Identification And Expression Analysis Of Saponin Biosynthesis-related Genes And Genome-wide Analysis Of MiRNAs In Asparagus Officinalis L.

Garden asparagus (Asparagus officinalis L.), belonging to Lily aspartic genera, is an important diecious plant specious. It is highly nutritional, containing high content of proteins, carotenes, vitamins and many kinds of mineral elements. Modern pharmacology study shows that asparagus is not only edible tender bine, but also have important medicinal value with the effects of antioxidant, anti-cancer, immunity, cardiovascular and cerebrovascular diseases which is because its abundance of flavonoids, many kinds of vitamins and especially with high content of steroidal saponin. But the research for asparagus steroidal saponins biosynthesis was still limited. To elucidate the molecular basis of asparagus saponin synthesis and the regulation of accumulation, the RT-PCR and Rapid-amplification of cDNA ends were used to clone the full length cDNA. of farnesyl pyrophosphate synthase (FPPS), squalene synthase (SQS), Squalene epoxidase (SE),2,3-oxidosqualene cyclases (OSCs). The gene expression profiles of FPPS, SQS, SE, CAS genes were characterized using qRT-PCR. in different organs and different developmental stage, On the other hand, increasing evidances have proved that the miRNAs play an important regulating role in many bioprocess, including growth, biological or abiotic stress resistance and so on. In our research, we constructed two sRNA libraries of female and male asparagus plants. With the highthroughput sequencing technology, we try to distinguish the difference between female and male asparagus in the level of miRNA, so as to find out the the case of asparagus sex differention, which may provide some theoretical basis for the research of asparagus sex differenation. Since asparagus has become a model for the investigation of early sex chromosome evolution for several dioecious species, the research may provide certain theoretical basis for exploring the evolution of plant sex chromosome.The main results are as fellows:(1) The enzyme genes of including farnesyl pyrophosphate synthase (FPPS), squalene synthase (SQS), Squalene epoxidase (SE),2,3-oxidosqualene cyclases (OSCs) were cloned from Asparagus officinalis L through RT-PCR and Rapid-amplification of cDNA ends methods. The cDNA of AoFPPS was speculated to code 349 amino acid flanked by a 54 bp 5’-untranslated region (UTR), and a 301bp 3’-UTR. The cDNA of AoSQS was speculated to code 409 amino acid flanked by a 88 bp 5’-UTR and a 481 bp 3’-untranslated region. The cDNA of AoSE was speculated to code 527 amino acid flanked by a 75 bp 5’-untranslated region, and a 253 bp 3’-untranslated region. The cDNA of AoCAS was 2556bp and could code 760 amino acid flanked by a 79 bp 5’-untranslated region, and a 194 bp 3’-untranslated region.(2) To evaluate the distribution of saponins in asparagus, the total saponins from root, steam, leaf, flower and spears with different length of asparagus were extracted using ultrasonic extraction method, and the content in different asparagus organs were compared using the Anisaldehyde-sulfuric acid colorimetry. The results showed that the content of saponins from different organs had significant difference. Among these organs detected, the leaf had the highest level of saponin content with 29.28 mg/g DW. During the development of spears, the cotent of saponin from middle spears was lower than top and bottom spears.(3) To investigate the expression pattern of AoFPPS, AoSQS, AoSE, AoCAS, qRT-PCR with specific primers was processed at different organs (root, stem, leaf, flower and fruit). The results show that the AoFPPS, AoSQS, AoSE, AoCAS genes exhibited different expression pattern in different organs and spears of different part in various developmental stages. All the four genes had a lower expression level in root. In steam and flowers, the expression level of AoFPPS was higher and the AoSQS, AoSE gene had the highest expression level in steam, and AoCAS had the highest expression level in flowers. All of these four genes had lower expression level in the spears with the length of 4-5 cm.(4) With high-throughput sequencing technology, about 153 known miRNAs and 39 new candidate miRNAs that had not been detected before were identified in garden asparagus. The 153 known miRNAs could be classified into 24 families and all of them may perform their respective functions in various life activities, eg:miR172a regulate cell differentiation and seed development, miR171 family regulate the cell divition, miR167a regulate flower development and the miR166d has an important role in defense response. Among the miRNA identified from asparagus, a total of 53 miRNA had different expression level between male and female plants. Basing on the results of degredome sequencing, a total of 35 miRNAs of asparagus found its target genes. And some of the had the function of floral organ differentiation or sex determination, such as miR159、miR167、 miR172. According to the analysis of Gene Othology (GO), among the 35 target gene found in asparagus,only 15 of them could map to Gene Othology, this 15 target gene could be clasfied into 21 different biological process, lo kinds of cell components and 18 Molecular Function.