Study on the Effect of Rhodococcus rhodochrous NB1 on the Tolerance to Salt and Growth-promoting of Maize and Its Whole Genome

doi:10.13560/j.cnki.biotech.bull.1985.2024-0503

Abstract

Abstract:

【Objective】To study the salt tolerance and growth-promoting characteristics of Rhodococcus rhodochrous NB1, analyze its whole genome information, and explore the salt tolerance promoting genes of NB1 strains.【Method】NB1 strain were identified using morphological observations and 16S rRNA gene sequence analysis. The nitrogen fixation, phosphorus lysis and ACC deaminase production were identified by modified medium, Pikovaskaia's medium, DF liquid medium and ADF liquid medium. The NB1 strain was inoculated to NB solid concentrations of 0, 5%, 10% and 15% on the trophic base, the tolerable concentration of the strain was determined after 48 h of incubation. Maize seeds treated with and without the NB1 strain were inoculated onto 1/2 MS medium, and the plant height, root length, fresh weight and root weight were determined after 15 d of continuous culture. Growth indicators of maize seedlings watered and untreated with NB 1 broth were measured in the form of pot experiments under the same salt concentration stress. Whole gene analysis of NB1 strain was conducted using Illumina second generation sequencing and PacBio third generation sequencing. 【Result】The NB1 strain belongs to Rhodococcus rhodochrous, it may produce ACC deaminase, has the ability of fixing nitrogen and dissolving phosphorus, and may tolerate the salt concentration of 5%. Under soil-free culture conditions, the plant height, root length, fresh weight and root weight of maize seedlings treated with NB1 strain increased significantly. After pot planting, the plant height and fresh weight were significantly higher than that of CK. The NB1 strain has 5 259 coding genes, the total length of coding genes is 5 230 674 bp, and the average GC content is 68.30%. They are annotated to 5 235, 4 379, 4 195, 3 758, 3 263, and 2 449 genes in NR, Pfam, COG, Swiss-Prot, GO, and KEGG databases, respectively. The protein structures encoded from 178 genes in NB1 strain belongs to the CAZy family and containing genes of chitinase,sucrase and other enzymes.Meanwhile, it is predicted that there are 14 secondary metabolite gene clusters, 457 virulence factors and 324 resistance genes in NB 1 strains, and related genes such as tetrahydropyrimidine and tetracycline antibiotics with salt resistance and promoting properties were found from the NB1 genome. 【Conclusion】Rhodococcus rhodochrous NB1 has salt resistance and growth promoting effect on maize, which provides new strain resources for plant salt resistance.

Key words: Rhodococcus rhodochrous, whole genome, maize, growth-promoting effect, salt-alkali tolerance

YIN Zi-wei, HONG Yu. Study on the Effect of Rhodococcus rhodochrous NB1 on the Tolerance to Salt and Growth-promoting of Maize and Its Whole Genome[J]. Biotechnology Bulletin, 2024, 40(12): 193-207.

Figures/Tables 13

Fig. 1 A phylogenetic tree based on the 16S rRNA gene of the strain NB 1

Fig. 2 Tolerance of NB 1 strains to salinity

Table 1 Effect of NB1 broth on the growth of maize seedlings

处理Treatment		株高Plant height/cm	根长Root length/cm	鲜重Fresh weight/g	根重Root weight/g
无土培养Soil-free culture	CK	7.21±1.33	6.21±1.25	1.50±0.13	0.40±0.07
无土培养Soil-free culture	NB1	10.45±1.12^**	11.76±1.13^**	1.72±0.16^*	0.52±0.07^*
盆栽实验Pot experiment	CK	24.97±0.96	15.98±2.38	4.33±0.59	2.14±0.32
盆栽实验Pot experiment	NB1	34.38±2.38^**	19.13±3.38	5.29±0.70^*	2.27±0.26

