Effects Of MiR159 Gene Knockout On Growth And Development Of Petunia Hybrida

MicroRNA(miRNA)is a kind of non-coding RNA with a length of about 20-24 nucleotides and regulatory function.Since the discovery of microRNAs,there have been many reports on the function and specific expression of microRNAs.Nowadays,microRNAs have been identified by scientists as important factors involved in various biological activities,including cell proliferation and differentiation,autoimmunity of human diseases,biological and abiotic stresses.MiR159 gene family is one of the oldest and most conservative miRNAs in botany.Its evolutionary ancestors can be traced back to bryophytes.Current studies have found that the family consists of three members,namely,miR159 a,miR159b and miR159 c.The main members of the family are miR159 a and miR159 b.The major target genes of miR159 are MYB transcription factors,such as MYB33,MYB65,MYB97,MYB101 and so on.MiR159 gene family is rich in functions,not only involved in vegetative growth and reproductive growth of plants,but also in biological and abiotic stresses.Overexpression of MYB33,a target gene of miR159,resulted in smaller leaves and shorter petioles.MiR159 a and miR159 b mutants with functional deletion could not form complete flowers.MiRl59 b regulated tomato seed development and so on.Although there are some studies on the function of microRNA159 in many plants,there are few studies on the role of microRNA159 in the development of plant ornamental traits.The purpose of this experiment is to explore the function of microRNA159 in petunia,an ornamental plant,by means of gene knockout and overexpression:1.Construction of petunia miR159 a and miR159 b gene knockout vectors to obtain transgenic plantsUsing CRISPR/Cas9 gene editing technology,two target sites were designed toconstruct miR159 a and miR159 b knockout vectors,and the petunia was transformed by Agrobacterium infection method.After Basta screening,resistant strains were obtained,using CTAB method.The DNA of the resistant strains was extracted,and the deletion of the fragments of the three resistant lines was detected by PCR and gel electrophoresis.Finally,the miR159 b gene knockout of the three deletion fragments was confirmed by sequencing.The mutant strains were named 639-2,5,and 6respectively(wherein the mutation types of 639-2 and 639-5 were identical)..2.Phenotypic observation of miR159 b knockout mutantThe tissue culture seedlings of the knockout plants were transplanted to the field management for preliminary observation and collection of seeds,followed by seeding to perform phenotypic observation and data analysis on the Genome Editing 1(E1)mutant: miR159 b knockout mutant and Compared with the wild type petunia,the leaf area became larger,the plant height became higher,the number of leaves in the bud stage increased,and the flowering time was slightly later.The total RNA of flower buds was extracted and quantitatively analyzed by fluorescence.The expression of MYB1 in miR159bE1 mutant was higher than that of wild type,while the expression of MYB2 in miR159bE1 mutant was lower than that of wild type.3.Construction of overexpression vector of miR159bAccording to the primer sequence miR159b-F/miR159b-R of miR159 b gene,the miR159 b gene was cloned from wild type petunia,and the target gene was ligated with the cloning vector pMD19-T to construct an intermediate vector,and then pG605 was digested separately.The intermediate vector overexpressing with the miR159 b gene was ligated to construct the miR159 b overexpression vector,which provided favorable conditions for further analysis of the function of the miR159 b gene.