PLANT MICROORGANISM INTERACTIONS Identification and expression analysis of BnaCNGC family gene in the response to phytohormones, abiotic and biotic stresses in Brassica napus

The cyclic nucleotide-gated ion channel (CNGC) family affects the uptake of Ca, regulates the growth and development, pathogen defense, and abiotic stress tolerance in plants. However, the systematic identification, origin, and function of the CNGC gene family has not been performed in Brassica napus. In the present study, we identified 61 putative BnaCNGC genes in the B. napus genome, which are non-randomly localized on 18 chromosomes, and could be classified into five major groups: Group I, II, III, IV-a, and IV-b. Gene structure, multiple sequence alignment, and MEME analysis showed that all the CNGC genes are intron rich and conserved. The expression analysis showed that the BnaCNGC genes have different expression patterns in B. napus, under different phytohormones, abiotic stresses (cold, hot, waterlogging), and infection of Sclerotinia sclerotiorum. Among them, four genes (BnaC03g31050D, BnaC03g31720D, BnaA05g01380D and BnaC04g01250D) from Group I and two genes (BnaCnng45430D and BnaA03g34680D) from Group IV-a, all were strongly induced by SA and infection of S. sclerotiorum, and reduced by cold and heat stresses, suggesting their importance in the abiotic and biotic stress responses in rapeseed. Our comprehensive genome-wide analysis represents a rich data resource for studying the CNGC gene family in B. napus. ARTICLE HISTORY Received 15 July 2021 Accepted 11 November 2021


