| Auxin regulates stem cell regeneration for the induction of somatic embryogenesis At the same time, auxin response factor which is located in the center of auxin signal transducti-on is regulated by the small RNA. Small RNAs and target gene auxi response factor (ARFs) were cloned from different embryogenic cultures in Dimocarpus longan Lour. by the RT-PCR combined with RACE method, and carries on the bioinformatics analysis.Then validate the cleavage sites of the target genes TAS3, ARF3, ARF4, ARF6, ARF8, ARF10, ARF16, ARF 17 with RLM-RACE method, and the mRNA transscripti-on level of Small RNAs and ARFs in the process of somatic embryogenesis was determined by qRT-PCR (real-time reverse transcription PCR) method, and analysis its regulation mechanism.ln this experiment,overexpression dlo-pri-TAS3 in Micro-Tom tomato and detect the expression of miR390, ARF3, ARF4 expressionThe main results were as follows:1 Obtaining the primary transcripts of miR160,miR167,miR390, TAS3 and analysis of bioinformation from longanThe pri-miR160, pri-miR167, pri-miR390, pri-TAS3 genes were cloned from different culture materials in Dimocarpus longan Lour "hong he zi". by the RT-PCR and they were named as dlo-pri-miR160, dlo-pri-miR167, dlo-pri-miR390, dlo-pri-TAS3 respectively; and the mRNAs full-length sequences of them were 441 bp,185 bp,785 bp,579 bp respectively. Bioinformatics analysis suggested that all of them had the typical secondary structure of miRNA, and their free energy of secondary structure were much higher than the average free energy of the general plants. The phylogenetic tree showed that dlo-pri-miR160, dlo-pri-miR167, dlo-pri-TAS3 were closed to Solanum lycopersicum, Citrus sinensis, Solanum lycopersicum in genetic relationship respectively, but dlo-pri-miR390 was far away from Arabidopsis thaliana, Vitis vinifera, Glycine max other plants in genetic relationship.2 Cloning and bioinformation analysis of target genes ARFs from embryonic callus in Dimocarpus longan Lour.The full-length cDNA of ARFs genes were cloned from embryogenic callus of Dimocarpus longan "hong he zi" by the RT-PCR combined with RACE method. Obtaining the target genes ARF3 (KJ200347.1), ARF4 (KJ461667.1), ARF6 (KJ410230), ARF8 (KJ410232),ARF10 (KJ410234), ARF16 (KJ410235), ARF17 (KJ410236), the mRNAs full-length sequences of them were 2561 bp,2698 bp, 3381 bp,2868 bp,2093 bp,2279 bp,2098 bp respectively. Bioinformatics analysis showed that protein D1ARF3, D1ARF6, D1ARF8, D1ARF10, D1ARF16, D1ARF17 were probably an soluble protein. D1ARF6, D1ARF8, D1ARF10, D1ARF16 consisted of a B3, Auxin-resp and AUX-IAA conservative district and structural function domain, but the protein D1ARF3 and D1ARF17 just consisted of a B3, Auxin-resp conservative district and structural function domain. Gln, Ser and Leu-rich middle region, which was considered as an activation domain.Phylogenetic analysis showed that auxin response factor family member of longan were closed to the auxin response factor family member of Arabidopsis thaliana in genetic relationship respectively,3 Validate the cleavage sites of the target genes TAS3 and ARFs5’-RACE PCR of mRNAs by using target genes specific primers from mixture of embryo callus, globular embryo, heart embryo, torpedo embryo and cotyledon embryo different culture materials in Dimocarpus longan Lour "hong he zi". we can found that the cleavage sites in dlo-pri-TAS3 was TTA/TCC,and cleavage sites in the ARF3, ARF4 recognition sequences are the second sites, GCA/AGG, CAA/GTT respectively. However, cleavage sites in ARF6 was found outside the recognition sequence of miR167, and only one of the cleavage sites in the ARF8 inside the recognition sequence of miR167, GCU/GGC.The same cleavage sites of ARF10, ARF16, ARF17 was AGG/GAG4 Analysis of small RNAs and ARFs expression profiles during Somatic Embryogenesis in Dimocarpus longan Lour.In this experiment, the expression patterns of