Cloning And Function Analysis Of Phosphorus Transport-Related Transcription Factor PmMYB In Pinus Massoniana

Phosphorus(P) plays an important role in the development and metabolism of plants, thus is one of the most important limited factors for agricultural and forestry production. The deficiency of available P in the soil severely restricted the forestry production. Cloning and characterization of genes related to the low-P tolerance is an efficient strategy for solving the problem via creating high P efficiency germplasms.Masson pine(Pinus massoniana Lamb.) as an important economic species tree in China, demonstrates huge value in forestry. However, the yields have been highly affected by the deficiency of available phosphorus in the soil. Therefore, it is essential to screening the key genes in response to low phosphorus stress.Based on the previous work, currently, a masson pine germplasm, which showed high tolerance to low P stress, was used as material, and the MYBs in response to low phosphorus stress were preliminarily screened by RT-PCR and quantitative PCR.Subsequently, the full-length sequence of a PmMYB in response to low P stress was cloned by RACE strategy. Finaly, the plant expression vector was constructed herein.The obtained results facilitated the better understanding for the molecular mechanism underlying the high tolerance to low-P stress, as well as the creation of high P utility germplasm in this species. The main results were as follows:1. By RT-PCR with UBC, GAPDH and 18 s rRNA, UBC and GADPH were proved to be the best reference genes. With the combination of RT-PCR and real-timeqPCR, the expression patterns of four MYB transcription factors were characterized in response to low P stress. The expression of MYB158 was persistantly down-regulated, conversely, that of MYB169 was progressively up-regulated under low P status. the expressions of MYB233 and MYB312 showed a fluctuation trend in exposure to low P stress.2. The full-length sequence of MYB169 was cloned by RACE strategy, which named as PmMYB169. The sequence analysis showed that the total length of PmMYB169 was 1407 bp, containing 187 bp untranslated 5'-UTR region and 293 bp untranslated 3'-UTR region, and the open reading frame(ORF) was 927 bp, encoding308 amino acids.3. Bioinformatic analysis revealed that PmMYB169 belonged to R2R3-MYB,which contained MYB family structure with the conservative areas. Phylogenetic analysis demonstrated that PmMYB169 was most close to the MYB gene of Picea sitchensis.4. Tissue specific expression analysis showed that PmMYB169 was a constitutive expression gene and expressed in root, stem and leaf, among which leaf showed the highest expression level, followed by root, and stem showed the lowest expression. In root and stem, PmMYB169 showed the lowest and highest expression in 36 d and 60 d, respectively; in leaf, PmMYB169 demonstrated the lowest expression in 24 d and highest expression in 60 d after the stress. All the expression patterns characterized in a up-down-up trend.5. To further unravel the function of PmMYB169, the plant expression vector(pCAMBIA1301-PmMYB169) was constructed, which also facilitated the creation of new germplasms with better tolerance to high P utility.