Introduction
Ca 2+ , an important second messenger, has been shown to be involved in plant response to environmental changes including abiotic and biotic stresses and developmental cues (Tuteja and Mahajan, 2007). However, the calcium-permeable channels in plants have remained largely unknown. One of the potential pathways for the uptake of Ca 2+ ions in signal transduction is via cyclic nucleotide-gated ion channels (CNGCs) (Chin et al., 2009;Jammes et al., 2011).
CNGCs, a kind of calcium-permeable cation channels, have been demonstrated to play a vital role in multiple molecular functions involved in plant development and environmental stress tolerance (Kaplan et al., 2007). In plants, CNGCs localized in the plasma membrane (Borsics et al., 2007), vacuole membrane (Yuen and Christopher, 2013), or nuclear envelope (Charpentier et al., 2016), are composed of six transmembrane (TM) domains with one pore region (P loop) between the fifth and sixth TM domains, one Cterminal cyclic nucleotide-binding domain (CNBD), one C-terminal CaM-binding domain (CaMBD), or isoleucineglutamine domain (IQD) (Chen et al., 2015;Hao and Qiao, 2018). CNGC activity has been reported to be regulated by cNMPs such as cAMP and cGMP binding to the highly conserved region CNBD . CNBD has a phosphate-binding cassette (PBC) and a hinge region which are plant CNGC-specific motifs (Saand et al., 2015). In Arabidopsis, AtCNGC1 and AtCNGC2 are permeable to both K + and Ca 2+ ,and AtCNGC5,6,7,8,9,10,14,16,and 18 are Ca 2+ -permeable cation-selective channels (Wang et al., 2019). CNGCs as Ca 2+ -permeable channels interacted with the ubiquitous Ca 2+ /calmodulin (CaM) via diverse CaMBDs, including IQD (Fischer et al., 2017). A latest study showed that CNGC12 gene channel functionality depends on the conserved IQ-motif in the C-terminus of the channel (DeFalco et al., 2016).
Previous studies have shown that CNGCs play an important role in plant growth and development. In Arabidopsis, 20 CNGC genes are differentially expressed in all tissues and their functions critically depend on the presence of cAMP and/or cGMP that function as signaling molecules (Nawaz et al., 2014). AtCNGC18 is expressed primarily in pollen, and the loss of AtCNGC18 causes abnormalities in pollen tube growth (Frietsch et al., 2007). AtCNGC7/8 and AtCNGC18 together reported as pollen-tube-specific CNGC proteins, interacting with calmodulin 2 (CaM2) constitute a molecular switch that either opens or closes the calcium channel depending on cellular calcium levels . OsCNGC13 is preferentially expressed in the pistils and facilitates the penetration of the pollen tube in the style for successful double fertilization and seed-setting in rice (Xu et al., 2017). AtCNGC1/10 has been shown to play a role in the roots of Arabidopsis (Ma et al., 2006;Borsics et al., 2007). AtCNGC14-dependent Ca 2+ signaling is essential for regulating the early posttranscriptional phase of auxin growth responses in Arabidopsis roots (Shih et al., 2015).
AtCNGC5 and AtCNGC6 genes encode unique cGMP-activated nonselective Ca 2+ -permeable cation channels in the plasma membrane of Arabidopsis guard cells (Wang et al., 2013).
In rice, OsCNGC genes were highly responsive to multiple stimuli including hormonal, biotic, and abiotic stress (Nawaz et al., 2014). For example, the function of two closely related CNGC genes, OsCNGC14 and OsCNGC16, are investigated for increasing temperature-stress tolerance in rice (Oryza sativa). A rice plasma membrane-localized Ca 2+ -permeable nonspecific cation channel OsCNGC9 enhances chilling tolerance in rice, through regulating cold-induced calcium influx and cytoplasmic calcium elevation . Arabidopsis ATCNGC2 and CNGCb gene from Physcomitrella patens act as the primary thermosensors of land plant cells. CNGCb loss-of-function causes a hyper-thermoresponsive Ca 2+ influx and altered Ca 2+ signaling (Finka et al., 2012). Arabidopsis plants may contain a cyclic nucleotide-based signaling pathway that directly affects Na + transport and improves Arabidopsis salinity tolerance (Maathuis and Sanders, 2001). The AtCNGC10 channel is involved in Na + and K + transport during cation uptake in roots and in long-distance transport, and the AtCNGC10 antisense lines were more sensitive to salt stress compared with the wildtype (Guo et al., 2008). AtCNGC19 and AtCNGC20 are also involved in salt stress responses (Kugler et al., 2009;Yuen and Christopher, 2013).
CNGCs also play important roles in the resistance to biotic stress. Loss-of-function AtCNGC2 exhibited increased resistance to pathogen infection after infection with some avirulent pathogens (Clough et al., 2000;Chin et al., 2013). AtCNGC4 gene expression is induced by pathogen infection and some pathogen-related signals (Balagué et al., 2003). AtCNGC11 and AtCNGC12 are involved in R gene-mediated resistance and exhibits increased resistance to pathogen infection (Yoshioka et al., 2006;Moeder et al., 2011). The mutation of AtCNGC20 partially restores disease resistance in eds1 (ENHANCED DISEASE SUSCEPTIBLITY1) (Zhao et al., 2021). In rice, OsCNGC9 positively regulates the resistance to rice blast disease (Wang et al., 2019). SlCNGC genes of Group IV-b regulate different types of resistance against diverse pathogens in tomato (Saand et al., 2015).
In this study, 61 putative CNGC genes were identified in B. napus and classified into five groups (Group I, II, III, IV-a, IV-b). The analysis of evolutionary and structure implied that the CNGC genes were conserved and there was an expansion of Group IV-a members in Brassicaceae. The expression analyses showed that BnaCNGC genes were particularly sensitive multiple stimuli including hormonal, abiotic, and biotic stress in B. napus. Our objective elucidated the expansion and functional diversification of the CNGC gene family and identified some novel genes potentially useful for breeding in B. napus.

Identification of CNGC in B. napus
To identify the CNGC in B. napus, 20 Arabidopsis CNGC protein sequences were used as queries to perform BLASTP for the B. napus genome in the Ensembl Plants database (http://plants.ensembl.org/index.html). All non-redundant protein sequences were retrieved, and the conserved domains were analyzed with Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/) and the Conserved Domains Database (CDD) (http:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).

Chromosomal locations and gene duplication events
Details regarding the chromosomal locations of the BnaCNGC genes were obtained from the Ensembl Plants database. TBtools (Chen et al., 2020) was employed to analyze chromosomal localization.

Analysis of gene structure, motif composition
Gene exon/intron structures were predicted with the TBtools (Chen et al., 2020), with genomic and coding sequences from the Ensembl Plants database (http://plants.ensembl.org/ index.html). The conserved motifs in the CNGC sequences were identified using the MEME (https://meme-suite.org/ meme/tools/meme) with the following parameters: optimal motif width: 6-50; maximum number of different motifs: 10.

