The Molecular Regulation Mechanisms Of Growth Of Lettuce Treated By TiO₂/ZnO Nanomaterials

Great progress has been made in the application of nanotechnology in the field of agriculture,but there are few reports about the regulation mechanism of plant growth and development.Our previous study found the appropriate dosage of nanomaterials in hydroponic lettuce,which could promote growth and improve quality.Therefore,on the basis of former researches,this paper aims to obtain digital gene expression profiling of lettuce leaf and root treated by nanomaterials using RNA-seq and the key relative-genes,and then validate them by real-time quantitative PCR,thus explore the molecular regulation mechanism of growth of lettuce treated by TiO₂/ZnO nanomaterials.The main results of this study are as follows:1.A hydroponic experimental study was performed to determine the effects of TiO₂/ZnO nanomaterials on the growth and major elements absorption of hydroponic lettuce with and without nanomaterials.The results showed that the fresh and dry weights of shoots and roots were significantly enhanced and the contents of soluble sugar,soluble protein,and Vc were increased in treatment with nanomaterials.Nanomaterials could improve the shoot and root absorption of N,P,and K.Moreover,the variation trend of N,P,K contents in nutrient solution was in accordance with the absorption amounts of lettuce plants as evidenced by the consumptions of major elements of the treatment group were higher than those of the control group in nutrient solution.2.A cDNA library of lettuce leaf and root in the middle growth stage?25th day after transplanting?was constructed.Approximately 10.4 GB clean reads were obtained,and a total of 36919 unigenes were formed by initial sequence splicing,with an average read length of 897 bp.29136 unigenes?78.92%?were annotated based on at least one database.509 genes were identified as differentially expressed genes?DEGs?in leaf treated by nanomateials,which comprised 223 up-regulated genes and 286 down-regulated genes,while 3666 genes were identified as DEGs in root,which comprised 844 up-regulated genes and 2822 down-regulated genes.Hundreds of genes potentially involved in nanomaterials were identified from these DEGs,including the genes related to photosynthetic metabolism,antioxidant enzymes,nitrogen metabolism and sucrose and starch metabolic pathways.Ultimately,a preliminary regulatory mechanism of growth of lettuce treated by TiO₂/ZnO nanomaterials was proposed based on transcriptome analyses.3.To determine the optimal reference gene for q-PCR analysis under nanomaterials,we analyzed six candidate reference genes and found that LsCAB was the best choice for normalization in different time treatments and different tissues.4.The full-length cDNA of AMT1 and PHT1 in lettuce were cloned using reverse transcription polymerase chain reaction?RT-PCR?and rapid-amplification of cDNA ends?RACE?.The results showed that LsAMT1 open reading frame is 1533bp which encoding a polypeptide of 510 amino acids with an estimated molecular mass of 54.36 kD.It has the highly conserved AMT1 family signatures,DFAGSGVVHMVGGIAGLWGALIEGPR.The similarity of amino acids between AMT1 with the homologous gene in Cynara cardunculus is 93%.Open reading frame of LsPHT1 is 1605bp which encoding a polypeptide of 534amino acids with an estimated molecular mass of 58.5 kD.It has the highly conserved PHT1 family signatures,GGDYPLSATIxSE.The similarity of amino acids between PHT1with the homologous gene in Chrysanthemum is 88%.The expression patterns were analyzed by q-PCR.The results showed that LsAMT1 and LsPHT1 were both expressed only in the roots of lettuce and the expression level of them in the whole growth period showed a tendency of increasing first and then decreasing.The expression in roots of lettuce treated with nanomaterials was significantly higher than that of control at the middle and late growth stage,indicating that the expressions of these two genes were related to nanomaterials.