Carboxymethyl chitosan-induced transcriptome WRKY genes analysis and resistance-related gene mining in grapefruit fruit

doi:10.3969/j.issn.1004-1524.2023.03.13

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (3): 598-614.DOI: 10.3969/j.issn.1004-1524.2023.03.13

• Horticultural Science • Previous Articles Next Articles

Carboxymethyl chitosan-induced transcriptome WRKY genes analysis and resistance-related gene mining in grapefruit fruit

YANG Qing¹(), LIU Shenghong¹, HUANG Erbin¹, DU Rongyu¹, WANG Fang¹^,², DENG Jia¹^,³^,^*()

1. College of Forestry, Southwest Forestry University, Kunming 650224, China
2. Key Laboratory of National Forestry and Grassland Administration on Biodiversity Conservation in Karst Mountainous Areas of Southwest China, Kunming 650224, China
3. Key Laboratory of Forest Resources Conservation and Utilization in the Southwest Mountains of China, Ministry of Education, Kunming 650224, China

Received:2022-09-16 Online:2023-03-25 Published:2023-04-07

Abstract

Abstract:

In this experiment, postharvest grapefruit fruits were treated with a 1.5% carboxymethyl chitosan (CMCS) solution and sterile water for 24 hours to study their resistance to green mould caused by Penicillium digitatum and Penicillium expansum. Results showed that the CMCS treatment group significantly reduced the incidence of green mould caused by P. digitatum and P. expansum and inhibited the growth rate of spot diameter.Non-reference transcriptome sequencing was conducted on CMCS and sterile water-treated grapefruit peels to screen for CMCS-induced differentially expressed genes (DEGs). A total of 85 DEGs were obtained, including 67 up-regulated genes and 18 down-regulated genes. GO and KEGG analyses were performed on the DEGs, and their functions were categorized into 37 GO entries in three major categories: biological processes, cellular components, and molecular functions. These DEGs were significantly enriched in 20 KEGG metabolic pathways. Furthermore, WRKY genes were mined, and five WRKY genes were screened as the most likely to be involved in the plant disease resistance process. All five WRKY genes were up-regulated, and the evolutionary tree analysis with Arabidopsis showed that the five WRKY genes were functionally similar to those of Arabidopsis genes. The functions of the involved DEGs were mainly related to secondary metabolism, phytohormone signaling, peroxisome, protein processing in the endoplasmic reticulum, glutathione metabolism, and phenylpropane biosynthesis pathways. qRT-PCR validation of the eight key differentially expressed genes showed that the regulatory trends were consistent with transcriptomic trends. These results may provide a theoretical basis for mining CMCS-induced resistance-related genes in grapefruit and studying postharvest molecular resistance mechanisms in grapefruit.

Key words: carboxymethyl chitosan, de novo RNA-Seq, biological information analysis, induced resistance

CLC Number:

Q943.2

YANG Qing, LIU Shenghong, HUANG Erbin, DU Rongyu, WANG Fang, DENG Jia. Carboxymethyl chitosan-induced transcriptome WRKY genes analysis and resistance-related gene mining in grapefruit fruit[J]. Acta Agriculturae Zhejiangensis, 2023, 35(3): 598-614.

Figures/Tables 17

References 34

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引物名称 Primer name	正向引物 Forward primer (5'→3')	反向引物 Reverse primer (5'→3')
WRKY40	TCTTGTGTTGGGATCGAGGC	CCCAAGCTGTCCCGTAAAGA
WRKY53	AGTTCTGCCGCATCAATGGA	TCCGAACTCCCTGAGCTACA
WRKY24	TAGAGCCAAAGACGAGCCAC	TGTCAAGTTCAGTGTGGCCT
WRKY54	GGAAAAGATGGCGAAGGAGC	CTCAGCCGAAACCAACCCAT
GGCT2;2	TCATCAAGGACTACAGGCGC	CCCCTGTGATCAATGCATGC
NCED1	CCGTTTGTTGGCCATGTCTG	TCATCGTGGACTTGAGCTGG
PME7	CTTTACCCACCATGCAAGCG	TGACTCTCCAAACGGAAGCC
LOX2.1	TACGTGCCCAGAGATGAAGC	TGGCCTCGAGTGATGGAAAC
Actin	CATCCCTCAGCACCTTCC	CCAACCTTAGCACTTCTCC