Plant materials and treatments, heat map analysis of the BnaCNGC transcriptome data
For different abiotic stress and phytohormones treatments, the ZS11 (B. napus L. cv. Zhongshuang 11) seeds were germinated and transplanted to pots containing soil or vermiculite. The growth conditions, phytohormones treatments, and abiotic stress conditions were as described previously (He et al., 2019).
For waterlogging treatment, the 15-day-old seedlings of winter-type rapeseed variety Avatar and semi-winter rapeseed variety ZS9 were submerged for 24 h, and the expression data of BnaCNGC genes were from the GEO database (GEO: GSE140828) (Wittig et al., 2021).
For infection with S. sclerotiorum, healthy leaves of susceptible (Westar) and resistant (ZY821: Zhongyou821) genotypes of rapeseed were inoculated at the bloom stage, and the expression data of BnaCNGC genes were from the GEO database (GEO: GSE81545) (Girard et al., 2017). A heat map was generated with the TBtools program (Chen et al., 2020).
For BnaCaM family genes classification refer to He et al. (2020).

Identification of CNGC genes in B. napus
Using the 20 AtCNGC proteins as queries in BLASTp search for the Ensembl Plants database, 61 putative CNGC proteins were identified in the B. napus genome. Among them, 51 members contain all the essential CNGC-specific domains (TM, CNBD, and CaMBD/IQD), and 10 members were truncated proteins lacking one or two of the CNGC-specific domains.
The physiological and biochemical properties of the putative 61 BnaCNGC proteins were determined by computing different parameters, and are tabulated in Table S1. These 51 typical BnaCNGC proteins varied in length from 600 to 801 amino acid residues in molecular weights from 70.24 to 90.74 kDa, and in pI from 8.35 to 9.88 (Table S1). The predicted subcellular localization showed that most of CNGC proteins were localized in the plasma membrane, while AtCNGC20 and its 11 orthologous BnaCNGC proteins were localized in the chloroplast membrane (Table S1).
According to the orthologous gene sets among the Ar (B. rapa), Co (B. oleracea), An-, and Cn-subgenomes of B. napus, 25 Ar-Co-An-Cn pairs were identified (Table S2). Fifty of 61 BnaCNGC genes with a subgenome assigned were found in the corresponding An-and Cn homoeologous chromosomes, and they had homoeologous genes both in Ar (B. rapa) and Co (B. oleracea). Three members (BnaC06g16330D, BnaC02g14560D, and BnaC09g29350D) from Group I had no orthologous genes in B. oleracea, though they had orthologous genes in both B. rapa and An-subgenomes of B. napus. BnaC01g42610D (homologous to AtCNGC19) had no orthologous genes in the corresponding An homoeologous chromosome, though it was found to have an orthologous gene in both B. rapa and B. oleracea (Table S2). Additionally, there were four members (BnaC03g31050D, BnaC06g16240D, Bra031515.1 and BnaAnng17920D) from Group I that had no orthologous genes.

Chromosomal distribution and diversification of BnaCNGC genes
The 61 BnaCNGC genes were mapped onto B. napus chromosomes and the position of each locus was determined (Figure 2(B)). The BnaCNGC genes were unevenly distributed (1-5 genes on one chromosome) on 18 chromosomes (i.e. BnaA01-07, BnaA09-10 and BnaC01-C09). Additionally, there was a tandem duplication (a three-gene cluster) in the orthologous regions between BnaA05 and BnaC05 chromosomes (Figure 2(B)). As expected, the distribution of CNGC genes in B. rapa and B. oleracea was similar to the distribution of the orthologous BnaCNGC genes in the B. napus An-subgenome and Cn-subgenome, respectively (Figure 2).

Gene structure and motif composition analysis
To characterize the structural diversity of the CNGC family members, we analyzed the exon-intron organization of individual CNGC genes. Closely clustered CNGC genes in the same clades were similar in the number of exons and intron. The majority of the CNGC genes from phylogenetic Groups I, II, III, IV-b contained 6-9 exons, while the Group IV-a members had 10-12 exons (Figure 3). A comparison between the exon-intron organizations of BnaCNGC and AtCNGC genes clustered in the same phylogenetic groups revealed that there was one more or less intron in BnaCNGC genes than that in AtCNGCs.

