Bioinfomatics Analysis Of Natural Antisense Gene And Alternative Splicing On Pepper

Pepper is an important vegetable crop,which is widely loved for its economic value and unique flavor.In the past few years,significant advances have been made in sequencing the pepper complex genome,with results published for ‘CM334’ from Mexico and the ‘Zunla’ from China,providing an opportunity for the development of pepper functional genomics.In order to better identify the complete gene structure of pepper and the regulatory region of pepper gene,we used Pac Bio full-length m RNA sequencing to obtain more complete information of gene un-translation region and more accurate genes splicing site,further improving the integrity and accuracy of pepper genome annotation.Natural Antisense Transcripts(Umezawa et al)are a kind of endogenous RNA with mutual regulation found in eukaryotes in recent years.In recent years,studies have found that NATs play an important regulatory role in different biological processes,such as gene transcription level,post-transcription level and RNA editing,which has attracted more attention.Transcriptome sequence is the basis of NATs identification,so complete genome annotation is particularly important for identifying more NATs and later exploring their functions.We used Pac Bio sequencing to obtain more complete and more accurate full-length m RNA data of pepper,providing an important data basis for the identification of NATs in pepper.On the other hand,alternative splicing(AS)of m RNA precursors plays an important regulatory role in response to biological and abiotic stresses and developmental signals in plants.Therefore,second-generation strand-specific sequencing results allow us to more easily explore the differences in the occurrence of alternative splicing.Based on the second and third generation sequencing technologies,we analyzed the transcriptome data of pepper at different stress,hormone treatment and development stages,and identified many cisNATs,trans-NATs and AS events in response to these biological processes,suggesting that nat-si RNA and alternative splicing play a wide regulatory role in these processes.The main results are:(1)Full-length transcriptome sequencing were used to promote pepper genome annotation.We mixed RNA samples from different stress treatments and different development stages of pepper,and obtained a total of 57,204 full-length transcript and5,769 were newly detected genes.In previous pepper genome annotation,a longer intron or a lower exon expression enables an entire gene to be annotated into multiple gene fragments.In this study,476 new complete genes in pepper were identified,and the accuracy of gene structure was identified by RT-PCR.(2)NATs mainly rely on s RNA for regulation.Based on the pepper new developed annotation in this study,we identified approximately 10,000 genes in pepper formed NATs,including 2057 pairs of cis-NATs(containing 4114 genes)and 635 groups of trans-NATs(containing 5880 genes).Further genome-wide prediction of repeat m RNA sequences showed that trans-NATs contained more transposon fragments(55.1%,3176 of 5766).Finally,s RNA enrichment analysis showed that s RNA could significantly enrich in the trans-NATs complementary sequence region and cis-NATs exon complementary sequence region.(3)cis-NATs play a major role in different abiotic stress,plant hormone treatment,and different developmental periods.We identified that 27 pairs of cis-NATs can respond to different biological processes.For example,formate dehydrogenase 1 Ca FDH1 is necessary for the disease-resistant response mediated by plant R gene,while the positive feedback regulation of the salicylic acid pathway involved by Ca FDH1 gene may be controled by nat-si RNA.For another example,Ca MPK4 and Ca ICU2,both biological and abiotic stress response genes,are dependent on the regulation of cis-NATs,and these two genes can be induced to express in the abiotic stress,hormone and plant development of pepper.Ros scavenging gene Ca DJ-1 is also involved in the response to capsaicin hormone treatment and development.Meanwhile,Ca BSK1,a kinase gene involved in Brassica Napus lipid signaling and plant innate immune response,forms an opposite gene expression regulation pattern in plant hormone treatment and pericarp development.Ca PDCT and Ca MBD10 showed opposite regulation patterns of cis-NATs gene expression during the development of pepper petal and placenta.We also found that Capana02g002369(Ca Cis UK1),a pepper specific gene,plays a negative regulatory role in the process of plant disease resistance by using the expression of tomato bushy stunt virus fused the green fluorescent protein.(4)The process of pepper color may be controled by trans-NATs.We analyzed the expression profiles of different biological processes and identified 81 pairs of trans-NATs subgroups with expression regulation function.Among them,PIP5 K,BARD1 and CER10 genes form the same expression trans-NATs subgroup,and all of them are involved in the development process of seeds.We also found that the catalytic lycopene to the δ-carotene of lycopene,cyclase Lcy E genes,as the change of plant growth period by trans-NATs inhibition of expression,the situation well explains the pepper fruit on a specific development directs the lycopene to capsanthin and capsaicin synthesis pathway to promote colouring phenomenon.(5)Using the comparative genomics method,we analyzed cis-NATs of dicots arabidopsis,tobacco,potato,tomato and pepper.We found that the number of cis-NATs increased significantly during the differentiation from brassica to solanaceae.Through the analysis of cis-NATs evolution,it is found that the increase of intergenic sequences rather than the insertion of large fragments is the main way to cause the disappearance of conservative cis-NATs.The insertion of transposons into longer introns is the main cause of new cis-NATs in pepper,while the insertion of transposon fragments into gene promoters and terminators or their attachment regions is the main cause of new cis-NATs in tomato.(6)AS regulated different biological processes of pepper.Through the analysis of genome-wide alternative splicing(AS)events and the difference analysis of AS in different biological processes,we found that the AS events of intron retention type(RI)presented a gradually changing trend in different development processes of pepper,and the AS events of RI type in pepper leaves and roots significantly increased under heat stress.Ca NAC82 and heavy metal transporter protein showed persistent exon loss(SE)changes during heat stress and placental development,respectively.