Circadian Expression Regulation Of One Alternatively Spliced Transcript By Another Of Glycine-Rich RNA Binding Protein To Control Flowering Time

There is a great expansion of transcriptome through alternative splicing（AS）,named spliceome,signifying its genetic importance for crop improvement.Flowering time is an important agronomic trait influencing the reproduction and yield of crops.The objectives of the study are to determine some of the AS characteristics of a flowering time control gene GRP8（Glycine-rich RNA-binding Protein）from Arabidopsis thaliana（A.thaliana）to Brassicas of the Brassicaceae family,the expressional and functional divergence of AS of GRP8 transcripts in flowering time control and regulation of AS events in Brassica napus（B.napus）.The following results were obtained.1.The conservation of ASGRP8 has RRM（RNA recognition motif）located at C terminus and glycine rich stretches at N-teminus.The gene full length is 1.388kb with two exons and one intron.In Brassicas,there are four orthologs of AtGRP8,two in the A genome of B.rapa and B.napus and two in the C genome of B.oleracea and B.napus.BnGRP8 has overall 88%sequence similarity with its orthologs in B.rapa and B.oleracea.One ortholog BnaC01g00280D（named as BnGRP8 hereafter）displays a predominant expression level（several folds）over the other three in different tissues.GRP8 produces multiple transcripts.In Arabidopsis,the TAIR database shows four transcripts from GRP8,and among them,except for the first full CDS（GRP8.1）of 510 bp,the other three transcripts are produced from exon skipping in the second exon.By RT-PCR,however,we detected the fifth transcript,named as AtGRP8.2,which is produced from an alternatively spliced first intron at the 5′cryptic splice site（158 bp of the intron retained to form a 668 bp transcript）.The splicing results in an interruption of a RRM domain of the gene and introduction of pre-termination codon（TGA）(16^thcodon)in the transcript.The RT-PCR results showed that these transcripts have differential transcript abundance.All Brassica orthologous GRP8 produce both GRP8.1 and GRP8.2.GRP8 in the A genome of B.rapa and B.napus produce an additional AS transcript which retains the full intron.GRP8.1 and GRP8.2 are conservative among B.rapa,B.oleracea and B.napus.These five transcripts from B.napus A and C genomes differentially express,and GRP8.1 is predominant over GRP8.2 in both A.thaliana and B.napus.Because the predominant expression of BnaC01g00280D（BnGRP8）and conservation of both GRP8.1 and GRP8 in the four species,the study just focused on BnGRP8.1 and BnGRP8.2 afterwards.2.The function of alternatively spliced transcripts of BnGRP8 in flowering time controlFirst GWAS（Genome-Wide Association Study）was performed on a 131 B.napus diverse accession panel in which genotypic data were from whole genome re-sequencing and phenotypic data from multiple environment assessment of flowering time.The results showed that SNP in BnGRP8（BnaC01g00280D）strongly associated with flowering time.Expression monitoring over the 24hrs time course showed that in a 16h light/8h dark scheme,expression levels of both BnGRP8.1 and BnGRP8.2 increase when light is on at 8:00 and reach their peaks at 17:00,and then sharply decrease.BnGRP8.1 expression abundance is much higher than that of BnGRP8.2 in all time points sampled.AtGRP8.1 and AtGRP8.2 display a similar expression trend but AtGRP8.2 is higher than AtGRP8.1 at the peak time.These led to further function investigations of BnGRP8 with overexpression plants ox BnGRP8.1,ox BnGRP8.2,knockdown plants（RNAi-AtGRP8.2）and the T-DNA insertion mutant Atgrp8 in Arabidopsis.The vector ox BnGRP8.1 was transformed into both wild type（WT）and Atgrp8 whereas ox BnGRP8.2 and RNAi-AtGRP8.2 into WT only.The comparison of the mutant and WT indicated that BnGRP8 promotes flowering and days to flowering among different transgenic plants are with a sequence:the mutant Atgrp8>ox BnGRP8.2/WT（WT background）>WT>ox BnGRP8.1/Atgrp8>ox BnGRP8.1/WT>RNAi-AtGRP8.2/WT in both long and short day conditions,indicating a strong negative correlation between flowering time and expression quantity of GRP8.1 where dynamic suppression of the transcript GRP8.1 by GRP8.2 plays a crucial role.Histochemical analysis with a GUS reporter where GUS was fused with intronless GRP8.1 and GRP8.2 and full length DNA of the gene under the native promoter confirmed the interfering role of GRP8.2 on GRP8.1.3.The regulation of transcript abundance and alternative splicing of BnGRP8A series of transgenic plants with different combinations of gene sequence fragments were produced to answer which fragment determines transcript abundance of BnGRP8.1 and BnGRP8.2.These sequence fragments to form constructs included 1)P_BnGRP8+BnGRP8.1（transcript sequence）+GUS,2)P_BnGRP8+BnGRP8.2（transcript sequence）+GUS and 3)P_BnGRP8+full gene sequence with 5’UTR and3’UTR.The results indicated that 1)Both BnGRP8.1 and BnGRP8.2 highly express in different tissues of transgenic plants with the constructs of either P_BnGRP8+BnGRP8.1+GUS or P_BnGRP8+BnGRP8.2+GUS,but expression is compromised in transgenic plants with P_BnGRP8+full gene sequence with 5’UTR and 3’UTR.Furthermore,the comparison of native P_BnGRP8+full gene sequence with 5’UTR and 3’UTR and 35S promoters P_35S+full gene sequence with 5’UTR and 3’UTR indicates promoter does not affect AS,suggesting that AS regulation is independent of a native promoter and UTRs of the gene.These suggest that only the intron of BnGRP8 gene has the signal for the production of BnGRP8.2.In this study,we determined that the time coursed AS transcripts of BnGRP8 work in coordination to control timely floral transition.The intron retained BnGRP8.2 regulates the abundance of constitutive transcript and spatiotemporal transcript abundance of the gene.This AS is conserved in Brassica species despite of long evolutionary history.The AS occurrence is dependent on splice signal present in intron.The findings in the study enhance our understanding of AS,its impact on gene expression regulation and phenotypes,and provide information for genetic improvement.