Identification and Expression Analysis of Tomato SA Signaling Pathway Genes in Response to Root Knot Nematode MiPDCD6 Protein Based on RNA-seq

doi:10.7668/hbnxb.20193473

Abstract

Abstract:

In order to reveal the molecular mechanism of MiPDCD6 protein suppressing tomato PTI immunity,the MiPDCD6 overexpression seedlings of tomato variety Xinjinfeng 1 were used as experimental materials,and the tissue culture seedlings of tomato variety Xinjinfeng 1 were used as control.Transcriptome sequencing was performed on tomato MiPDCD6 overexpressing seedlings and control seedlings,respectively.With tomato cultivars Heinz 1706,comparing the genome as a reference genome,FPKM method was used to calculate quantity of gene expression,set parameters(|log₂FC|>1 and P<0.05)in screening the differentially expressed genes(DEGs).Gene ontology(GO)database was used to analyze the GO functional enrichment of DEGs,count the number of DEGs in each GO term,calculate the significance of gene enrichment,and find out the functional term with significant enrichment.KEGG database was used for Pathway enrichment analysis of DEGs,and hypergeometric distribution test was used to calculate the significance of enrichment of DEGs in each Pathway.The enrichment degree of KEGG was measured by FDR and gene number.Combined with gene differential expression analysis and functional enrichment analysis,the effect of MiPDCD6 protein on tomato PTI immune-related pathway genes was studied.The results showed that there were 2 366 DEGs in MiPDCD6 overexpressed tomato plants compared with wild-type tomato,including 1 354 up-regulated genes and 1 012 down-regulated genes.In these DEGs,a large number of differentially expressed genes were concentrated in KEGG pathways such as plant hormone signal transduction(sly4075),plant-pathogen interaction(sly04626),plant MAPK signal pathway(sly04016)and procycloid biosynthesis(sly00940)through GO and KEGG annotation.SA biosynthesis pathways included ICS and PAL.In the MiPDCD6 overexpressed tomato plants,PAL1 and PAL-like genes in SA synthesis pathways and TGA9,TGA10-like and PR1a2 genes in SA signal transduction pathways were significantly down-regulated,suggesting that MiPDCD6 may inhibit SA synthesis thus inhibiting plant PTI immunity.

Key words: Tomato, Root knot nematode, Transcriptome, Differentially expressed genes, SA signal transduction pathways, MiPDCD6

DENG Xiaoda, YUAN Yongqiang, CAI Shujing, ZHENG Lijun, XU Chunling, WANG Xinrong. Identification and Expression Analysis of Tomato SA Signaling Pathway Genes in Response to Root Knot Nematode MiPDCD6 Protein Based on RNA-seq[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(1): 184-195. doi: 10.7668/hbnxb.20193473.

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Figures/Tables 14

Tab.1 Primers used for RT-qPCR

基因名称 Gene name	引物序列(5'—3') Primer sequences(5'—3')	参考文献 References
PAL1	F: GTCAAGGCAGCTCAGAAGCT	[23]
	R: GCAAGGAATTGAAGTTCTGAGC
TGA9	F: ACGAGCAAAGCAGACGAAGA	本研究
	R: AAACTTGCCAGGGTACCACC
TGA10-like	F: GGCTTTGTTAGACAGGCGGA	本研究
	R: TATTCATGTCGAGGCCGAGC
WRKY33B	F: AATGGCTTCTTCAGGTGGAAA	本研究
	R: AAGGCGAAGCAGGTGATGTT
RIN4	F: ACCACTTCACCCCAATTCCC	本研究
	R: TCATTGTCATCCCATCCGCC
β-Actin	F: TGTCCCTATTTACGAGGGTTATGC	[24]
	R: CAGTTAAATCACGACCAGCAAGAT

Tab.2 Transcriptome sequencing data of MiPDCD6 overexpression tomato

样本名 Sample name	净读数 Clean reads	序列长度/bp Sequence length	GC含量/% GC	Q20/%	Q30/%
WT1	24 143 599	150	42	99.991 20	98.645 45
WT2	23 297 585	150	42	99.991 13	98.709 50
OE1	16 287 433	150	42	99.988 38	98.524 16
OE2	19 089 630	150	42	99.990 79	98.501 17

