Functional Analysis Of Auxin Response Factor Gene CcARF In Hickory (Carya Carthayensis) During Graft Process

Hickory（Carya cathayensis Sarg.） is the important woody oil trees, which is specialty dried fruitin northwestern Zhejiang, having high economic value, and one of the economic pillars to the farmer.The problems are the long juvenile period, not easy to harvest, and difficult to horticultural cultivate ofhickory becoming a huge bottleneck of industry development. Plant grafting is the main method to solvethose problems of cultivation of Hickory. With the progressive development of the grafting technique,there are more and more study about the deeper level of grafting techniques in order to better guide thegrafting.According to the auxin response factor (CcARF) gene fragment of cDNA-AFLP, we get thefull-length of CcARF cDNA. Using the techniques of qRT-PCR and Western blotting, we analysed thefunction of CcARF during the grafting process. The IAA oxidase activity was detected. The main resultsare as follows:1. The total RNA with high quality, integrality and no-impurity extracted with modified CTAB+Trizolmethod was better than the method of trizol kit and RNAiso-mate for Plant Tissue kit, which wassuitable to the full length cDNA clone and Fluorescence Real Time RT-PCR analysis.2. There were2337bp of CcARF from the initiation codon to the termination codon and encoding778amino acids. The cDNA sequence homology and amino acid sequence homology was similar with theother19plants, especially in B3-DNA binding domain (DBD). The phylogenetic tree showed CcARFwas closest to ricin gene, followed by grapes gene, but far from poplar gene.3. To determine the special-temporal (0d,1d,3d,5d,7d,14d) expression pattern of CcARF underdifferent treatments (control,0.3μg/l NPA,4mg/l IAA) in nickory grafting, Fluorescence Real TimeRT-PCR techniques were applied. The results showed that: under control, the expressions of CcARFdecreased significantly in the scion1day after grafting and increased sharply in the following2daysafter grafting, then decreased. In the rootstock, the expressions of CcARF decreased significantly1dayafter grafting and increased in the following6days after grafting, then decreased. Under4mg/kg IAAtreatment, the expressions of CcARF from the beginning of grafting to3days after grafting decreasedsignificantly and increased in the following11days in the scion. However, in the rootstock, theexpressions of CcARF decreased1day after grafting and increased in the following13days aftergrafting. Under NPA treatment, the expressions of CcARF in both rootstock and scion decreased sharply1day after grafting and increased in the following13days after grafting. The expression of CcARFunder IAA treatment was higher than that in the control, while the expression of CcARF under NPAtreatment was lower than that in the control.4. Using Western blot hybridization technique to determine the expression of the auxin response factor(CcARF) protein. The results showed that: the CcARF protein expression in the translational level wasdifferent during the grafting under different treatment. The expression of CcARF protein under IAA treatment was higher than that under control. But the expression of CcARF protein under NPA treatmentwas inhibited.5. The indoleacetic acid oxidase enzyme activity was detected. The results showed that: the activity ofIAA oxidase enzyme increased during the grafting under control. The activity both in the scion androotstock increased1.6times and1.3times from the beginning of grafting to14days after grafting. Theactivity of IAA oxidase enzyme under IAA treatment was lower than that under control during thegrafting. However, the activity of IAA oxidase enzyme under NPA treatment was higher than that undercontrol during the grafting.