Construction Of Yeast Engineering Strain Of OsTIP2-1 And Its Application In The Detoxification Of Aluminum

Aluminum(Al)toxicity significantly restricts the growth of plants in acid soil,so the mitigation and remediation of Al toxicity in acid soil has become an important research field.In this study,according to the Al detoxification mechanism of rice,the yeast engineering strain carrying the aquaporin OsTIP2-1 gene was successfully constructed by genetic engineering technology,and the strain was used to treat Al wastewater and improve the Al resistance of crops in acidic soil.At the same time,Os NIP1-2 yeast engineering strain,which is located in the cell membrane of rice root cells and has been verified to have Al toxicity,was added at the same time to improve the treatment effect of Al containing wastewater,and lay the foundation for an effective method to solve the Al pollution problem in the environment.First,we analyzed the expression patterns of rice OsTIP gene family members in response to aluminum toxicity.The results showed that OsTIP2-1 expression was significantly induced by aluminum toxicity.The localization of OsTIP2-1 on the vacuole membrane was determined by laser confocal microscopy.Then CRISPR/Cas9 gene editing technology was used to edit the OsTIP2-1 gene in wild type rice,and OsTIP2-1loss-of-function mutant rice was obtained.Compared with wild type rice,the growth status and Al content of the mutant rice under Al stress were analyzed.Al stress specifically and rapidly induced OsTIP2-1 expression in roots,and in two independent rice lines,functional mutations in OsTIP2-1 significantly increased the sensitivity of rice to Al but not to other metals.Compared with wild-type rice,ostip2-1 mutants accumulated less Al in roots and more Al in stems and leaves,indicating that OsTIP2-1 is involved in the excess accumulation of Al in rice roots and limits its migration to the stem.Moreover,the Al accumulation in cell wall was almost unchanged in the ostip2-1 mutant compared with the wild type rice,while the Al accumulation in cell sap was significantly reduced,indicating that OsTIP2-1 plays an important role in Al transport and storage in root vacuoles.Heterologous expression of OsTIP2-1+GFP fusion protein in Saccharomyces cerevisiae BY4741 revealed that OsTIP2-1 was also localized to the vacuole membrane in yeast.Further results showed that the OsTIP2-1 strain was more tolerant to aluminum toxicity than the control strain,indicating that OsTIP2-1 located on the vacuole membrane could promote Al sequestration from yeast cytoplasm to vacuoles,which could be used as a future engineering strain to alleviate aluminum toxicity.When the constructed OsTIP2-1 strain was applied to Al-contaminated wastewater,the Al removal rate and absorption rate were increased but not significantly in the strain overexpressing the OsTIP2-1 gene compared with the wild type(p YES2)yeast.In contrast,the engineered strain transformed with Os NIP1-2 gene significantly increased Al removal and absorption,which might be related to the localization of Os NIP1-2 on the cell membrane and OsTIP2-1 on the tonoplasmic membrane.However,the removal and absorption of Al were much greater when the two engineered bacteria were added at the same time than when they were added alone.The reason for this may be that the Os NIP1-2 gene is located in the plasma membrane and can directly contact Al in Al-contaminated water,thus having a more direct absorption or transformation effect on Al.However,OsTIP2-1 gene is located in the cell vacuole,and it cannot directly absorb or transform Al in the environment;therefore,its removal effect on Al is less obvious.Furthermore,Os NIP1-2 and OsTIP2-1 showed similar cell-specific localization,and Os NIP1-2 and OsTIP2-1 may act synergistically when added together,that is,Al transported by Os NIP1-2 is sequestered into cellular vacuoles by OsTIP2-1.The low Al removal rate of OsTIP2-1 strain was solved by adding two engineered strains simultaneously.In the hydroponic study of two different varieties of wild-type rice(R1 and R2),the addition of wild-type yeast strain,Os NIP1-2 strain and OsTIP2-1 engineering strain promoted the root growth of wild-type rice.The effect of OsTIP2-1 engineering strain was more significant,followed by Os NIP1-2 engineering strain.Addition of the engineered strain reduced the Al content in the roots of both wild type rice strains.These results indicated that Os NIP1-2 and OsTIP2-1 endowed the yeast strains with the ability to resist Al virulence,and thus enabled the two engineered strains to promote wild type rice grown in Al environment.The possible mechanism may be that Os NIP1-2 and OsTIP2-1 have a coordination effect in transporting Al in plant cells,which transport Al to yeast cytoplasm and vacuole,respectively,to reduce the toxicity of Al to rice.