| Self-incompatibility(SI) is a special reproduction barrier and adopted by many flowering plants in evolution which is a flowering plant promote cross-pollination of reproductive isolation mechanism to maintain the genetic diversity of plants, which played a very important role in the evolution of angiosperms. At present, most orchids are self-compatibility, but there are also self-incompatibility which Dendrobium accounted for about 3/4.To understand the SI mechanism between pollen and stigma, we detected the expression of the stigma differentially expressed transcriptome between self-pollination and cross-pollination by Illumina technology. The Dendrobiums we used were provided by Orchid Conservation & Research Center of Shenzhen.The results were as follows:1)Based on the RNA-seq about Dendrobium, we received a total of 51,577,372 clean reads(about 6.45G). Finally 91,457 unigenes with certain reliability were assembled by SOAPdenove with an average length of 1105 bp.2)By similarity search against protein databases, a total of 25,208 unigenes matched to gene models, most of which were functionally classified into 44 GO terms, 25 COG classes and 256 KEGG pathways.3)Unigenes including three transcriptome were ranking according to expression(RPKM value), in order to screening the high abundance gene between crosspollination 4 hours(YH4) and self-pollinated 4 hours(ZH4). Compare to selfpollination and the control group, we screened a total of 2056 differentially expressed genes, of which there are 1238 genes showed up-regulated and 818 genes downregulated. Cross-pollination comparison to the control group, we selected 2284 differentially expressed genes, including 1634 genes up-regulated and 650 genes downregulated. In the cross-pollination and self-pollination comparison, we selected a total of 1856 genes, of which there are 586 genes showed up-regulated and 1270 genes down-regulated.4) Eight unigenes were amplified verification by RT-PCR method, which were consistent with the trend DEG sequencing results, so the qualitity of this transcriptome were better.Which we found that DC-ADK2(Unigene1595) in the expression of selfpollination were eight fold change compare to cross-pollination in the transcriptome using RT-PCR validation. In order to study the role of DC-ADK2, we cloned it and analysis its express pattern. Thus we found that during the period of pre-pollination DC-ADK2 gene have no significant effects in Arabidopsis, but in the late pollination it affect the growth of the pod, resulting in shorter pods and the number of seeds in single pod is reduced. Also we found that DC-ADK2 affect the growth and development in Arabidopsis.5) The conservative and self-incompatibility related miRNA of Dendrobium were also studied in this paper. We use Solexa sequencing to obtain a sequence length of small fragments 18-30 nt, where in the measured maximum number of small RNA is 24 nt, total clean reads in the final three groups were obtained 7,933,907?8,324,131?7,497,645 clean reads and then use Rfam(10.1) database comparison, the final classification s RNA comments screening, the total number of three groups respectively miRNA is 77,015?59,882?44,994. The resulting analysis is known, novel miRNA target gene prediction were a total of 9,829 pieces of predicted target genes. We selected 11 from the difference table to perform miRNA RT-PCR validation, including seven known miRNA and four novel-miRNA. Results are consistent with the results of highthroughput sequencing. |
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