| Hickory(Carya cathayensis Sarg.)has the problem of long juvenile period, high tree, harvesting and horticultural cultivation difficulty as one of the major economic trees for farmers to take off the poverty, which becomes a bottleneck. The plant grafting is a specific method to resolve these problems. With the development of grafting technology, people begin to study the grafting mechanism in order to guide grafting. Based on the original clone to get the primary auxin-responsive Aux/IAA gene(CcIAA) fragment cDNA, we obtained the full length cDNA by RACE technology, and did the homology alignment and phylogenetic tree analysis. Using qRT-PCR and Western blot the function of CcIAA gene at the transcriptional and transnational levels in the grafting process was analyzed. MicroRNAs(miRNAs) play crucial roles in plant growth and development negative regulation.To understand the roles of miRNAs involved in the hickory graft process, we constructed three small RNA libraries from hickory root-stock(2 years old) and scion(1 year old) of 0, 7, and 14 days post-graft. The main findings are as follows:1. There were 591 bp of CcIAA from the initiation codon to termination codon and encoding 196 amino acids. CcIAA was similar to the other 17 Aux/IAA genes from the sequence homology and amino acid sequence homology. The phylogenetic tree indicates CcIAA was closest to castor RcIAA gene and relatively far away from CaIAA gene.2. We used qRT-PCR technology to determine the different periods grafting(0d, 1d, 3d, 5d, 7d, 14d) expression pattern of CcIAA under the different hormone treatments(control, 4mg/l IAA and 0.3ug/l NPA). The results showed that under control, the expression of CcIAA genes in the root-stock and scion decreased sharply after grafting until 7 day and its expression level at 7 day after grafting was almost zero, but till to 14 day after grafting, the expression was also transiently increased and higher than that before grafting. Under 4 mg/kg IAA treatment, the expression of CcIAA sharply increased one day after grafting and decreased significantly in the following 4 days, then up to 14 day, whose expression level was increased. The expression of CcIAA in root-stock and scion are the same trend. Under NPA treatment, the expression of CcIAA was relatively low in 7 day after grafting and significantly increased until to 14 day after grafting not only in scion but also in root-stock. However, the expression of CcIAA under the IAA treatment was higher than that in the control, while the expression of CcIAA under the NPA treatment was less than the control.3. We used Western blot technique to determine the expression of CcIAA protein of different periods(0d, 1d, 3d, 5d, 7d, 14d) under the different treatments(control, 4mg/l IAA and 0.3ug/l NPA).The study showed that the expression of CcIAA proteins expression in the transnational level was not the same in the grafting process under the different treatment. Under Control, regardless of the root-stock or scion, the expression of CcIAA in the grafting is relatively low, and the expression significantly increased until to 14 days after grafting. Under 4 mg/kg IAA treatment, the expression of CcIAA protein expression is obviously relatively higher than that in control, especially the expression in the root-stock is higher than that in scion. Compared to the control, the expression of CcIAA protein was increased under the NPA treatment. But that expression was inhibited significantly under NPA treatment when comparing to the IAA treatment..4. Sequences analysis of the three small RNA libraries identified 21 conserved miRNAs belonging to 13 families, 10 novel and 8 potential novel miRNA belonging to 15 families. Among these miRNAs, 12 miRNAs were differentially expressed during the graft process in hickory and two-thirds were down-regulated. Quantitative real-time polymerase chain reaction(qRT-PCR) validated 14 miRNAs and their expression trends were similar to the results obtained by Soelxa sequencing. Further, a total of 89 target genes for conserved and 26 target genes for novel miRNAs were predicted. To validate the relationship of miRNAs and their targets, we selected four miRNA target genes(cca-miR156, cca-miR159, cca-miR390 and cca-miR827) to perform qRT-PCR. The results suggested an inverse relationship between the expression of the miRNAs and the regulation of their corresponding target genes. This evidence further validated the reliability of mi RNA sequences generated by Solexa sequencing. |
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