Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (19): 3751-3766.doi: 10.3864/j.issn.0578-1752.2022.19.006

• PLANT PROTECTION • Previous Articles     Next Articles

Genome-Wide Identification and Analysis of CC-NBS-LRR Family in Response to Downy Mildew and Powdery Mildew in Cucumis sativus

KANG Chen(),ZHAO XueFang,LI YaDong,TIAN ZheJuan,WANG Peng,WU ZhiMing()   

  1. Institute of Cash Crops, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050051
  • Received:2022-04-28 Accepted:2022-05-29 Online:2022-10-01 Published:2022-10-10
  • Contact: ZhiMing WU E-mail:kangchen19910228@163.com;zhiming71@126.com

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Abstract:

【Objective】 The bioinformatics and expression pattern analysis of the CC-NBS-LRR (CNL) gene family of the whole cucumber (Cucumis sativus) genome was carried out to provide a reference for further study of the functions of the CsCNL gene family in growth, development and disease stress response. 【Method】 Taking the CNL gene sequence of Arabidopsis thaliana as the reference, the genome of cucumber ‘9930’ was searched by local Perl language and Pfam software, and the members of CsCNL gene family were identified. CsCNL gene family was analyzed by some bioinformatics tools, such as ExPASY, GSDS2.0, MEGA, MEME, Tbtools and Mev. Transcriptome data, inoculating with downy mildew and powdery mildew pathogens (Pseudoperonospora cubensis and Podosphaera xanthii) and real-time quantitative PCR (qRT-PCR) were used to evaluate the expression of the genes. 【Result】 A total of 17 CsCNL genes were identified from cucumber genome. CsCNL genes are distributed on 6 chromosomes except chromosome 1. The encoded proteins are similar in structure to other plant CNLs. All CsCNL proteins contain conserved domains of CC, NBS and LRR. The amino acid sequence size ranged from 197 to 1 148 aa, with a molecular weight (MW) of 22.6 to 131.3 kD, and a protein isoelectric point (PI) ranged from 5.71 to 8.38. Collinear analysis showed that there were no segmental duplication and tandem duplication genes, and the multi-species phylogenetic relationship showed that CNL gene family members had high structural and functional similarities among Cucurbitaceae plants. There were many cis-acting elements related to disease resistance in the promoter of CsCNL genes. The expression of CsCNL genes was tissue-specific. At 2 d and 5 d after inoculation with P. cubensis, Csa3G815400, Csa3G822360 and Csa7G420890 were all significantly up-regulated in resistant variety, Csa4G015850 was down-regulated in resistant variety, and significantly different in susceptible variety. At 2 d and 5 d after P. xanthii inoculation, Csa2G008000 and Csa3G684170 were significantly up-regulated in susceptible variety and significantly down-regulated in resistant variety. Csa4G016360, Csa7G420890 and Csa7G425940 were significantly up-regulated in resistant variety, while unchanged or significantly down-regulated in susceptible variety. 【Conclusion】 CsCNL gene family has specific expression pattern in different tissues, and most of the CsCNL genes were induced by P. cubensis and P. xanthii. It is speculated that the increased expression of Csa3G815400, Csa3G822360 and Csa7G420890, and the decreased expression of Csa4G015850 can induce downy mildew resistance in resistant cucumber varieties. The increased expression of Csa4G016360, Csa7G420890 and Csa7G425940, and the decreased expression of Csa2G008000 and Csa3G684170 can induce the resistance to powdery mildew in resistant cucumber varieties, while the increased expression of Csa7G420890 can simultaneously induce disease resistance to downy mildew and powdery mildew in resistant cucumber varieties.

Key words: cucumber (Cucumis sativus), CC-NBS-LRR (CNL), downy mildew, powdery mildew, gene expression

Table 1

The primer sequences for qRT-PCR"

