Functional Identification Of Long Non-coding RNA-LncRNA170 Regulating Trichome Formation In Tomato

Trichomes are the outermost specialized structures originating from plant epidermal cells and play an important role in the resistance against insect and virus.Meanwhile,due to their simple structure,trichomes are also used as a model system for studying the regulation of plant cell fate determination.At present,many transcription factors regulating trichomes formation have been cloned,including MYB transcription factors,b HLH transcription factors,and WD40 transcription factors,etc.Recently,it has been suggested that long non-coding RNAs(lnc RNAs)function in many developmental processes and stress responsesin Arabidopsis,tomato,rice,and maize.However,the role of Lnc RNAs in trichome formation remains largely unknown.Therefore,it is worthwhile to identify the biological function of Lnc RNAs in tomato glandular trichome formation.Our previous studies showed that 1303 Lnc RNAs are detected based on the RNA-seq data of glandular hairs isolated from Wo gene mutant LA3186,Ailsa Craig and LA3186.Moreover,we determined that 30 lnc RNAs are obviously up-regulated in tomato glandular trichomes and 29 lnc RNAs are down-regulated,suggesting that lnc RNAs may be involved in the regulation of trichome formation.Based on these results,we identified the regulatory roles of the Lnc RNAs highly expressed in trichomes through transgenic analysis in this study.We found that overexpression of lnc RNA170 in the Wo gene mutant LA3186 resulted in the disappearance of trichomes,indicating that lnc RNAs might be essential for tomato trichomes formation.Our findings sheds new light on the molecular mechanism underlying trichomes formation.Main results are as follows:1.Based on our previous transcriptome data,we further determined that Lnc RNA170 is obviously up-regulated in trichomes by QRT-PCR.In addition,bioinformatics analysis revealed that Lnc RNA170 is located on the complementary strand of a protein-coding gene Solyc10g006360.Therefore,it is speculated that both may be involved in the trichomes formation.2.Based on the c DNA sequence and genomic sequence of Lnc RNA170 and Solyc10g006360,we designed primers for targets of CRISPR vectors,overexpression vectors as well as promoter vectors by using the online software.The double-target and three-target CRISPR/Cas9-Lnc RNAs vectors are constructed by using PTX and p201 N Cas9,respectively.Overexpression vectors of Lnc RNAs are constructed by using p Hellsgate8,and the Lnc RNAs promoter is used to drive the GUS vector by p MV2-GUS.3.The vectors containing the target gene fragments are transformed into Wo gene mutant LA3186 using the Agrobacterium-mediated genetic transformation.PCR results indicated that T-DNA is integrated into the tomato genome.After acquiring the transgenic plants,the positive rate of the transgenic plants is detected by PCR.The results indicated that the foreign gene is integrated into the tomato genome.4.Through the phenotype analysis of transgenic plants,it is found that overexpression of Lnc RNA170 resulte in a dramatically decrease in the number of type I glandular hairs of tomato stems,and a small layer of hair is attached to the surface.In addition,overexpression of Solyc10g006360 resulted in a similar phenotype,indicating that Lnc RNA170 and Solyc10g006360 played a key role in the formation of this type of glandular hair.Q RT-PCR showe that the expression level of Lnc RNA170 is only slightly increased,while the expression level of overexpressed transgenic plant Solyc10g006360 is significantly increased.5.The background material of the transgenic plants is the Wo gene mutant LA3186,the phenotypic separation appear in the offspring of the LA3186 due to the Wo gene homozygous embryo lethal.Therefore,in order to confirm that whether transgenic material contains the Wo gene,we performed background detection.Mutation of the Wo gene leads to changes in the cleavage site,and PCR amplification combined with enzyme digestion is used to detect the results.The results showed that they all contained Wo gene mutation sites.6.The target site amplification primers are designed based on the genomic sequence information of the location of the target site.The fragment containing the target site was amplified by PCR and submitted to the biotechnology company for sequencing.The alignment of the sequencing results with the reference genome sequence reveal that the T0 generation of the CRISPR Cas9 transgenic plants have different types of mutations or deletions.Strangely,In the unedited Lnc RNA898 transgenic plants observed that the density of type I hair was significantly reduced and the density of short hair on the stem surface was significantly increased.It is speculated that this phenomenon may be caused by off-targeting lead to non-target genes being edited.