果树学报2025，Vol.42Issue(10)：2262-2272,11.DOI:10.13925/j.cnki.gsxb.20250158
香蕉A、B基因组胆碱单加氧酶基因功能验证
Functional validation of choline monooxygenase genes in banana A and B genomes
摘要
Abstract
[Objective]Osmotic stress is a major environmental constraint that severely limits plant growth,development,and agricultural productivity.Glycine betaine(GB),a compatible osmolyte syn-thesized in response to abiotic stresses,plays a pivotal role in osmotic adjustment,with choline monoox-ygenase(CMO)acting as the rate-limiting enzyme in its biosynthetic pathway.Banana(Musa spp.),a globally significant crop with diverse genomic compositions derived from M.acuminata(A genome)and M.balbisiana(B genome),exhibits genotype-specific variations in stress tolerance.Previous stud-ies have identified structural and functional differences in CMO genes across banana genotypes,sug-gesting potential divergence in their roles during stress adaptation.This study aims to functionally vali-date CMO genes derived from the A and B genomes of banana,elucidate their contributions to osmotic stress tolerance,and explore the genomic basis for stress resilience in different banana genotypes.[Methods]Four banana genotypes[Zhanjiang AA(AA genome),Brazilian banana(AAA genome),Guangdong plantain(AAB genome),and Fenjiao(ABB genome)]were selected for their distinct ge-nomic compositions.Tissue-cultured seedlings at the five-leaf stage were used for gene cloning and stress treatments.CMO genes were amplified using genotype-specific primers designed based on prior sequencing data.The four CMO gene coding sequences(CMO-A,CMO-H,CMO-B1 and CMO-B2)were recombined into the pYES2-NTB yeast expression vector and the pGFPGUSplus plant overexpres-sion vector by homologous recombination method.The yeast transformation experiment was carried out by lithium acetate conversion method.Yeast functional complementation assays were conducted using INVSC1(for osmotic,salt,and low-temperature stress)and ycfl(for heavy metal stress)strains.Trans-genic Nicotiana benthamiana lines overexpressing banana CMO genes were generated through Agro-bacterium-mediated transformation(GV3101 strain)and validated by PCR and hygromycin resistance screening.For stress assays,yeast transformants were grown in SG-Ura media containing 1.0 mol·L-1 NaCl(salt stress),1.0 mol·L-1 mannitol(osmotic stress),40 μmol·L-1 CdCl2(heavy metal stress),or in-cubated at 18℃(low-temperature stress).Growth was monitored via serial dilution spot assays.In to-bacco,seed germination rates and root elongation were evaluated under 150 mmol·L-1 mannitol-in-duced osmotic stress,with or without 20 mmol·L-1 exogenous GB pretreatment.Subcellular localiza-tion of CMO-GFP fusion proteins was transiently expressed in tobacco leaves,and visualized via confo-cal microscopy using chloroplast autofluorescence(640 nm excitation).[Results]Overexpression of ba-nana CMO genes significantly enhanced yeast tolerance to multiple stresses.Under 1.0 mol·L-1 NaCl,1.0 mol·L-1 mannitol,and 18℃ conditions,when INVSC1 strains expressing CMO-A(AA genome),CMO-H(hybrid A/B genome),CMO-B1,and CMO-B2(B genome)were compared with the empty ve-hicle control,the survival rate of INVSC1-CMO recombinant yeast significantly increased.No signifi-cant differences were observed among the four CMO variants under these stresses.Under 40 μmol·L-1 CdCl2,ycf1 strains expressing CMO-H and CMO-B1 showed superior growth,and colony growth was significantly better than that of ycfl-CMO-A and ycfl-CMO-B2 recombinant yeasts,respectively,indi-cating enhanced heavy metal tolerance linked to B genome-derived CMO variants.Osmotic stress(150 mmol·L-1 mannitol)reduced seed germination rates in wild-type(WT)tobacco to 42.3％,whereas transgenic lines overexpressing CMO-B1 and CMO-B2 maintained germination rates of 68.9％and 71.2％,respectively-a 30％-33％improvement over WT.Exogenous GB further increased germina-tion rates to 85％-89％in transgenic lines,demonstrating synergistic effects between endogenous and exogenous GB.Root elongation under osmotic stress revealed genotype-specific effects:CMO-B1 and CMO-B2 transgenic seedlings exhibited root lengths of 2.57 cm,1.43-fold longer than WT(1.80 cm),whereas CMO-A and CMO-H lines showed no significant improvement(2.03-2.07 cm).Confocal mi-croscopy confirmed that all four CMO-GFP fusion proteins were localized exclusively to chloroplasts,as evidenced by complete overlap with chloroplast autofluorescence.This finding aligns with the role of CMO in chloroplastic GB biosynthesis.[Conclusion]B genome-derived CMO genes(CMO-B1/B2)confer superior osmotic/heavy metal stress tolerance compared to A genome variants,highlighting func-tional diversification shaped by genomic ancestry.Chloroplastic localization underscores their role in stress-responsive GB synthesis.These findings identify CMO-B1/B2 as key targets for breeding stress-resilient banana cultivars and provide a molecular framework for understanding A/B genome functional divergence.This study pioneers the functional dissection of CMO allelic diversity in banana,linking ge-nomic ancestry(A vs B)to stress-responsive gene performance.The development of a dual-model vali-dation system(yeast and tobacco)provides a scalable framework for screening stress-tolerance genes in polyploid crops.Additionally,the chloroplast-specific localization of CMO resolves long-standing ambi-guities about its subcellular activity in monocots,offering insights for engineering GB biosynthesis path-ways in non-accumulator species.关键词
香蕉/A、B基因组/胆碱单加氧酶合成基因/非生物胁迫/功能差异Key words
Musa spp./A,B genome/CMO genes/Abiotic stress/Functional differences分类
园艺学与植物营养学引用本文复制引用
朱博为,于佳玄,刘菊华,李新国..香蕉A、B基因组胆碱单加氧酶基因功能验证[J].果树学报,2025,42(10):2262-2272,11.基金项目
国家自然科学基金项目(No.32160679) （No.32160679）
