Study On The Role Of Post-transcriptional Regulation Of Genes In The Vivipary Developmental Process Of Kandelia Obovata

Plant vivipary refers to the phenomenon that the offspring germinates only after a short period of dormancy or without dormancy in the process of plant reproduction,which is most typical in some mangrove plants.Kandelia obovata has a typical viviparous phenotype in mangrove.It is generally believed that viviparous is a unique way of reproduction evolved during adaptation to the harsh environment in the intertidal zone of coastal wetland.Therefore,the study of viviparous process of K.obovata is helpful to understand the molecular mechanism of its adaptation to harsh environment.At the same time,alternative splicing(AS)and alternative polyadenylation(APA)play important regulatory roles in plant development.However,up to now,there is no report on the analysis of AS and APA during the viviparous development.Based on the transcriptome sequencing data of K.obovata,the AS and APA patterns in different stages and tissues were analyzed,and the differential AS genes(DASG)and differential expressed APA genes(DEAPAG)were investigated.The potential functions of AS and APA models were explored by functional enrichment analysis.Secondly,Key DASG and Key DEAPAG related to plant development were further analyzed.Then,through the functional enrichment analysis of Key DASG and Key DEAPAG,we explored the potential regulatory role of AS and APA in the seed development of K.obovata.Finally,selected target genes were verified by in vitro experiments.Our results provide a foundation for the understanding of molecular mechanism of vivipary in K.obovata and in mangroves.The main results are as follows.AS and APA patterns were very common in the process of viviparous development of K.obovata(the number of DASG was about 455-1976,and the number of DEAPAG was about 522-1374).For AS,the main types of AS were skipped exons,alternative 3’ splice sites and alternative 5’ splice sites,while retained introns and mutually exclusion exons were less.In terms of APA pattern,the expression of DEAPAG using the proximal poly(A)sites(short 3’UTR)was generally higher than that using the distal poly(A)site(long 3’UTR).It should be noted that APA pattern showed a certain trend in the development of hypocotyl(ax/hy),that is,the number of DEAPAG using the proximal poly(A)site(short 3’UTR)first increased and then decreased,while the number of DEAPAG using the distal poly(A)site(long 3’UTR)first decreased and then increased.For AS,the GO enrichment analysis results of DASG at different stages showed that the functions regulated by DASG were closely related to the biological processes necessary for the viviparous growth of K obovata,such as the metabolic process of macromolecules,enzyme activities and catalytic functions.The results of KEGG enrichment analysis also showed that the nucleic acid metabolic pathway necessary for the vivipary of K.obovata was regulated by DASG.In addition,GO and KEGG enrichment analyses of DASG show that they were mainly enriched in metabolism related processes,such as protein hydrolysis metabolism.Combined with the above two comprehensive analyses,it can be seen that the AS mode in the viviparous process of K obovata mainly plays a role through metabolism,enzyme activity and catalysis.As far as APA mode is concerned,functional enrichment analysis of DEAPAG at different stages showed that these genes were mainly enriched in transcriptional regulation,such as DNA binding and RNA binding.Compared with different tissues,DEAPAG was enriched in different signaling pathways.This suggested that the APA pattern in the process of viviparous process of K.obovata was closely related to the mechanism of post transcriptional regulation.In conclusion,the AS patterns of K.obovata during the viviparous process was related to metabolism and enzyme catalysis,while APA patterns was related to post transcriptional regulation.In the aspect of AS,the number of genes regulated by AS was increasing with the development of both axis(ax/hy)and cotyledon tissues(from 34 to 147).There was no significant change in the number of AS genes(about 46-89)related to plant development among different tissues.Compared with each other,the development related AS model was stronger than that caused by tissue differences,which indirectly suggested that AS played an important role in development.In the aspect of APA,the genes regulated by APA were mainly concentrated in the late stage of hypocotyl development(DEAPAG in S4ax-S5hy and S5hy-S6hy groups were more than that in S3ax-S4ax groups).In terms of cotyledon(co)development,APA regulated genes were mainly concentrated in the early stage of development(DEAPAG in S3co-S4co group was more than that in S4co-S5co and S5co-S6co groups).However,the number of DEAPAG in different tissues did not change significantly(about 69 to 139).This suggested that the trend of APA pattern was different for different tissues.The results of functional enrichment analysis showed that the functions or signaling pathways annotated/enriched by Key DASG or DEAPAG were relatively conservative(broad-spectrum effect)and specific for a certain group(tissue-specific).For example,in the case of DASG produced between different developmental stages and tissues,or DEAPAG produced between different developmental stages and tissues(broad spectrum),these genes were mainly annotated into plant hormone related pathway,metabolism related pathway and biosynthesis related pathway,such as plant hormone signaling pathway,carbon metabolism and flavonoid biosynthesis.It was worth noting that DEAPAG of hypocotyls was uniquely annotated into MAPK signaling pathway(specific)from S3 to S4.RT-PCR and RT-qPCR results showed that the gene expression levels and AS types of four DASG(HAB1,FC2,BHLH47,GAMMACA1)were consistent with our transcriptome analysis results.The results of 3’RACE and RT-qPCR show that the usage and expression trend of poly(A)sites of four DEAPAG(EMB3003,ARF8,AFB2,PP2AA)were consistent with our prediction.That was,PP2AA has different PA sites in S5 and S6 hypocotyls,AFB2 has different PA sites in S4 testa and S4 axis.In summary,there are few studies on vivipary of mangroves.Thus,it is important to analyze the transcriptome data during the viviparous process of K.obovata.Based on the transcriptome sequencing data of different stages and tissues of K.obovata viviparous development,the possible AS and APA patterns were analyzed,which can help researchers to further understand the transcriptome changes during the viviparous development and provide innovative ideas and theoretical basis for future exploration on the molecular mechanisms of the viviparous development and adaptation of mangrove to their environment in general.