引物名称 Primer name	正向引物 Forward primer (5'→3')	反向引物 Reverse primer (5'→3')
WRKY40	TCTTGTGTTGGGATCGAGGC	CCCAAGCTGTCCCGTAAAGA
WRKY53	AGTTCTGCCGCATCAATGGA	TCCGAACTCCCTGAGCTACA
WRKY24	TAGAGCCAAAGACGAGCCAC	TGTCAAGTTCAGTGTGGCCT
WRKY54	GGAAAAGATGGCGAAGGAGC	CTCAGCCGAAACCAACCCAT
GGCT2;2	TCATCAAGGACTACAGGCGC	CCCCTGTGATCAATGCATGC
NCED1	CCGTTTGTTGGCCATGTCTG	TCATCGTGGACTTGAGCTGG
PME7	CTTTACCCACCATGCAAGCG	TGACTCTCCAAACGGAAGCC
LOX2.1	TACGTGCCCAGAGATGAAGC	TGGCCTCGAGTGATGGAAAC
Actin	CATCCCTCAGCACCTTCC	CCAACCTTAGCACTTCTCC

样品名称 Sample	原始数据 Raw data	纯净数据 Clean data	Q20/%	Q30/%	GC含量 GC content/%
CK-24 h-1	6 375 427 500	6 268 838 074	98.29	94.57	43.92
CK-24 h-2	6 494 895 900	6 402 506 126	98.22	94.30	43.74
CK-24 h-3	5 569 391 400	5 487 236 235	98.07	93.94	43.70
CMCS-24 h-1	6 237 662 100	6 161 803 163	98.30	94.56	43.82
CMCS-24 h-2	5 987 908 500	5 917 842 274	98.14	94.10	43.65
CMCS-24 h-3	5 925 497 100	5 842 851 149	98.11	94.07	43.78

样品名称 Sample	原始数据 Raw data	纯净数据 Clean data	Q20/%	Q30/%	GC含量 GC content/%
CK-24 h-1	6 375 427 500	6 268 838 074	98.29	94.57	43.92
CK-24 h-2	6 494 895 900	6 402 506 126	98.22	94.30	43.74
CK-24 h-3	5 569 391 400	5 487 236 235	98.07	93.94	43.70
CMCS-24 h-1	6 237 662 100	6 161 803 163	98.30	94.56	43.82
CMCS-24 h-2	5 987 908 500	5 917 842 274	98.14	94.10	43.65
CMCS-24 h-3	5 925 497 100	5 842 851 149	98.11	94.07	43.78

样品名称 Sample	总碱基数 Total	Unmapped/%	Unique mapped/%	Multiple mapped/%	Total mapped/%
CK-24 h-1	42 052 500	18.32	79.85	1.83	81.68
CK-24 h-2	42 897 144	17.75	80.50	1.75	82.25
CK-24 h-3	36 761 526	17.92	80.25	1.83	82.08
CMCS-24 h-1	41 287 708	18.13	80.12	1.75	81.87
CMCS-24 h-2	39 616 648	17.89	80.37	1.74	82.11
CMCS-24 h-3	39 131 608	17.76	80.36	1.89	82.24

Carboxymethyl chitosan-induced transcriptome WRKY genes analysis and resistance-related gene mining in grapefruit fruit

