Study On The Expression Profile And Function Of MicroRNA In Cytoplasmic Male Sterile Line And Maintainer Line Of Kenaf

MicroRNAs are a kind of endogenous,non-coding small molecule RNA with a length of about 21nt.Studies showed that miRNA were widely involved in various processes of plant growth and development as a class of negative regulatory factors.In recent years,by using high-throughput sequencing technology,a large number of miRNAs in plants,including conservative miRNAs and species-specific miRNAs,were found and identified,which has greatly expanded the types and quantities of miRNAs in plant miRNA populations.However,as an important bast fiber crop,there has been no reports on the type and function of miRNA in kenaf so far.In present study,we collected anther samples,for to construct small RNAs libraries,from two Kenaf lines(UG93A a male sterile line and UG93B a maintainer line respectively.We used high-throughput sequencing to compare the expression profiles of miRNA in both(UG93A)and(UG93B)lines,and we also used targetfinder to predict the target genes of miRNA.Further,we will study the functions of miR394,miRn20 and miR167 in Kenaf.The results of the preliminary studies were as follows:1.We used high-throughput sequencing to obtain 42 conserved miRNA,which belonged to 14 miRNA families and 41 novel miRNAs.Among them,14 miRNAs were differentially expressed between anther of UG93A and UG93B.The target genes of miRNA were predicted by Targetfinder software,and a total of 79 genes were found the tatget of those differentially expressed miRNAs.Bioinformatics analysis,revealed that most of those target genes were found to be transcriptional factors,such as transcription factor SPL,GATA,leucine zipper,and NAC.In addition,some target genes were found as enzymes,such as calcium transportase,cinnamyl alcohol dehydrogenase and glycosyltransferase,etc.2.We selected 10 miRNAs ramdomly,and employed fluorescent dye embedding(qRT-PCR)method for to varify the results of high throughput sequencing and kenaf H3 gene was used as internal reference gene.The(qRT-PCR)results showed that high throughput sequencing results were reliable.3.Cloning miR394,miRn20,miR167 precursors gene sequence with DNA,and using mfold online software analysis,the precursor sequence can form a stable secondary structure.The over expression vector of miR394a,miRn20 and miR167b was successfully constructed by double-enzyme method.4.Using improved CRISPR/Cas9 multi-target vector system,Golden Gate Cloning method was used to construct miR3 94、miRn20 and miR167 pYLCRISPR/Cas9 carrier respectively.They were named pcas9-mir394,pcas9-mirn20 and pcas9-mir167 respectively.The protoplasts from kenaf leaves were separated by enzymatic hydrolysis,and three cas9 vectors were transferred into the protoplasts.The results showed that pCas9-miR394,pCas9-miRn20 carrier had the activity of target cutting in the protoplast of kenaf.5.By using the genetic transformation system of tobacco and agrobacterium tumefaciens mediated leaf disc method,the constructed miR394,miRn20 and miR167 over expression vectors were transferred into tobacco for to carry out functional research.A number of miR394,miRn20 and miR167 transgenic plants were obtained through molecular detection.The current phenotypes of those 3 types of plants were observed.In addition,two seedlings from the miR394 transgenic lines,were small in size,bearing small,thick and dark green leaves.butthe other transgenic seedlings were not significantly different from each other,and all the vegetative growth was normal.