTTGCTGGTTTTGGCGTT GCAAACAAGGCTACCAGTATGAT
LusKUP12-1 TGGTGGGACTATGAGGAGGAG CCATGTCGCCATAGACCACT
LusKUP12-2 TGGTGGGACTATGAGGAGGAG ACTCCAAGCGTTTGAAAGGC
LusKUP12-3 AGGAGGAGGTTGGTGAAGAAG CCATGTCGCCATAGACCACT
LusKEA5-1 ATGAGAAGGGCAAGAAAAATGA GACGTTATCCTTTTTGTCAATGA
LusKEA5-2 ATGAGAAGGGCAAGAAAAATGA CAGCACAGGATATTTAGATTTCTTG
LusKEA5-3 AGGGCAAGAAAAATGACACACA GACGTTATCCTTTTTGTCAATGA
LusCHX17-1 CTCACTCGCATCCTCGCTTT ACTGTGAGAGACTTATGTGGGAA
LusCHX17-2 CTCACTCGCATCCTCGCTTT TATGTGGGAAGATAGTGTTGAGG
LusCHX17-3 ATCCTCGCTTTCCTCCTC ACTGTGAGAGACTTATGTGGGAA
LusEF1A GCTGCCAACTTCACATCTCA GATCGCCTGTCAATCTTGGT
LusUBI CCTCCTTGATAGCAGCCTTG CTCCGTGGAGGTATGCAGAT
LusTUA CCTGTTGGGAGCTTTACTGC AAGGTGTTGAAGGCATCGTC
LusEF2 GTGGTGCTGAGATCACGAAA AGACGGTTATGCTTGTTGGG
LusActin GGTGTTATGGTTGGAATGGGTC CCTCAGTGAGAAGTACAGGGTG

Fig. 1

Plant phenotype of before and after treatment of low-K+ in flax A: growth in vermiculite; B: plant phenotype before treatment; C: growth condition of control and treatment under low-K+ stress; D: plant phenotypes of low-K+ and control plants after treatment; E: single plant phenotype of low-K+ and control after treatment. a: plant phenotype under low-K+; b: plant phenotype of control."

Fig. 2

Screening of marker genes for low-K+ stress in flax M: DL2000; 1: LusKC1-1; 2: LusKC1-2; 3: LusKC1-3; 4: LusSKOR-1; 5: LusSKOR-2; 6: LusSKOR-3; 7: LusAKT2-1; 8: LusAKT2-2; 9: LusAKT2-3; 10: LusHAK5-1; 11: LusHAK5-2; 12: LusHAK5-3; 13: LusKUP2-1; 14: LusKUP2-2; 15: LusKUP2-3; 16: LusKUP3-1; 17: LusKUP3-2; 18: LusKUP3-3; 19: LusKUP12-1; 20: LusKUP12-2; 21: LusKUP12-3; 22: LusKEA5-1; 23: LusKEA5-2; 24: LusKEA5-3; 25: LusCHX17-1; 26: LusCHX17-2; 27: LusCHX17-3; 28: LusEF1A; 29: LusUBI; 30: LusTUA; 31: LusEF2; 32: LusActin."

Fig. 3

Effects of different time on the relative expression of LusKC1, LusSKOR, and LusHAK5 under low-K+ treatment in flax Different lowercase letters mean significant differences at the 0.05 probability level during the same gene."

Table 2

Main characteristics of 12 transcriptome data in flax"

样品
Sample		 总的原始读数
Total raw reads (Mb)		 总的过滤读数
Total clean reads (b)		 比对读数
Compared reads		 比对特异性
Compared specificity (%)		 比对基因Compared genes
12h-CK-1 38.07 36,976,258 32,381,211 87.57% 82.01 35,281
12h-CK-2 38.07 36,547,552 31,737,232 86.84% 81.82 34,782
12h-CK-3 38.07 36,309,778 31,468,060 86.67% 80.67 35,320
12h-KS-1 38.07 36,507,590 32,071,565 87.85% 83.38 35,352
12h-KS-2 38.07 36,763,226 32,059,054 87.20% 81.62 35,351
12h-KS-3 38.07 36,513,816 31,749,080 86.95% 81.41 35,429
96h-CK-1 38.07 36,886,386 32,109,249 87.05% 81.79 35,608
96h-CK-2 38.07 36,515,094 31,576,196 86.47% 81.72 35,270
96h-CK-3 38.07 36,494,576 31,449,058 86.17% 81.15 35,260
96h-KS-1 38.07 36,956,330 32,146,308 86.98% 81.33 35,807
96h-KS-2 38.07 36,737,316 32,133,744 87.47% 82.20 36,263
96h-KS-3 38.07 36,409,348 31,493,300 86.50% 81.31 35,141

Fig. 4

Venn diagram of DEGs under low-K+ in flax A: the number of DEGs at 12 h and 96 h after low-K+ treatment in flax; B: the number of DEGs in control and low-K+ treatment."