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基因编号 Gene ID	对照组表达量均值 CK_mean	处理组表达量均值 CMCS_mean	差异倍数 log₂(FC)	P	FDR	基因名称 Gene name
Unigene 0016512	5.20	33.64	2.69	0.001	0.091	WRKY40
Unigene 0022945	2.24	14.47	2.69	0.008	0.326	WRKY53
Unigene 0004228	0.61	3.18	2.38	0.009	0.340	WRKY24
Unigene 0030218	0.78	2.25	1.53	0.002	0.170	WRKY54
Unigene 0015122	4.29	9.36	1.13	0.027	0.572	WRKY30

代谢通路 Pathway	通路编号 Pathway ID	P	上调基因 Up-regulated gene	下调基因 Down-regulated gene
戊糖、葡萄糖醛酸转换 Pentose and glucuronate interconversions	ko00040	0.010	—	PME44、PME7
亚油酸代谢 Linoleic acid metabolism	ko00591	0.026	—	LOX2.1
维生素B6代谢 Vitamin B6 metabolism	ko00750	0.041	—	PS2
油菜素类酯生物合成 Brassinosteroid biosynthesis	ko00905	0.044	—	CYP92C6
代谢途径 Metabolic pathways	ko01100	0.048	AGT1、NCED1、PS2、 TENA_E、GGCT2;2	PER63、LOX2.1、PME44、 PME7、CYP92C6
硫胺素代谢 Thiamine metabolism	ko00730	0.066	TENA_E	—
类胡萝卜素生物合成 Carotenoid biosynthesis	ko00906	0.122	NCED1	—
植物昼夜节律 Circadian rhythm-plant	ko04712	0.122	APRR5	—
α-亚麻酸代谢 α-Linolenic acid metabolism	ko00592	0.132	—	LOX2.1
丙氨酸、天冬氨酸和谷氨酸代谢 Alanine, aspartate and glutamate metabolism	ko00250	0.150	AGT1
内质网中的蛋白质加工 Protein processing in endoplasmic reticulum	ko04141	0.179	HSP17.4B/HSP22.0	—
甘氨酸、丝氨酸和苏氨酸代谢 Glycine, serine and threonine metabolism	ko00260	0.207	AGT1	—
谷胱甘肽代谢 Glutathione metabolism	ko00480	0.266	GGCT2;2	—
乙醛酸和二羧酸代谢 Glyoxylate and dicarboxylate metabolism	ko0063	0.267	AGT1	—
次生代谢产物生物合成 Biosynthesis of secondary metabolites	ko01110	0.270	AGT1、NCED1	PER63、LOX2.1、CYP92C6
过氧化物酶体 Peroxisome	ko04146	0.271	AGT1	—
代谢通路 Pathway	通路编号 Pathway ID	P	上调基因 Up-regulated gene	下调基因 Down-regulated gene
苯丙烷生物合成 Phenylpropanoid biosynthesis	ko00940	0.295	—	PER63
MAPK植物信号通路 MAPK signaling pathway-plant	ko04016	0.322	PP2C	—
植物激素信号转导 Plant hormone signal transduction	ko04075	0.387	PP2C	—
碳代谢Carbon metabolism	ko01200	0.657	AGT1	—

处理 treatment	基因表达量Gene expression level
处理 treatment	PP2C	AGT1	HSP17.4B	HSP22.0	GGCT2;2	NCED1
CK	4.11±0.66 b	7.01±1.22 b	1.70±0.20 b	8.50±0.41 b	1.83±0.28 b	1.12±0.41 b
CMCS	8.72±1.49 a	24.10±3.86 a	5.88±1.17 a	25.16±2.00 a	9.25±0.44 a	6.34±1.50 a

处理 treatment	基因表达量Gene expression level
处理 treatment	PME44	PME7	LOX2.1	PER63	CYP92C6
CK	9.31±1.43 a	177.55±33.56 a	188.20±24.90 a	18.11±1.93 a	166.46±25.77 a
CMCS	4.05±0.86 b	64.91±17.77 b	80.45±16.96 b	8.12±2.10 b	78.37±14.12 b