基因名称 Gene name 正向引物序列 Forward sequence (5′-3′) 反向引物序列 Reverse sequence (5′-3′)
ACTIN TCCACGAGACTACCTACAACTC GCTCATACGGTCAGCGAT
Csa2G008000 GCCGCTCAATTTTCTGGAGA GAGTAAACCCGTGTGCCATC
Csa2G074130 GATGGATTGGCAACCTCACC AGGCCAATCTTCTCGTCTGT
Csa2G433340 TGTTCATGAGGCTGACGACT TTGCGCAACTTCAGTCTCTG
Csa3G172400 GCAACCCAACAAATCCGTCT CTCGCCTAAGGTCCTCGTAG
Csa3G684170 GCTGACAAAACACCTCCACA ATCTGCTCCCTTTGCTTCCT
Csa3G815400 GGCTTGAAAACTCGGCAGAA ACTGCCAAATTCCGGTGTTC
Csa3G822360 TGGCATGGGAGTTGGAGAAT TATGCTCCACTGTTCGTCGA
Csa4G015840 CCGAGATTGACGAGCATTCC TGCAAGCTTGGAGGGTAGTT
Csa4G015850 TGGGTGGAGAAGCTTGAACA CCATGATACTCGCTGCACAA
Csa4G016360 GTGAACGTTGCAGCTCAGAA CCCATCTGCCCATCTCTTCA
Csa4G016430 GGGGTCTACGAGAAATGGGT TTGAATGAACCCTTGTGCCA
Csa4G016460 TGGGTGCCGTAAAATGACAC GAAGTTGCTCAGTCACGCTC
Csa5G266890 ACCCAACTTCCCCAACAACT TGGCCTCTCTCGAAGGTAAC
Csa6G375730 AGGACCATGAACTCGACACA GCCTTGTGCCATCCATTGTT
Csa7G420890 CGAATTTTGGGGCAAGGACA CTGTTGTCCTTGCTGCCATT
Csa7G425930 TGTTCAACGTTGCTGCAAATG CAATCTTTGACTGCGTGGCT
Csa7G425940 TGAGTTCTTGCCAGTGGAGT AAGTGTCCGGAGATTGCTGA

Fig. 1

The chromosome location of the CsCNL gene family"

Table 2

Genome-wide identification and molecular characterization of CsCNL gene family"

基因编号
Gene ID		 染色体位置
Genomic location		 氨基酸数量
Number of amino acid (aa)		 外显子
Exon		 分子量
MW (Da)		 等电点
PI
Csa2G008000 Chr02：1351990-1356361 823 5 95328.28 6.80
Csa2G074130 Chr02：5810883-5814239 915 2 105955.85 8.29
Csa2G433340 Chr02：22624852-22628165 966 2 111929.62 8.38
Csa3G172400 Chr03：11532697-11536241 1148 1 131344.13 7.38
Csa3G684170 Chr03：26192533-26195851 1089 1 125618.99 6.10
Csa3G815400 Chr03：31465330-31468278 892 1 103116.33 6.43
Csa3G822360 Chr03：32245772-32249528 1053 2 120249.54 6.27
Csa4G015840 Chr04：2102886-2106281 1087 1 124356.65 6.84
Csa4G015850 Chr04：2107386-2110628 1080 1 123310.24 7.36
Csa4G016360 Chr04：2113346-2116883 958 2 109571.71 7.09
Csa4G016430 Chr04：2146414-2149099 840 3 96126.70 6.14
Csa4G016460 Chr04：2153828-2157080 947 2 108232.33 7.33
Csa5G266890 Chr05：11239765-11242908 1047 1 119379.10 6.04
Csa6G375730 Chr06：16934210-16937464 917 2 106784.55 8.21
Csa7G420890 Chr07：16247369-16250149 555 2 64716.85 7.61
Csa7G425930 Chr07：16263128-16264206 197 2 22566.76 5.71
Csa7G425940 Chr07：16268256-16272547 1020 5 116400.68 6.86

Fig. 2

The gene structure (A) and conserved motif (B) of CsCNL gene family members"

Fig. 3

Phylogenetic relationships of CNL family protein in different species"

Fig. 4

Syntenic analyses of the CNL genes between cucumber and other species The gray lines in the background indicate the collinear blocks, while the red lines highlight the syntenic CNL gene pairs"

Fig. 5

The disease resistant related cis-elements on CsCNL promoters The numbers represent the amount of cis-elements"

Fig. 6

The 3D structure modeling of CsCNL family proteins"

Fig. 7

The expression profiles of the CsCNL family genes"

Fig. 8

Expression analysis of CsCNLs in response to P. cubensis by qRT-PCR Significance differences between treatment and control were analyzed by t-test. * α=0.05,**α=0.01。The same as Fig. 9"

Fig. 9

Expression analysis of CsCNLs in response to P. xanthii by qRT-PCR"

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