Expression patterns of BnaCNGC in different rapeseed tissues
We investigated the expression patterns of BnaCNGC in 12 tissues (i.e. root, stem, leaf, bud, sepal, stamen, new pistil, blossom pistil, wilting pistil, ovule, pericarp, and silique) using RNA-sequencing data. It was found that BnaCNGC genes were differentially expressed in the 12 tissues at different developmental stages (Figure 4). Eighteen BnaCNGC genes from Group I (10/15), II (3/10) and IV-b (5/6) members were constitutively expressed (FPKM>10) in all 12 rapeseed tissues. In contrast, the majority of BnaCNGC genes from Group III and IV-a were lowly or not expressed (FPKM<3) in any of the tested 12 rapeseed tissues.
From Group I, BnaA04g15220D and BnaC04g38170D (a pair of genes homologous to AtCNGC11/12) were predominantly expressed in root. BnaC04g01250D and BnaA05g01380D (a pair of genes homologous to AtCNGC3) were predominantly expressed in pericarp and sepal. BnaC03g31720D and BnaA03g26780D (a pair of genes homologous to ATCNGC13) were predominantly expressed in leaf and sepal. BnaA02g10440D and BnaC02g14560D (a pair of genes homologous to AtCNGC1) had high transcription levels in almost all tissues. In particular, BnaA10g06480D and BnaC09g29350D (another pair of genes homologous to AtCNGC1) were expressed specifically in the stamen.
From Group II, BnaC02g11090D (homologous to ATCNGC5) was relatively highly expressed in root, sepal, and blossom pistil, and its expression level is higher than BnaA02g07990D (another homologous to AtCNGC5) in all rapeseed tissues. BnaA03g50250D (homologous to AtCNGC9) was relatively highly expressed in newpistil and blosomypistil. BnaC04g36890D (homologous to AtCNGC6) was expressed specifically in root, sepal and ovule.
From Group III, BnaC09g42720D and BnaA10g19030D (a pair of genes homologous to AtCNGC18) were predominantly expressed in bud and newpistil tissues. BnaC01g08020D and BnaA01g06670D (a pair of genes homologous to AtCNGC17) were relatively highly expressed in root.
From Group IV-a, BnaA05g22550D, BnaCnng48280D, and BnaC05g35860D were predominantly expressed in root, and BnaCnng45430D was predominantly expressed in leaf, sepal, and pericarp. BnaC05g35840D was relatively highly expressed in root and leaf and BnaCnng48270D was relatively highly expressed in leaf, stem, pericarp, and silique. These genes in pairs had no similar expression patterns.

Expression patterns of BnaCNGC in response to abiotic stresses and phytohormones
We also examined the BnaCNGC expression levels under abiotic stresses (NaCl, PEG, cold, heat, waterlogging) and phytohormones treatments (ABA, MeJA, Ethylene, and SA).  Figure 5, the majority of members from Group II and III were slightly or not responsive to all the tested stresses and photohormone treatments, while the majority expressed members from Group I, IV-a, and IV-b were regulated by temperature stresses and SA treatment. For example, the expressions of BnaA05g01380D/ BnaC04g01250D (a pair of genes homologous to AtCNGC3) and BnaA03g26780D/BnaC03g31720D (homologous to AtCNGC13) from Group I all were significantly up-regulated with SA treatment and down-regulated with ABA treatment and cold/heat/NaCl stresses. BnaC03g31050D (from Group I) and BnaCnng45430D/BnaA03g34680D (from Group IVa) were strongly induced with SA (at 6 and 12 h treatment point) and reduced with cold/heat stresses, and down-regulated with ABA and ethylene after 3 h treatment but up-regulated with ABA and ethylene after 12 h treatment. However, another pair of genes from Group IV-a (BnaCnng48280D/ BnaC01g34940D) was induced by SA, cold, and heat treatments.

