Construction Of Yeast Engineering Strain Of OsNIP1-2 And Its Application In The Detoxification Of Aluminum

Aluminum toxicity is a key factor that affects plant growth and results in crop failures in acid soil.The harm of aluminum toxicity in acidic soil in South China is becoming more and more serious.Traditional physical and chemical methods such as liming will cause irreversible damage to land resources.Therefore,exploring and developing new biological methods is the only way to solve the impact of soil aluminum toxicity on crop growth.Plants will evolve a series of aluminum resistance mechanisms in the process of growth,and aquaporin(AQP)family have been proved to play the role of aluminum transport and reducing aluminum toxicity,so it is of great practical significance to clone related aquaporins gene,construct gene engineering strains and apply them to environmental remediation damaged seriously by aluminum toxicity.In this paper,we used OsNIP1-2 as the research object,use the mutant obtained by knocking out the OsNIP1-2 in rice by CRISPR-Cas9 and wild-type rice as materials to analyze the difference of growth status between them under aluminum stress and the content of aluminum in various parts of plant,and elucidate the function of OsNIP1-2 on resistance to aluminum toxicity in rice.Furthermore construct the gene engineering strain of OsNIP1-2 and analyze its function and application in detoxification of aluminum.The main results obtained are as follows:(1)After knocking out OsNIP1-2,the aluminum transport pathway is blocked,leading to the accumulation of aluminum in the most sensitive cell wall,and reducing the transport efficiency of aluminum from sensitive root to above-ground part,suggesting that OsNIP1-2 is involved in the transport of aluminum in rice.(2)Construct the yeast expression vector of OsNIP1-2,and successfully transform the yeast BY4741 strain,and obtain OsNIP1-2 engineering strain.The analysis results of the heterologous expression and growth curve of the gene engineering strain under aluminum stress show that the strain expressing OsNIP1-2 increases the accumulation of aluminum ions in the strain,resulting in a slower growth rate than the strain expressing pYES2,indicating that the gene engineering strain expressing OsNIP1-2 is successfully constructed.(3)In order to further explore the changes of metabolic pathway in gene engineering strain expressing OsNIP1-2,through identity of metabolites with different concentration in strains expressing OsNIP1-2 before and after test under the condition of growth without aluminum by LC-GC method,after joint calculation of univariate analysis and multivariate analysis,screen 151 differential metabolite composition and their corresponding metabolic pathway.The results showed that the expression of OsNIP1-2 can significantly change the amino acid metabolism and nucleotide metabolism of yeast,which suggests that the expression of the target protein accelerate the pathway of amino acid metabolism and nucleotide metabolism of yeast,which might be related to its involvement in the aluminum transport process.(4)Through the functional verification analysis of the engineering strain,it is showed that the optimum condition for the engineering strain to absorb aluminum is that the amount of inoculation is 5%,p H=4.2,initial aluminum concentration is 50 μM and absorption time is2 h.(5)The absorption of aluminum ions by engineering strain and the removal rate of aluminum in polluted water are higher than that of control group.(6)The co-culture experiment of engineering strain with maize showed that the engineering strain had a certain alleviating effect on aluminum toxicity,and could promote the growth of maize under the condition of aluminum toxicity stress.The study on the strain before and after adsorption of aluminum by infrared absorption spectrum showed that the removal function of OsNIP1-2 engineering strain mainly by absorbing Al3+ in polluted water,and the adhesion of Al3+ is less helpful,and its structure and composition keep intact before and after absorbing Al3+.(7)This engineering strain can be used to remove aluminum from polluted water and alleviate the harm of aluminum toxicity to crop growth in acid soil.It provides an effective strategy,and the engineered strain constructed has shown great potential as a plant growth-promoting agent under aluminum stress.