The Functional Characterization Of BrLMaP,a Long Non-coding RNA In Brassica Rapa

Long non-coding RNAs(lnc RNAs)is a class of RNAs whose transcripts are greater than 200 nt and have no coding potential.They function in the form of RNAs to regulate gene expression through epigenetic,transcriptional and post-transcriptional regulation levels.Some studies have shown that lnc RNA is involved in the regulation of plant growth and development.In our previous study,we performed RNA-sequencing(RNA-seq)on the flower buds at five developmental stages of a genetic male sterile systerm ‘Bcajh97-01A/B’ in Brassica rapa,and identified a candidate lnc RNA,BrLMaP(lnc RNA of BrMYB80 a promoter),which is specifically expressed in male sterile line of ‘Bcajh97-01A/B’.The transcription factor MYB80 is a key regulator in pollen extine formation in Arabidopsis.The knock-out of MYB80 resulted in completely pollen abortion.Two genes encoding MYB80 are present in the genome of B.rapa,they are Bra002847 and Bra035604(named BrMYB80 a and BrMYB80 b,respectively),which are located on chromosome A10 and chromosome A02,respectively.The non-coding BrLMaP was found to be specifically located in the promoter region of BrMYB80 a.We speculate that BrLMaP may participate in the pollen development through regulating the expression of BrMYB80 a and/or BrMYB80 b.In this study,the function of BrLMaP in pollen development were explored.Firstly,the fulllength sequence of BrLMaP was obtained by rapid amplification of cDNA ends(RACE),and the coding potential of BrLMaP was analyzed.Then,the expression of BrLMaP in different tissues were investigated.Finally,the over-expression and RNAi constructs of BrLMaP were constructed and transformed into Chinese cabbage ‘Youqing 49’ to explore its function in pollen development,and the changes in expression level of BrMYB80 a and BrMYB80 b were detected in the transgenic plants.The main findings of this study are as follows:(1)The full length of BrLMaP transcript is 557 nt,and three introns were found in the BrLMaP sequence.Coding potential analysis showed that BrLMaP has very low coding potential,and there was no open reading frame(ORF)great than 200 nt was found in BrLMaP sequence.BrLMaP is a sense long non-coding RNAs.(2)The specific expression of BrLMaP in the srerile ‘Bcajh97-01A’ line was verified by realtime quantitative PCR(qRT-PCR).The expression diversity in the two copies of MYB80(BrMYB80 a and BrMYB80b)in B.rapa were also identified in ‘Bcajh97-01A/B’.The results showed that BrLMaP,BrMYB80 a and BrMYB80 b were sepecic to infloresence and highly expressed at the uninucleate microspore stage of ‘Bcajh97-01A’,however,compared with the expression of BrMYB80 a and BrMYB80 b in ‘Bcajh97-01B’,the high expression period of them in ‘Bcajh97-01A’ was delayed.(3)The over-expression vector of BrLMaP was transformed into Chinese cabbage ‘Youqing 49’,and the cytopathological and morphological observation of the transgenic plants showed that overexpressing of BrLMaP resulted in giant pollen with four apertures.Start at the binucleate microspore stage,the cytoplasm of pollen began to degrade.In vitro germination assay showd that the 32% of the transgenic pollen could not germinate and 35% burst at the tip of pollen tube.The expression levels of BrMYB80 a and BrMYB80 b were all down-regulated in BrLMaP overexpressing transgenic plants,suggesting that BrLMaP might participate in pollen development by regulating the expression of BrMYB80 a and BrMYB80 b.(4)The RNAi vector of BrLMaP was constructed and transformed into Chinese cabbage ‘Youqing 49’.The pollen viability of transgenic plants with down-regulation of BrLMaP was found to be normal through Alexander staining.However,by scanning electron microscopy,a more dense structure of pollen surface of transgenic plants was found than that of control plants.An increasing expression level of BrMYB80 b was detected in the transgenic lines,while no significant change in the expression of BrMYB80 a was detected.The up-regulation of BrMYB80 b may cause excessive accumulation of sporopollenin on pollen surface.(5)Overall,regarding the expression change patterns of BrMYB80 a and BrMYB80 b in BrLMaP over-expressing and RNAi transgenic plants,we found that the expression of BrMYB80 b was negatively correlated with BrLMaP.It is speculated that BrLMaP might regulate the expression of BrMYB80 b in trans.However,the expression of BrMYB80 a was also negatively correlated with BrLMaP in the over-expressing transgenic plants,but there was no significant change detected in the BrLMaP silenced plants.The reasons need to be further studied.