Fig. 3 Effect of strain NB1 on the growth of maize seedlings

Fig. 4 Phylogenetic tree based on the genome sequence of the strain NB1

Fig. 5 Genome map of strain NB1 The outermost circle of the circle diagram is the genome size logo. The second and third turns are the CDS on the positive and negative strand. Different colors indicate the functional classification of different COG in CDS. The fourth circle is for the rRNA and the tRNA. The fifth circle shows the GC content. The red portion outward indicates a higher GC content in this region than the genome-wide average GC content. A higher peak indicates a larger difference from the mean GC content. The inward blue part indicates that the GC content in this region is lower than the genome-wide average GC content. A higher peak indicates a greater difference from the mean GC content. The innermost lap is the GC-Skew value, the specific algorithm is G-C/G + C, may assist in the judgment of the leading and lagging strand. The general leading strand, GC skew> 0, the lagging strand GC skew <0, you may also help in judging the replication origin（cumulative offset minimum）and the endpoint（cumulative offset maximum）, especially most important for the circular genome

Fig. 6 Distribution of gene functional annotation database in strain NB1 genome

Fig. 7 Results of COG database alignment analysis of strain NB1 genome The abscissa indicates the different COG types, and the ordinate indicates the number of genes. Specific functional description of each COG type is provided in the legend on the right

Fig. 8 Results of GO database alignment analysis of strain NB1 genome

Fig. 9 Results of KEGG database alignment analysis of the strain NB1 genome

Table 2 Secondary metabolite synthesis gene cluster of strain NB1

基因编号Cluster ID	位置Location	类型Type	登录号MIBiG accession	相似基因簇Similar gene cluster	相似度Similarity/%	基因数量Gene No.
Cluster1	Plasmid B	Butyrolactone	-	-	-	8
Cluster1	Chromosome	Terpene	BGC0001456	Isorenieratene	25	17
Cluster2		T1PKS	-	-	-	29
Cluster3		Betalactone	-	-	-	30
Cluster4		NAPAA	-	-	-	30
Cluster5		NRPS	BGC0000371	Heterobactin A / Heterobactin S2	36	40
Cluster6		NRPS	-	-	-	37
Cluster7		Ectoine	BGC0000853	Ectoine	75	8
Cluster8		Terpene	BGC0000269	SF2575	6	16
Cluster9		NRPS	BGC0000368	Streptobactin	11	43
Cluster10		NRPS	BGC0000893	Chloramphenicol	17	41
Cluster11		Terpene	BGC0000664	Isorenieratene	42	19
Cluster12		NRPS	-	-	-	39
Cluster13		NRPS	-	-	-	29

Fig. 10 Prediction analysis of virulence factors in strain NB1

Table 3 Statistics on prediction analysis of resistance genes in strain NB1

药物类别Drug class	基因数量Gene No.	药物类别Drug class	基因数量Gene No.
Disinfecting agents and antiseptics	57	Polyamine antibiotic	6
Tetracycline antibiotic	51	Elfamycin antibiotic	5
Macrolide antibiotic	42	Pleuromutilin antibiotic	4
Fluoroquinolone antibiotic	31	Streptogramin antibiotic	4
Peptide antibiotic	30	Fusidane antibiotic	3
Penam	24	Sulfonamide antibiotic	3
Cephalosporin	22	Thioamide antibiotic	3
Rifamycin antibiotic	22	Diaminopyrimidine antibiotic	3
Phenicol antibiotic	21	Penem	3
Aminoglycoside antibiotic	20	Salicylic acid antibiotic	3
Phosphonic acid antibiotic	18	Nitroimidazole antibiotic	3
Glycopeptide antibiotic	16	Mupirocin-like antibiotic	3
Aminocoumarin antibiotic	15	Nucleoside antibiotic	2
Isoniazid-like antibiotic	13	Sulfone antibiotic	2
Carbapenem	13	Pyrazine antibiotic	2
Oxazolidinone antibiotic	13	Antibacterial free fatty acids	2
Cephamycin	10	Nitrofuran antibiotic	1
Glycylcycline	9	Streptogramin B antibiotic	1
Monobactam	7	Streptogramin A antibiotic	1
Lincosamide antibiotic	7	Nybomycin-like antibiotic	1

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