dlo-pri-miR160, dlo-pri-miR167, dlo-pri-miR390, dlo-pri-TAS3 and target genes ARFs were analyszed by qPCR under the hormones 2,4-D. The main results were as follows:firstly, dlo-pri-miR160 and its target genes ARF10, ARF16, ARF17 have expression and split in globular embryo period; but undetect the expression signal of dlo-pri-miR167, it’s target genes ARF6 and ARF8 have the opposite change tendency. There was a significant difference expression between dlo-pri-miR390 and dlo-pri-TAS3 in the globular embryo period, and the expression pattern of ARF4 was close to the dlo-pri-TAS3 gene, the expression levels of ARF3 during longan EC were stable. In addition, dlo-pri-miR160 has high expression levels than it’s target genes ARF10, ARF16, ARF17 in seed and root of longan "si ji mi". dlo-pri-miR390, dlo-pri-TAS3, ARF3 and ARF4 were exprissed in nine tissues and organs, and has the highest expression of the quantity in the roots. Although undetect the expression signal of dlo-pri-miR167, it’s target genes auxin response factor ARF6, ARF8 in bud, root had high expression levels. The expression patterns of dlo-pri-miR160, dlo-pri-miR167, dlo-pri-miR390, dlo-pri-TAS3 and target genes ARFs under different concentrations of 2,4-D.Such as the relative expression level of ARF10 activity rose quickly and went to the peak at 2.0 mg/L, the expression levels of dlo-pri-miR160 andARF16, ARF17 were low and the difference were not significan. Then auxin response factor ARF6, ARF8 activity rose quickly with the increase of auxin concentration, the significant expression difference between the two genes was at the concentration of 0.5 mg/L. We can found that the expression levels of dlo-pri-miR390 slightly low and stable, dlo-pri-TAS3 has a higher expression level in 1.0,2.0 mg/L, and the relative expression level of ARF3,ARF4 had a peak value,when the concentrations of 2,4-D was at 1.0 mg/L. At the same time, ARF3.ARF4 activity dropped quickly at 1.0 mg/L of 2,4-D.5 Genetic transformation into Micro-Tom tomato and function identification of dlo-pri-TAS3The pCAMBIA1301-dlo-pri-TAS3 expression vector was constructed by digestion technology, and transformed into Agrobacterium EHA105. Agrobacter ium-mediatedtransformation of Micro-Tom tomato plants. The results of PCR detection showed that dlo-pri-TAS3 genes have been successfully transferred into Micro-Tom tomato.Compared with the control group of normal Micro-Tom tomato, the transgenic tomato plant phenotypic exists obvious difference, such as the curley leaves, stout stems and thick roots. The expression levels of miR390, ARF3, ARF4 in transgenic tomato plants by qPCR method.The results showed that the expression levels of auxin response gene ARF3, ARF4 in transgenic tomato were significantly lower than the control level. It was speculated that overexpression of dlo-pri-TAS3 gene in transgenic tomato plants inhibit strongly the expression of auxin response factor ARF3, ARF4, and dlo-pri-TAS3 played a important role during the development of plant roots, stems, leaves.Above all, small RNAs in longan,cleave the target tanscripts directly to influence somatic embryogenesis. We show that negative regulation of ARF10, ARF16, ARF17 by miR160, ARF6 and ARF8 are targeted by miR167, ARF3 and ARF4 are targeted by TAS3, and TAS3 is regulated by miR390 negativly. In the bud, flower, leaves,small fruit, big fruit, fruit pulp, fruit, seeds and roots of longan "si ji mi", the qPCR analysis showed that longan small RNAs and their target genes had a certain expression, and had a higher level of transcription in the roots. However, in the expreriment, the expression signals of pri-miR167 was still undetected. The root, stem and leaf of transgenic plants are different from the normal tomato plants.Therefore, small RNAs and their target genes ARFs form a network control system to effect the development of plants. |
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