Identification and Expression Analysis of the CBL-CIPK Gene Family in Sugarcane

doi:10.13560/j.cnki.biotech.bull.1985.2023-0830

Abstract

Abstract:

【Objective】 Sugarcane is an important sugar crop, and temperature, salinity, water and other factors are the key environmental factors restricting its growth and development. Calcineurin B-Like protein（CBL）family represents a group of Ca²⁺ binding proteins and plays a key role in decoding calcium signals by specifically interacting with and regulating a family of protein kinases CIPKs（for CBL-interacting protein kinases）in abiotic stress signal transduction pathway. At present, the whole genome of sugarcane has been sequenced, but its CPL-CIPK gene family members have not been identified, and the interaction regulation mechanism is still unknown. Here, the members of CBL and CIPK in sugarcane were identified and the CPL-CIPK interaction relationship was revealed, which may provide gene resources and theoretical basis for studying the mechanism of CPL-CIPK interaction in sugarcane.【Method】 The expression levels of CBLs and CIPKs under low temperature, high temperature, NaHCO₃ and PEG were analyzed by RT-qPCR from sugarcane GT58. The interactions between SsCBLs and SsCIPKs were analyzed by yeast two-hybrid assay.【Result】 There were 19 CBL genes and 82 CIPK genes in the whole genome of sugarcane, which distributed in different evolutionary branches with gene replication. The physicochemical properties of gene family members were different, the structural domains and protein motifs were highly conserved, and the distribution of cis-acting elements was diverse. SsCBL7/SsCBL12/SsCIPK1/SsCIPK5 were specifically expressed under low temperature, high temperature, drought and high salt and other abiotic stress regulation. Meanwhile, SsCBL1 interacted with SsCIPK47 and SsCIPK81, and SsCBL8 interacted with SsCIPK47 and SsCIPK81.【Conclusion】 The CBL-CIPK signaling system may play an important role in response to abiotic stress during the growth and development of sugarcane.

Key words: sugarcane, SsCBL, SsCIPK, abiotic stresses, yeast two-hybrid

XIN Qi, LI Ya-fan, YIN Zheng, ZHANG Xiao-dan, CHEN Ting, LIU Xiao-hua. Identification and Expression Analysis of the CBL-CIPK Gene Family in Sugarcane[J]. Biotechnology Bulletin, 2024, 40(2): 197-211.

Figures/Tables 9

Table 1 Statistical table of physico-chemical properties of 19 CBLs of sugarcane

基因 Gene	基因ID Gene ID	基因长度 Gene length /bp	正负链 Strand	mRNA长度 mRNA length/bp	氨基酸数目 Amino acid/aa	等电点 pI	外显子个数 Exon number	染色体定位 Chromosome	相对分子质量 Molecular weight/kD	亚细胞定位预测 Subcellular localization
SsCBL1	Sspon.01G0014730-1A	3 124	＋	579	193	5.00	7	Chr.1A	22.26	Cyto
SsCBL2	Sspon.01G0023690-1A	3 641	＋	675	225	4.82	8	Chr.1A	25.77	Cyto
SsCBL3	Sspon.01G0014730-2B	3 431	-	639	213	4.75	8	Chr.1B	24.49	Cyto
SsCBL4	Sspon.01G0014730-3C	3 431	-	639	213	4.75	8	Chr.1C	24.49	Cyto
SsCBL5	Sspon.01G0014730-4D	3 431	-	639	213	4.75	8	Chr.1D	24.49	Cyto
SsCBL6	Sspon.01G0023690-3D	3 641	＋	675	225	4.82	8	Chr.1D	25.77	Cyto
SsCBL7	Sspon.02G0030420-1A	3 582	＋	660	220	4.92	9	Chr.2A	25.43	Chlo
SsCBL8	Sspon.02G0030420-2C	3 640	-	675	225	4.79	8	Chr.2C	25.84	Cyto
SsCBL9	Sspon.03G0011980-1A	4 077	-	738	246	6.14	10	Chr.3A	28.83	Cyto
SsCBL10	Sspon.03G0012700-1A	4 617	-	882	294	4.84	9	Chr.3A	33.11	E.R.
SsCBL11	Sspon.03G0031480-1B	4 573	-	864	288	4.84	8	Chr.3B	32.40	Chlo
SsCBL12	Sspon.03G0011980-2B	4 758	＋	882	294	5.28	10	Chr.3B	33.94	Cyto
SsCBL13	Sspon.03G0011980-1P	3 620	-	660	220	6.19	8	Chr.3B	25.66	Cyto
SsCBL14	Sspon.04G0013450-2B	3 432	＋	639	213	4.77	8	Chr.4B	24.34	Nucl
SsCBL15	Sspon.07G0003410-1A	3 363	-	633	211	4.88	8	Chr.7A	24.11	Chlo
SsCBL16	Sspon.07G0018960-1A	3 619	-	669	223	4.82	8	Chr.7A	25.68	Chlo
SsCBL17	Sspon.07G0018960-2B	3 363	-	618	206	4.99	7	Chr.7B	23.77	Chlo
SsCBL18	Sspon.07G0018960-3C	3 324	＋	612	204	4.91	7	Chr.7C	23.52	Mito
SsCBL19	Sspon.07G0018960-4D	3 485	＋	642	214	4.92	8	Chr.7D	24.69	Chlo

Fig. 1 Domain and motif analysis of CBL-CIPK gene family A: Analysis of motif, domain of sugarcane CBL family members. B: Analysis of motif, domain of sugarcane CIPK family members. From left to right are the evolutionary relationship tree, motif and domain, with different colored modules representing different motifs and domains

Fig. 2 Analysis in the gene structures of CBL-CIPK gene family A: Gene structure of sugarcane CBL family members. B: Gene structure of sugarcane CIPK family members. Evolutionary relationship tree on the left, gene structure of family members on the right, UTR in green and CDS in yellow

Fig. 3 Analysis for their positions on the chromosomes in CBL-CIPK gene family A: Chromosomal localization of sugarcane CBL family members. B: Chromosomal localization of sugarcane CIPK family members

Fig. 4 Predicted statistics for cis-acting elements of the sugarcane CBL gene family

Fig. 5 Phylogenetic tree and covariance analysis of the CBL-CIPK gene family A: Phylogenetic evolutionary tree of the multispecies CBL family. B: Phylogenetic evolutionary tree of the multispecies CIPK family. C: Analysis of intraspecific CBL-CIPK gene family covariance in sugarcane. All gene pairs in the whole genome are gray, CBL-CIPK family members form gene pairs in red, sugarcane is a heterotetraploid, and 1A-8D are the 32 chromosomes of sugarcane

Fig. 6 RT-qPCR analysis of SsCBLs, SsCIPKs genes involved in different temperature stresses response A: Gene expression profiles of SsCBLs under 4℃ treatment. B: Gene expression profiles of SsCIPKs under 4℃ treatment. C: Gene expression profiles of SsCBLs under 45℃ treatment. D: Gene expression profiles of SsCIPKs under 45℃ treatment

Fig. 7 RT-qPCR analysis of SsCBLs, SsCIPKs genes involved in NaHCO3/PEG stresses A: Gene expression profiles of SsCBLs under170 mmol/L NaHCO3 treatment. B: Gene expression profiles of SsCIPKs under 170 mmol/L NaHCO3 treatment. C: Gene expression profiles of SsCBLs under 150 g/L PEG-6000 treatment. D: Gene expression profiles of SsCIPKs under 150 g/L PEG-6000 treatment

Fig. 8 Interaction between SsCBLs and SsCIPKs

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