Construction Of Mutant Library Of Helminthosporium Spp. And Functional Study Of BsTup1 Gen

Sleep exists in vertebrates and invertebrates,and it plays an indispensable role in the evolution and entire life of animals.Sleep disorders often mean the abnormal function of organisms,which can even cause shock or death.However,there are huge sleep differences in different animals.For example,the giraffe only needs two hours of sleep to maintain daily vitality,but the bat requires twenty hours of sleep.In addition,in the same population,a small number of individuals have different sleep patterns,such as people with narcolepsy and familial advanced sleep phase syndrome.In the long-term genetic evolution,these sleep patterns have been deeply written into the genomes of different species,and some special genetic mutations can also explain the abnormal sleep of a few individuals in the same group.Therefore,it’s very important to understand and control sleep by exploring the genetic mechanism of sleep regulation.In the past few decades,through the forward and reverse genetic research in Caenorhabditis elegans,Drosophila melanogaster,and mouse,many sleep regulation genes have been found.Also,in whole genome sequencing research in patients with abnormal sleep,some genes that regulate sleep time were reported.However,these sleep regulation genes have almost not been reported that they have a relationship between upstream and downstream regulation.Traditional mammalian genetic research usually requires a lot of time to mate and reproduce.It also requires precise micro-operating technology or some dangerous mutations-induced drugs.At present,this is not enough to support the molecular genetic research of the brain nerve system.The optogenetics and chemogenetics that have been developed in recent years have found many sleep-regulation brain regions and neural circuits,but no one has determined a core sleep-regulation center.These studies were performed at the cell level and can’t further analyze the changes in genetics at the molecular level.And these studies show that sleeping research at this stage requires more in-depth molecular genetic exploration.Benefiting from a new-type adeno-associated virus vector AAV-PHP.eB which has a high brain tendency,we first realize the rapid genetic manipulation of adult mice at the molecular genetics level.This not only avoids that some sleep regulatory genes may participate in the important development function of the body,but also avoid the waste of genetic mating time,funds,and manpower.At the same time,through the multi-virus delivery method,some research on redundant genes with the same function can be performed simply.In addition to the over express ion genes in wild-type mice,we also realized multiple gene knockouts in Cas9 transgenic mice by CRISPR/Cas9 system.Using this adult brain chimeric(ABC)expression/knockout(KO)system,we found that Hdac4 and Hdac5 genes which were the member of class Ⅱa histone deacetylases family can specifically regulate the sleep time of mice,and do not affect other phenotypes.Through multiple virus delivery methods,we also found that the sleep regulation of Hdac4 and Hdac5 genes could be regulated by the Crebl gene which was one of the downstream transcription factors of Hdac4 and Hdac5 genes.These studies have shown that our ABC-expression/knockout(KO)system can greatly accelerate the study of the core genes of sleep regulation,and contribute to the field of brain genetics research.