| Torreya grandis ‘Merrillii' is an improved variety of Torreya grandis that is screened through artificial breeding and propagaed through grafting.Seeds of T.grandis ‘Merrillii' is precious nuts native to China and is one of the financial sources that can help farmers in mountainous areas to get rid of poverty.Cultivation of T.grandis ‘Merrillii' has encountered the problems of low survival rate and long seedling growth duration.As one of the most widely used propagation methods,grafting can not only enlarge the cultivation scale but also increase survival rate of T.grandis ‘Merrillii'.In this paper,grafted seedlings of T.grandis ‘Merrillii' were used as materials.Seedlings were treated with different plant growth regulators and shade.The intents of this paper were to: determine the changes of endogenous hormone contents along with treatments,graft time and graft locations during the graft process of T.grandis ‘Merrillii';clone an auxin responsive factor(ARF)gene;screen metabolic pathways related with graft;analyze the expression differences of members in ARF gene family during the graft process of T.grandis ‘Merrillii'.This paper would provide some theoretical references for revealing the regulatory mechanism of ARF during the graft survival of T.grandis ‘Merrillii'.The main results were as follows:1.The changes of endogenous hormone contents,including IAA,GA,CTK and ABA,were determined using ELISA.Results showed that IAA and GA contents increased first and then decreased,ABA contents decreased first and then increased,while ABA contents increased with the time of graft increased in CK group.The contents of CTK,GA and IAA were increased while the content of ABA was decreased under external IAA treatment.The contents of the 4 hormones were all decreased under external IAA treatment NPA treatment,meaning that NPA could repress the expression of endogenous hormones.Under shade treatment,the contents of ABA and GA were increased,while the contents of CTK and IAA were decreased.2.The opening reading frame(ORF)of TgARF cDNA was 2676 bp in length,which encoded 891 amino acids.Phylogenetic analysis between Tg ARF and ARFs from 15 plant species showed that TgARF was closely related with ARFs of Picea abies,followed by Pseudotsuga menziesii.3.The expression profiles of TgARF among different treatments at transcription level weredetermined by qRT-PCR.Results showed that the expression of TgARF in scions of CK group was decreased 3 days after grafting,increased and peaked 7 days after grafting and decreased again 14 days after grafting,while TgARF expression was steadily increased in the rootstocks of CK group.Hormone and shade treatments could influence the expression of TgARF to some degree.Shade treatment could significantly increase the expression ranges of Tg ARF.4.The expression profiles of genes during the graft process of T.grandis were analyzed based on transcriptome sequencing.A total of 1.78 billion short reads were obtained and 370,791 unigenes were assembled.A total of 84468 and 135625 differentially expressed genes(DEGs)were detected after 7and 14 days of graft.These DEGs involved in a number of metabolic pathway.344 genes were functionally screened for ARF and the expression profiles of these genes during the graft process of T.grandis ‘Merrillii' were analyzed.12 out of the 344 ARF candidate genes were further screened and the expression changes of them during graft process were verified by qRT-PCR.Results showed that these involved in the graft process of T.Grandis.However,the express patterns of these genes were different between the rootstocks and the scions. |
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