Construction And Verification Of Maize Drought Resistance High Throughput FOX Library

As the raw material for the food,animal feed,biofuels and chemical products,maize(Zea mays)which has the highest yield is one of the most important crops in the world.Drought is the major abiotic stress that affects crop yield and plant growth.Full-length cDNA Over-eXpressing gene hunting system(FOX)was proposed in 2006 which using the heterotopic expression of quantitative cDNAs in organisms constructs a gain-of-function gene library to identify genes in bulk.In eukaryotes,the response of cells to osmotic stress are conserved.Therefore,screening the yeast library of expressing heterologous cDNAs is an efficient method for studying drought-related genes.In this study,a drought-related cDNA yeast library(FOX)was constructed.By using sorbitol simulated drought,candidate genes were obtained and the gene family of CBS domain in maize was identified.The main research results are as follows:(1)By using the drought-resistant inbred line KA105,which was selected from Shaanxi A and B groups and optimizing the steps,such as extracting RNA,cDNA double-strands synthesis cycle number,cDNA length fractionation,vector linearization,etc.,we have constructed a yeast library.The results showed that the capacity of the prokaryotic library finally reaches 6.5×10~6 cfu,and the size of the majority of the inserted gene is 0.75 kb-2.00kb,the average length is more than 1.00 kb,also the recombination rate is 95%,and the capacity of the eukaryotic library is 2.8×10~6 cfu.These demonstrate that we have constructed a qualified yeast FOX library.(2)By screening the entire maize drought-resistant FOX yeast library,analyzing the candidate genes and using the transcriptome data of inbred line KA105 for expression analysis,the results showed that under the condition of 2 mol/L sorbitol stress,very few wild-type yeasts can survive,and 38 non-redundant genes are screened from the entire library by 2 mol/L sorbitol.These genes are involved in signal transductions(9),transcription factors(5),antioxidants(2),metabolisms(5),heat shock protein/molecular chaperones(2),cell structures(4),hormone responses(2),redox homeostasis(1),material transport(1),hypothetical proteins(5),and the other two genes participate in drought resistance through multiple pathways.Subsequently,the obtained 38 candidate yeasts were re-infiltrated and confirmed again.Most genes can respond to drought stress,and multiple genes are up-regulated in specific tissues(leaves or roots).The genes,which encode NAC transcription factor and ABA response protein,can respond to drought strongly in the whole plant.Four of the five genes encoding unknown proteins can respond to drought stress,which suggests that the new pathways with unknown functions may mediate drought resistance in maize.(3)There is one gene which was screened from the 38 genes that contains the CBS domain,so the CBS gene family was identified,and the expression profiles of B73 inbred lines under normal and drought stress conditions were analyzed.The results showed that we identified 33 CBS family genes of maize,and constructed a phylogenetic tree,which was divided into 10 subgroups by combining the information of conserved domains and conserved motifs of ZmCBS family genes.Under normal condition the expression of members,which from the same subgroup,is both conserved and variant.And under the drought stress,multiple genes are upregulated in specific tissues.The ZmCBS17 can respond to drought stress at the whole plant level.