Analysis Of Banana CBL Family Genes And Study On The Function Of MaCBL2 Gene In Response To Potassium Deficiency

Calcineurin B-like proteins（calcineurin B-like proteins,CBLs）are the main Ca²⁺signal transduction receptors in plants,which can sense calcium signals generated under a variety of environmental stimuli and transmit them to downstream effectors to activate Adjust resistance to respond to various environmental stimuli.In this study,a systematic analysis of CBL family genes in bananas was conducted.Through transcriptome data analysis,it was found that the expression of MaCBL2gene in banana roots was significantly increased by potassium deficiency,and its response to low potassium stress was verified by q RT-PCR.The MaCBL2 gene was cloned from banana and analyzed by bioinformatics and subcellular localization.The gene was transferred into potassium-deficient yeast mutant R5421,yeast strains Invsc I and Nicotiana benthamiana for functional studies.The specific results are as follows:1.A total of 11 CBL genes MaCBL1～MaCBL11 were identified,and their physical and chemical properties were analyzed.Cluster analysis divided MaCBLs into four groups:Group I～Group IV.10 motif motifs were analyzed for each MaCBL protein,and all MaCBLs contained motif 1,motif 2 and motif 4 motifs,and the distribution of other motifs was different.Most of MaCBLs contain 8 exons,while MaCBL1 contains 7exons and MaCBL4 contains 9 exons.Transcriptome data analysis found that the expression of MaCBL5 in each potassium stress treatment was very low or undetectable.The expression of MaCBL10 was the highest under each treatment,but there was no significant difference in expression between different treatments.The expression difference of MaCBL2 under0 and 0.03 mmol/L K⁺treatments was 2.28 times and 3.66 times that between 3 mmol/L K⁺treatments,reaching a significant difference level.2.The banana seedlings were treated with potassium deficiency,and the results showed that from 3 mmol/L to 0 mmol/L K₂SO₄,the growth of banana leaves and roots were inhibited by potassium deficiency.q RT-PCR analysis showed that the expression level of MaCBL2 under 0,0.03 and 0.3mmol/L K⁺treatment was significantly higher than that under 3 mmol/L K⁺treatment.This result is consistent with the results of transcriptome analysis,indicating that MaCBL2 may play an important role in coping with low potassium stress.3.The MaCBL2 gene was cloned from Brazilian banana.The CDS region is 651 bp in length,which encodes 216 amino acids.Amino acid sequence analysis showed that MaCBL2 contains the conserved Ca²⁺binding-related EF-hand domain,the N-terminal myristinization domain"MGCXXS/K（T）"and"FPSF"motifs.4.The results of subcellular localization analysis show that MaCBL2is distributed in the nucleus,cell membrane and cytoplasm of tobacco leaf cells.5.Yeast experiments show that overexpression of MaCBL2 can restore the growth of K⁺uptake-deficient yeast mutant strain R5421 in low potassium medium.Overexpression of MaCBL2 gene improves the tolerance of transgenic yeast（Invsc I）to high temperature,Na Cl and sorbitol stress.6.Overexpression of MaCBL2 gene makes the germination time of transgenic tobacco seeds earlier than wild-type tobacco seeds.The root growth of transgenic tobacco under the stress of potassium deficiency,Na Cl,and mannitol was significantly better than that of the wild-type control.Some stress-related genes in transgenic tobacco,such as Nt KT12,Nt KT4,Nt CBL1 and Nt CIPK2,were significantly up-regulated under potassium deficiency,Nt NAC072 and Nt SKOR genes under Na Cl stress,and Nt NAC072 and Nt SAP5 under mannitol stress.The MDA content of transgenic tobacco under Na Cl and mannitol stress was significantly lower than that in the control.It shows that overexpression of MaCBL2 improves the tolerance of transgenic tobacco to potassium deficiency,drought and salt stress.