Study On The Preliminary Function Of DfNACs And DfMYB3 Genes In Dendrocalamus Farinosu

Dendrocalamus farinosus is an important bamboo specie for industrial raw material production in China.Its improved development is of great significance in promoting the sustainable development of the paper industry,and the improvement of the ecological environmentwhile using bamboo resouces in effective way.With the upgradation of the bamboo industry,the demand for bamboo raw material for high-tech products is increasing.As to meet the demand of high quality product,the demand for high-fiber and high-quality bamboo resources is becoming more and more obvious.However,it is difficult to improve the bamboo throught the traditional crossbreeding because of the special biological characteristics.With the rapid development of biotechnology,it is more and more payattion to studying on the secondary growth and development of bamboo through transgenic modification in recent years.NAC and MYB are important members in the plant transcription factor family.It is found that they play an important role in the secondary development of plants.In this study,Df NAC1,Df NAC2,and Df MYB3 transcription factors were cloned from Dendrocalamus farinosus.Their preliminary biological functions were studied.The main results are as follows:The transcription factors Df NAC1,Df NAC2,and Df MYB3 were cloned based on the transcriptome database of Dendrocalamus farinosu.Their full length sequences are 1179 bp,846bp,and 1287 bp,encoding the 392,280,428 amino acids,respectively.Their registration numbers of Gen Bank are KY963356,KY963357,KY963358,respectively.The results of bioinformatics analysis showed that both Df NAC1 and Df NAC2 have the typical conserved domains of NAM.They belonged to the NAC superfamily.Df MYB3 has the typical SANT domain with the functions of DNA binding domain.Df MYB3 is a typical R2R3-MYB protein.The results of phylogenetic tree showed that Df NAC2 could be regarded as a VND Like transcription factor.The Df NAC1 and Df NAC2 were related to the secondary development.The Df MYB3 transcription factor could be involved in plant growth,development and abiotic stress.Physicochemical properties predicted that Df NAC1 and Df MYB3 had more acidic residues and Df NAC2 had more basic residues,and both were unstable hydrophilic proteins.The secondary structures of three proteins showed that the content of ?-helices and random coils were abundant.The predicted results of tertiary structure show that Df NAC1 and Df NAC2 mainly consist of extended chains and random coils,the Df MYB consists of mainly ?-helix.The results of subcellular localization showed that Df NAC1,Df NAC2,and Df MYB are mainly expressed in the nucleus.The transcriptional self-activation activity test by Yeast one-hybrid showed that both the C-terminus and the full-length of Df NAC1 and Df NAC2 proteins have the transcriptional self-activation activity.However,the Df MYB3 had no transcriptional self-activation activity.The 35S:Df NAC1/2 was transformed into the embryo of phyllostachys edulis embryo and the leaves of Populus tomentosa.The transgenic plants of Df NAC1 and Df NAC2 in Populus tomentosa were successfully obtained by agrobacterium-mediated transformation,The transgenic plants of Df NAC2 in phyllostachys edulis were successfully obtained by particle bombardment.The results of semi-quantitative PCR displayed that Df NAC1 and Df NAC2 was successfully expressed in the transgenic plants of Populus tomentosa,the Df NAC2 was successfully expressed in transgenic plants of phyllostachys edulis.It was found that the plant height and internode increased in the transgenic plants of Populus tomentosa with the higher expression level of Df NAC,compared with CK.The results of morphology and anatomy in the stem of Df NAC2 transgenic plant showed that the there were the decrease of vessel numbers and the significant increase of parenchymal cells and layers in the stem transgenic plants,compared with the control plants.The promoter Dfp MYB3 was obtained by Genome Walking,linked to p BI121-MCS vector.The results of GUS staining in the Df MYB3 transgenic plant of Nicotiana tabacum showed that GUS gene expression mainly expressed in the stem and leaf veins.It was found that GUS gene expression was significant in the closely arranged parenchyma cells of stem xylem.The results of Dfp MYB3 and Df NAC2 co-transformation into the callus of wheat showed that the expression of Dfp MYB3 could be in the lignified tissue,and the color of callus in wheat increased by staining.It indicated that Df NAC2 could enhance the activation of Dfp MYB2.