As shown in
In addition, BnaA05g22570D and BnaC05g35860D (a pair of genes homologous to AtCNGC19) from Group IV-a were strongly induced by NaCl and PEG treatments. From Group IV-b, BnaC02g44680D and BnaA02g09790D (a pair of genes homologous to AtCNGC4) were up-regulated with cold treatment for (at 3 h treatment point) and heat treatment (at 6 h treatment point).
As shown in Figure 5, 16 members were up-regulated by submergence treatment. BnaA10g18740D and BnaC09g42460D (a pair of genes homologous to AtCNGC2) from Group IV-b were up-regulated in both two B. napus cultivars ZS9 and SAV with submergence treatment. Meanwhile, BnaC02g44680D and BnaA02g09790D (a pair of genes homologous to ATCNGC4), from Group IV-b, were downregulated after submergence stress. B. oleracea genome chromosomes; BnaA01-10: B. napus An-subgenome chromosomes; BnaC01-09: B. napus Cn-subgenome chromosomes; Random means genes were randomly distributed to a specific chromosome. BnaAnn and BnaCnn were unanchored scaffolds that could not be mapped to a specific chromosome from the A-and C-subgenomes, respectively.

Expression patterns of BnaCNGC in response to the infection of Sclerotinia sclerotiorum
As shown in Figure 5, all 20 SA-responsive BnaCNGC genes were regulated with the infection of S. sclerotiorum. For example, four genes (BnaC03g31050D, BnaC03g31720D, BnaA05g01380D and BnaC04g01250D) from Group I and three genes (BnaCnng45430D, BnaA03g34680D, and BnaC03g40070D) from Group IV-a, all were strongly induced by SA and the infection of S. sclerotiorum, but the change-fold (24 hpi/control) in a susceptible rapeseed cultivar (Westar) was higher than that in a tolerant one (Zhongyou 821). Moreover, all the members from Group IV-b were reduced by SA, and were reduced in the susceptible cultivar Westar but induced in the tolerant cultivar Zhongyou 821 after the infection of S. sclerotiorum.

Clustering analysis of expression with BnaCNGC and BnaCaM
Since a large number of studies showed that the interaction between CNGC and CaM plays an important role in plant growth and development (Fischer et al., 2017), the cluster analysis of gene expression patterns of BnaCNGC and Bna-CaM was performed as shown in Figure 6. A pair of BnaCNGC11/12 (BnaA04g15220D/BnaC04g38170D) and BnaCaM7 (BnaA06g20230D) had the same expression pattern and were predominantly expressed in the root.

