Identification Methods Of Long Non-coding RNAs During Pollen Development And Fertilization In Brassica Campestris Ssp. Chinensis

Pollen formation, pollination and fertilization are very important processes for seed plants to complete sexual reproduction and alternation of genarations, which is closely related to the use of male sterile in production and to the yield and quality of seed. Revealing the molecular mechanisms of them has great significance for in-depth understanding of basic phenomenon of plant life, extensively earring out crossbreeding in practice, and improving yield and quality of seed.In recent years, the rapid development of high-throughput sequencing technology makes it possible for us to understand and grasp the plant reproductive development process from the genomic level, with Arabidopsis and rice as the representative species.Long non-coding RNAs (lncRNAs) as a class of non-coding molecules widespread in the genome, have been proved to be an important element in the regulation of gene expression in eukaryotes, however, about whether and to what extent they are involved in reproductive development processes such as pollen development, pollination and fertilization, there are ralely reports.According to the previous study, one hncRNA-BcMFll associated with Brassica campestris pollen development, we try to further study its expression and mechanism of action on the basis; at the same time, in order to filter more lncRNAs related to pollen development, pollination and fertilization, we use diffrent stage flower duds and different time pistils after pollination of’Aijiaohuang’genie male sterility (ajhGMS) AB line (’ajh97-01A/B’) in Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensisas as materials. Adopt high-throughput transcriptome sequencing (RNA-seq) technology to identify the candidate lncRNAs from genomic level; then make analysis of the characteristics of these lncRNAs including the sequence, the distribution in each chromosome and expression characters; on these basises we compare the differential expression of lncRNAs in I-V stage buds of male sterile line’Bcajh97-01’A1and fertile line iBcajh97-01B’; make analysis of the expression of lncRNAs throughout the processes of pollination and fertilization; at the same time, we make use of bioinformatics tools to analog the obtained lncRNAs which may serve as endogenous target mimic of miRNA and predict die target genes of lncRNAs as well as make the co-expression analysis of them; make similarity comparison and analysis of Brassica campestris and Arabidopsis lncRNAs; at last, dig the candidate lncRNAs that may be functional in the flower and pollen development acting according to the function of the target genes. The main results are as follows:(1) We cloned the BcMFll gene sequences, and found that it has multiple homologous sequences in B.rapa, B.oleracea and B.nigra genomes; By RT-PCR technique, we make analysis of the expression in different tissues of Brassica campestris, and found that it was not the same as previous study which believes that it specific expressed in the bud, but bias expressed in root.(2) Based on the use of high-throughput transcriptome sequencing (RNA-seq) technology in diffrent stage flower duds and different time pistils after pollination of ajhGMS’Bcajh97-0JA/B\we build up the unigenes library. And with further use of bioinformatics tools, we filter and identify12051highly credible Brassica campestris candidate lncRNAs. After verifing the expression of some lncRNAs by RT-PCR, we can come to the conclusion that both of these two methods have nearly the same results. After the characteristics analysis of these lncRNAs, we found that more than98%of the lncRNAs length are within1000bp, they uniformly distributed in each chromosome, though these lncRNAs are with low expression levels, their expression are correlated with the organizations and their development state.(3) After analyzing the expression of lncRNAs in I-V stage buds (1stage bud period corresponds to the pollen mother cells formation period of pollen development, II stage bud period corresponds to the meiosis period, III stage bud period corresponds to the mononuclear pollen formation period, IV stage bud period corresponds to the two nuclear pollen formation period, V stage bud period corresponds to the mature pollen period) of ajhGMS "Bcajh97-01A/B" male sterile line and fertile line and comparing the differential expression in the same stage buds, we found that during the development process of male sterile line’Bcajh97-01^’floral buds, stage IV bud are with the maximum expression of lncRNAs, stage V bud with the largest specific expression amount; during the development process of fertile line iBcajh97-OIB’’buds, stage III bud are with the maximum expression of incRNAs.stage V bud with the the largest specific expression amount; as for the overall lncRNAs expressed in the whole bud development process(I-V stage buds), fertile line iBcajh97-01B’are more than sterile line’Beqjh97-01’A’;as for the bud at the same stage, except stage IV, the fertile line lBcajh97-01B’’express more than the sterile line’Bcqjh97-01’A’’in other stages,in addition, stage I with the most common expression, while stage IV with the least.(4) After the expression analysis of IncRNAs in different time after pollination pistils(1h after pollination is the pollen germination on stigma phase,3h after pollination is the pollen tube growth in stylar canal phase,10h after pollination is the pollen tube reaches the ovule and fertilization phase), we found that3h after pollination pistil are with the maximum expression of lncRNAs; while compared to non-pollination control, part of IncRNAs expression was inhibited in1h after pollination, lead to the reduction of IncRNAs number in pistil.(5) After the sustained expression analysis of IncRNAs from the buds to the fertilization development processes,we found that about1/10of IncRNAs continued to express from bud formation to10h after polination, others are stage-specific expression.(6) After the use of bioinformatics means to search for endogenous target mimi (eTM) in all these Brassica campestris IncRNAs for miRNA, which preciously obtained in ajhGMS’Bcajh97-01A/B’floral buds, and get15candidate IncRNAs that may function as the endogenous pseudo target genes of miRNA.(7) After predicting of the Trans target genes of IncRNAs and co-expression analysis of them, a total of904IncRNAs and6236Trans target genes and found, among which144IncRNAs with their706target genes have co-expression relationship.(8)According to the functional annotation of the706target genes which has co-expression relationships with144LncRNAs, we find18candidate IncRNAs members may associated with flowers or pollen development.(9) All these Brassica campestris IncRNAs were used to make similarity ratio and conservative analysis with Arabidopsis IncRNAs downloaded from the Arabidopsis thaliana database. The result shows that only9percent of all these Brassica campestris IncRNAs exist similarities with Arabidopsis IncRNAs, and the match lengths rang from28bp to1657bp with relatively high sequence similarity matching (an average of87%).