Tab.3 Statistical analysis of transcriptome sequencing genes from MiPDCD6 overexpressed tomato

样本 Sample	已知基因数 Number of known genes	新基因数 Number of novel genes	已知转录本数 Number of known transcripts	新转录本数 Number of novel transcripts
WT1	22 490	1 791	31 497	13 337
WT2	22 473	1 774	31 296	13 129
OE1	22 556	1 811	31 929	13 100
OE2	23 024	1 824	32 884	13 114

Fig.1 Detection of gene expression level of transcriptome from MiPDCD6 overexpression tomato A.The number of gene expression within five ranges of FPKM value of gene expression level;B.Boxplot of log₂(FPKM+1)values of gene expression levels among 4 samples.

Fig.2 Correlation analysis of transcriptome sequences from four samples related to MiPDCD6 overexpression tomato A.Spearman correlation heat map;B.Pearson correlation heat map.

Fig.3 Analysis of differentially expressed genes of transcriptome from MiPDCD6 overexpression tomato A.Statistical histogram of differentially expressed genes.B.Volcano map of differentially expressed genes:red represents up-regulated genes,blue represents down regulated genes,gray represents genes with no significant differential expression,each point represents a gene,and the abscissa is the logarithm of the difference multiple of the expression of a certain gene in the two samples(log₂ (FC));the ordinate is the negative logarithm(-log₁₀(P-value))of the statistical significance of the change in gene expression.C.For differential gene cluster analysis,each column represents a sample,each row represents a DEG,orange represents up regulation,and blue represents down regulation.

Fig.4 GO term analysis of different expressed genes in the transcriptom from MiPDCD6 overexpressed tomato

Tab.4 Results of KEGG pathway enrichment analysis of DEG from MiPDCD6 overexpression tomato plant

通路号 KO ID	通路名称 Pathway name	基因比例 Gene ratio	校正后P值 P-adjust
sly00196	光合作用-天线蛋白	21/934	0.000 000 000 487 6
sly00195	光合作用	41/934	0.000 000 003 452 6
sly04016	植物MAPK信号通路	67/934	0.000 000 190 593 6
sly04626	植物-病原互作	70/934	0.000 007 169 939 6
sly04075	植物激素信号转导	72/934	0.003 692 774 659 5
sly00860	卟啉和叶绿素代谢	18/934	0.024 461 163 393 7
sly00561	甘油酯代谢	24/934	0.059 662 578 388 4
sly00940	苯丙酸生物合成	56/934	0.060 904 395 160 7
sly00564	甘油磷脂代谢	29/934	0.068 028 570 366 4
sly00270	半胱氨酸和蛋氨酸代谢	31/934	0.153 741 213 184 3
sly00073	角质、软木脂和蜡的生物合成	12/934	0.153 741 213 184 3
sly00910	氮代谢	11/934	0.227 817 972 577 2
sly00400	苯丙氨酸酪氨酸和色氨酸	14/934	0.325 873 798 172 2
sly00906	类胡萝卜素生物合成	11/934	0.338 278 128 256 7
sly00920	硫代谢	11/934	0.338 278 128 256 7
sly03440	同源重组	15/934	0.338 278 128 256 7
sly00500	淀粉和蔗糖代谢	35/934	0.345 821 690 083 9
sly00450	硒化合物代谢	6/934	0.430 445 770 585 5

Fig.5 Analysis of KEGG pathway of differential expression gene from MiPDCD6 overexpressed tomato plant Functional analysis point diagram of differentially expressed gene KEGG pathway;P-adjust is the corrected P-value,the smaller the P-adjust value the more significant the enrichment.

Fig.6 DEG heat map of plant pathogen interaction pathway in MiPDCD6 overexpression tomato

Fig.7 DEG heat map of biosynthesis pathway of phenylalanine,tyrosine and tryptophan in MiPDCD6 overexpressed tomato

Fig.8 DEG heat map of phenylpropionic acid biosynthesis KEGG pathway in overexpressing MiPDCD6 tomato

Fig.9 DEG heat map of hormone signaling pathways in MiPDCD6 overexpressed tomato

Fig.10 RT-qPCR of some differently expressed genes from MiPDCD6 overexpression tomato Data were analyzed by SPSS software for significance;a,b and c indicated significant difference at P<0.05(Duncan's test).

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