Discussion
As an important calcium channel protein, CNGCs participate in plant immunity and the tolerance to salt and temperature stress in many crops (Saand et al., 2015;Hao and Qiao, 2018;Meena et al., 2019;Wang et al., 2019;Wang et al., 2021). However, a functional identification of CNGC genes has not been reported in B. napus. In this study, we identified 61 putative B. napus CNGC genes, and determined that the BnaCNGC gene family is larger than the CNGC families of most of the reported crops, such as 18 in tomato (Hao and Qiao, 2018), 21 in pear (Chen et al., 2015), and 28 in rice (Nawaz et al., 2014). The results of synteny analysis indicated that the significantly higher number of BnaCNGC genes may be attributed to the expansion of members in Group IV-a (CNGC19/20) in B. napus. Meantime, the results showed that all the BnaCNGC members (18) from Group IVa in B. napus had corresponding orthologous genes in B. rapa (9) and B. oleracea (9), which implied that CNGCs in Group IV-a were expanded both in B. rapa and B. oleracea, and all were retained after the whole-genome duplication (WGD) event in B. napus (allopolyploidy rapeseed). Like AtCNGC19/20, most of the Br/Bo/BnaCNGCs from Group IV-a were tandem genes ( Figure 3). Gene duplications during evolution increase the genomic content and expand gene functions to optimize the adaptability of plants (Zelman et al., 2012). In Arabidopsis, AtCNGC19/20 have been found to regulate cell death and respond to various biotic and abiotic stresses, such as salinity, Pb 2+ and Cd 2+ stress, herbivory insect, plant pathogenic fungi and bacteria, and endophytes. We presume that the expansion of CNGC19/ 20 in Brassicaceae could greatly increase the adaptation of plants to stress conditions. Homologous genes within the same taxonomic group are assumed to exhibit similar structural, functional, and evolutionary properties (Zelman et al., 2012), which may help clarify the roles of BnaCNGC genes. Similar to the orthologous CNGC proteins in Arabidopsis, the 51 typical BnaCNGCs proteins had conserved gene structures and conserved domains including six membrane-spanning regions, pore region, CNBD, CaMBD, and/or IQD ( Figure  3 and Figure S1). Since CaMBD and/or IQD were the CaM-binding sites, the proteins lacking these two domains may lose the function in regulating Ca 2+ signaling (Fischer et al., 2013;Fischer et al., 2017). Therefore, it is reasonable to presume that the absence of CaMBD and IQ domain in six BnaCNGC members (BnaCnng45430D, BnaA03g34680D, BnaC03g40070D, BnaA03g34700D, BnaC03g31050D, and BnaC02g44680D) raised the possibility that they were function abnormal CNGC proteins.
Though the BnaCNGCs were similar with AtCNGCs in the gene sequences and structures, their spatiotemporal and stress-responsive expression patterns were quite different. There are many reports showed that most of AtCNGCs from Group II and Group III in Arabidopsis are mainly involved in the growth of pollen tube , guard cells (Wang et al., 2013), root hair (Tan et al., 2020), auxin signaling, salt, and Pb 2+ /Cd 2+ tolerance (Moon et al., 2019). Transcriptional data showed that the majority of BnaCNGCs from Group III were low-or no-expression genes, members from Group II and III all were slightly or not responding to all the tested stresses and photohormone treatments. Only BnaC09g42720D and BnaA10g19030D (a pair of genes homologous to AtCNGC18) from Group III were expressed predominantly in bud and new pistil, while a pair of genes homologous to AtCNGC1 (BnaA10g06480D and BnaC09g29350D) from Group I were expressed predominantly in stamen (Figure 4). Unlike AtCNGCs of Group II and III, the members from Group I, IV-a, and IV-b are mainly involved in plant immunity and cell death (Kugler et al., 2009;Chin et al., 2013;Yu et al., 2019).
During the process of plant immune response, there is a complex genetic regulatory network that affects SA-mediated signaling (An and Mou, 2011). The application of SA enhanced the resistance to S. sclerotiorum infection in B. napus (Wang et al., 2012). In our study, most of expressed BnaCNGCs from Group I, IV-a, and IV-b were induced and/ or reduced strongly by SA treatment and infection of S. sclerotiorum indicating that those BnaCNGCs play important roles in the SA-mediated response to S. sclerotiorum in B. napus. Additionally, studies showed that the members from Group IV-b are involved in thermotolerance in Arabidopsis (Finka et al., 2012) and rice . Our study showed that only two (BnaC02g44680D/ BnaA02g09790D) of six members from Group IV-b were slightly induced with cold and heat stresses (about twofold change). However, 16 BnaCNGCs from the other four groups were induced and/or reduced by cold and/or heat stresses.
Meanwhile, a large number of studies have shown that the interaction between CNGC and CaM plays an important role in plant growth and development (Fischer et al., 2017). For example, in Arabidopsis, AtCNGC7/8 and AtCNGC18 together interacting with calmodulin 2 (CaM2) constitute a molecular switch that controls the calcium channel during the pollen tube growth . The activity of AtCNGC12 was significantly enhanced when CaM1 was co-expressed in oocytes . Cluster analysis results showed that some BnaCNGC and BnaCaM gene had the same and specific expression patterns and were clustered together. For example, a pair of BnaCNGC11/12 (BnaA04g15220D/BnaC04g38170D), and BnaCaM7 (BnaA06g20230D) had the same expression pattern and were predominantly expressed in the root. BnaCNGC9 (BnaA03g50250D) and BnaCaM7 (BnaA06g19660D) were both predominantly expressed in new pistils. A pair of Bna-CaM2/3/5 (BnaA03g19320D/BnaC03g23130D) and a pair of BnaCNGC19 (BnaA05g22570D/BnaC05g35860D) were expressed in roots and strongly reduced by salt and PEG treatments. The generated data may be useful for constructing the CNGC-CaM interaction networks. Further characterization of the interactions between BnaCNGCs and BnaCaMs will expand our understanding of the functions of calcium oscillations in the development and adaptation of B. napus.
This work is the first comprehensive and systematic analysis of CNGC gene family in B. napus. There are 61 BnaCNGC genes that are identified in B. napus, and are classified into five major groups (i.e. Groups I, II, III, IV-a, IV-b), and Group IV-a appears to have expanded through WGT. Our results display that BnaCNGC have a very conservative domain, exon-intron structure, and this study assists to elucidate the functional diversity of BnaCNGC genes in the regulation of plant development and stress response in B. napus, and provides valuable information for the genetic and breeding improvement of rapeseed.