Fig. 5

GO enrichment of the DEGs under low-K+ treatment after 12 h in flax"

Fig. 6

KEGG pathway enrichment of the DEGs under low-K+ treatment after 12 h in flax"

Table 3

Screening of 7 genes directly related to low-K+ treatment in flax"

基因收录号
Gene ID		 基因名称
Gene name		 KS/CK的log2值
log2 FC		 注释
Annotation
MSTRG.20565.1 Potassium transporter 5-like 2.64 钾转运蛋白Potassium transporter
MSTRG.24915.2 Potassium transporter, putative -1.21 钾转运蛋白Potassium transporter
MSTRG.10817.1 Hypothetical protein EUGRSUZ_E04300 -1.23 钾转运蛋白Potassium transporter
MSTRG.6817.1 Potassium transporter 7-like -9.57 钾转运蛋白Potassium transporter
MSTRG.4695.1 Uncharacterized protein isoform 2 1.75 钾电压通道Potassium voltage-gated channel
MSTRG.30540.1 Potassium channel SKOR-like isoform X1 -1.40 钾通道Potassium channel
MSTRG.14498.2 Nucleolar protein nop56, putative -3.01 钠钾钙交换器Sodium potassium calcium exchanger

Fig. S1

Signal transduction and ion transporter regulation in responses to low-K+ stress in flax [22] a: plant phenotype of low-K+ treatment; b: plant phenotype of control. Plants are able to perceive external low-K+ stress and generate low-K+ signals in plant cells. The signals (Ca2+, ROS, etc.) can be transducted in cytosol, and eventually regulate the downstream targets (particularly K+ channels and transporters) at transcriptional and posttranslational levels. P represents phosphorylation and X indicates inhibition effect."

Table 4

Screening of 13 hormone related genes under low-K+ treatment in flax"

激素种类
Hormone kinds		 基因登录号
Gene ID		 基因名称
Gene name		 KS/CK的log2值
log2 FC		 注释
Annotation
Auxin MSTRG.4702.1 AUX -1.44 生长素诱导蛋白Auxin-induced protein
MSTRG.13772.1 AUX -1.51 生长素诱导蛋白Auxin-induced protein
MSTRG.7397.8 TIR 8.52 Toll/interleukin-1受体Toll/interleukin-1 receptor
MSTRG.6782.4 ARF 9.62 含家族蛋白的ARF-GTPase激活域
ARF GTPase-activating domain-containing family protein
MSTRG.29326.1 GH3 -3.02 吲哚-3-乙酸酰胺合成酶GH3
Indole-3-acetic acid-amido synthetase GH3
MSTRG.14003.4 GH3 -3.58 吲哚-3-乙酸酰胺合成酶GH3
Indole-3-acetic acid-amido synthetase GH3
Cytokinine MSTRG.25736.1 AHP1 -1.81 含组氨酸磷酸转移蛋白1
Histidine-containing phosphotransfer protein 1
Ethylene MSTRG.16160.5 CTR1 -6.23 丝氨酸/苏氨酸蛋白激酶CTR1亚型X1
Serine/threonine-protein kinase CTR1 isoform X1
MSTRG.19624.1 CTR1 -1.90 丝氨酸/苏氨酸蛋白激酶CTR1亚型X1
Serine/threonine-protein kinase CTR1 isoform X1
MSTRG.15232.4 CTR1 -7.72 丝氨酸/苏氨酸蛋白激酶CTR1亚型X1
Serine/threonine-protein kinase CTR1 isoform X1
MSTRG.36331.6 EIN2 -10.02 假定蛋白Hypothetical protein
MSTRG.40444.1 EBF1 -1.20 假定蛋白Hypothetical protein
MSTRG.12066.1 EBF1 -3.10 假定蛋白Hypothetical protein

Fig. S2

Genes related to auxin, cytokinin and ethylene synthesis pathway under low-K+ in flax [23] a: plant phenotype of low-K+ treatment; b: plant phenotype of control. Rectangular boxes indicate proteins be involved in these pathways. Protein-protein interactions are represented by arrows. Arrows represent an activation, bar-headed lines an inhibition, crossed lines a dissociation, dotted arrows an indirect effect, +u denotes ubiquitination."

Table 5

Screening of 6 genes directly related to cellulose under low-K+ in flax"

基因收录号
Gene ID		 基因名称
Gene name		 KS/CK的log2值
log2 FC		 注释
Annotation
MSTRG.20574.4 Exo70 -8.70 胞外复合蛋白Exocyst complex protein
MSTRG.29262.1 Exo70 -2.13 胞外复合蛋白Exocyst complex protein
MSTRG.16464.2 COB23 -2.03 外被体亚单位Coatomer beta subunit
MSTRG.16464.1 COB23 -1.73 外被体亚单位Coatomer beta subunit
MSTRG.18050.1 COB21 -1.94 外被体亚单位Coatomer beta subunit
MSTRG.5636.3 FEI1 -1.86 双特异性蛋白激酶Dual specificity protein kinase

Fig. S3

Genes related to cellulose synthesis pathway under low-K+ in flax [24] "

Fig. 7

Correlations between RNA-Seq and qRT-PCR"

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