The Cloning And Establishment Of Overexpression/RNAi Transgenic Plants Of PdapARF1 Gene In Populus Davidiana × P. Alba Var. Pyramidalis Responding To Trichoderma Induction

Trichoderma spp. is a fungal genus that plays an important role in biological control. Some of them could promote plant growth by secreted the plant growth regulators, such as IAA and so on. Auxin plays an important regulatory role throughout the growth and development process of the plants. Auxin response factor (ARF) is a transcription factor regulating the expression of the early auxin response genes, which plays a key role in auxin signal transduction pathway. In order to further study the molecular mechanism of ARF transcription factors on the growth and disease resistance of Populus davidiana×P. alba var. pyramidalis (Padp seedlings) induced by Trichoderma, the leaves of Padp seedlings were used as the material in this study. Plant genetic engineering technology was employed to construct the expression vectors of PdapARF1 gene overexpression and RNA interference (RNAi). And the plants of PdapARF1 gene overexpression and silencing were obtained by Agrobacterium-mediated genetic transformation. The main results are summarized as follows:1. According to the data of transcriptome library of Padp seedlings, the DNA and cDNA sequences of PdapARF1 were cloned successfully, and the obtained accession numbers of the PdapARF1 DNA and PdapARF1 mRNA in GenBank were KP165071 and KM113035.1, respectively. The full-length of PdapARF1 DNA was 5,390 bp, which containing 14 extrons and 13 introns. The full-length of PdapARF1 mRNA was 1,983 bp, which encoding 660 amino acids. The results of bioinformatic analysis showed that PdapARF1 protein had the physical properties of a molecular weight of 73.4 KDa, a theoretical isoelectric point of 5.81, and three family conserved domains including BfiⅠ_EcoRⅡ_N_B3, Auxin_resp and AUX_IAA. The result of multiple sequence alignment showed that the similarity was 88% between the amino acid sequence of PdapARF1 in Padp seedlings and the sequences of the other 11 plant species. The result of the homologous sequence search for PdapARF1 gene in the data base of the Populus trichocarpa genome obtained 10 homologous sequences. A three-dimensional structure of the DBD functional domains of PdapARF1 protein had been predicted using the tools on SWISS-MODEL2. The differential expression of PdapARF1 gene in the leaves and roots of one-year-old tissue culture Padp seedlings transplanted into the pots under natural conditions by Trichoderma asperellum (Ta) treatment has been analyzed using qRT-PCR. The results showed that the expression of PdapARF1 gene in the leaves and roots of Padp seedlings was all upregulated compared with that of the control during 72 h induced by Ta536, Ta429 and mixed Ta strains (mixing equal proportions of Ta536, Ta429, T650 and T4), respectively. In addition, the upregulated expression folds of PdapARF1 gene in the leaves and roots of Padp seedlings was maximum induced by mixed Trichoderma strains.3. The expression vectors of PdapARF1 gene overexpression and RNAi were constructed successfully, and they were transformed respectively into Agrobacterium tumefaciens EHA105 by electroporation technology. Two pairs specific primers of the gene were employed to validate the two expression vectors by PCR technology, the results showed that the two expression vectors of PdapARFl gene overexpression and RNAi were transferred successfully into Agrobacterium.4. pROK2-ARF1 and pFGC-arf1s expression vectors were transferred into Padp seedlings by Agrobacterium-mediated transformation. Fouty transgenic plants of eight plants of pROK2-ARF1 (gene overexpression) and 32 plants (gene RNAi expression) of pFGC-arfls (including six of pFGC-arf1B, eight of pFGC-arf1R, twelve of pFGC-arf1BR and six of pFGC-arf1A), were obtained and they were screened on the media with antibiotic. The results of PCR assay confirmed that one transgenic plant of pROK2-ARF1 and 5 transgenic plants of pFGC-arf1s (one pFGC-arf1B, two pFGC-arf1R, one pFGC-arflBR and one pFGC-arflA) were obtained. The conversion rate of Agrobacterium-mediated transformation was 12.5% and 15.6%, respectively.