Cloning And Preliminary Analysis Of TIR1 And AUX / IAA Family Genes Of Masson Pine

Auxin signal transduction pathways plays a crucial role in plant growth and development, TIR1 gene plays an important role in auxin signal transduction pathway, AUX/IAA family genes can be directly connected with the auxin and play a role. The study to TIR1 gene and AUX/IAA family genes can help to discover how the auxin regulating growth and development of Pinus massoniana.We used primer5 to design primers, through PCR and RACE techniques, the TIR1 gene from P. massoniana named PmTIR1 was cloned. PmTIR1, having 2446 bp in full length, contains an opening frame of 1725 bp that encodes a protein of 574 amino acids with predicted relative molecular mass of 64240.3u and PI of 6.76. Its nucleotide sequence and the encoded amino acid sequence were analyzed with softwares of ClustalX, DNAMAN and ANTHEPROT,the results showed that, the homology of PmTIR1 protein from P. massoniana and TIR1 protein from P. taeda was as high as 99%, PmTIR1 contained the F-box protein domains and multiple LRR domain; F-box protein domains in PmTIR1 may combine with SKP1 in SCFTIR1, it was consistent with the characteristics of F-box family. Using real-time quantitative techniques, we identified the amount of PmTIR1 expression, which were the highest in leaf and the lowest in stem from P. massoniana.Three genes from AUX/IAA gene family were cloned, named PmIAA1, PmIAA2 and PmIAA3.PmIAA1, having 1330 bp in full length, contains an opening frame of 711 bp that encodes a protein of 236 amino acids with predicted relative molecular mass of 26015.6u and PI of 7.01; PmIAA2, having 1628 bp in full length, contains an opening frame of 909 bp that encodes a protein of 302 amino acids with predicted relative molecular mass of 41176.1u and PI of 5.25; PmIAA3, having 1316 bp in full length, contains an opening frame of 759 bp that encodes a protein of 252 amino acids with predicted relative molecular mass of 28060.1u and PI of 6.01. the homology of PmIAA1 protein from P. massoniana and IAA1 protein from P. taeda was as high as 95%, the homology of PmIAA2 protein from P. massoniana and IAA2 protein from P. taeda was as high as 97%, the homology of PmIAA3 protein from P. massoniana and IAA3 protein from P. taeda was as high as 98%. PmIAA1, PmIAA2 and PmIAA3 contain AUX/IAA domain, The domain may combine with ARFs(auxin response factors), which is consistent with the characteristics of the family of AUX/IAA. Using real-time quantitative techniques, we identified the amount of PmIAA1, PmIAA2 and PmIAA3 expression, the amount of PmIAA1 expression were the highest in leaf and the lowest in stem from P. massoniana, the amount of PmIAA2 and PmIAA3 expression were the highest in root and the lower in leaf and stem from P. massoniana.PmTIR1 plant expression vector was constructed, then transformed it into tobacco, through the tobacco phenotypic changesthe function of PmTIR1 gene was discussed. Through observation of phenotypic, compared to wild-tobacco, transgenic tobacco had a dwarf characteristic; the leaf color of transgenic tobacco was greener and the leaf length/ width ratio of transgenic tobacco was smaller than that of the wild type; The amount of adventitious roots from transgenic tobacco were more than that of the wild type; which confirm that PmTIR1 gene plays an important role in the growth process of P. massoniana, and excessive expression of PmTIR1 gene can increase the amount of adventitious roots.