Cloning And Functional Analysis Of Transcription Factor NnMYB4 ORF In Nelumbo Nucifera

MYB transcription factor genes are widely distributed in higher plants and play important roles in the regulation of secondary cell wall formation in plants.Nelumbo nucifera is a perennial aquatic plant,and it is also one of the most widely cultivated and most widely used economic crops and ornamental plants in China.However,the current research on molecular breeding of Nelumbo nucifera is still at the initial stage.In this study,a R2R3-MYB transcription factor was isolated from Nelumbo nucifera.In order to understand the role of this gene in the synthesis of lignin and it was functionally characterized in Arabidopsis thaliana.These results are expected to provide theoretical basis for further research of MYB transcription factors on regulation of the development of secondary cell wall and genetic breeding of Nelumbo nucifera.The main results are as follows:A MYB gene was screened by BLAST analysis of the Nelumbo nucifera genome in terms of a high identity with AtMYB4.According to the cDNA full-length open reading frame(ORF),specific primers were designed,with 'Heilongjiang Guren' young leaves as materials,the full-length open reading frame was amplified by RT-PCR,designated as NnMYB4.The full length of the open reading frame was 726 bp,encoding 241 amino acids.It belongs to the R2R3-MYB transcription factor G4 subgroup and it was closely related to AtMYB4,CmMYB1and ZmMYB31.Quantitative real-time PCR showed that NnMYB4 were expressed in roots,stems,petioles,flag leaves,young leaves and climax leaves,but most strongly in the young leaves and most weakly in the flag leaves.It showed that the expression of NnMYB4 In different organs and tissues is different.The yeast effect expression vector pGBKT7-NnMYB4 was constructed,then transformed it into yeast strain Y2H.The active yeast transformants were obtained first by using SD/-Trp medium,and then by using SD/-His-Ade medium.Results show that,NnMYB4 has no transcriptional activation activity.The green fluorescent protein fusion expression vector pMDC43-GFP-NnMYB4 was constructed and transformed into the onion epidermal cells by using Agrobacterium-mediated techniques.The results of fluorescence microscopy showed that NnMYB4 protein was localized to the nucleus.The pMDC43-NnMYB4 overexpression vectors were constructed and transformed into Arabidopsis thaliana by Agrobacterium-mediated transformation.The NnMYB4 transgenic lines 35S::NnMYB4-1 and 35S::NnMYB4-2 were obtained by using 1/2MS medium containing hygromycin B and RT-PCR.The transgenic plants showed inflorescence stems shorter,flowering delay,xylem stain area and the degree of staining shallow smaller,lignin content was significantly reduced when compared to wild-type A.Thaliana.It indicated that the NnMYB4 can inhibit the growth of plant stem and lignin biosynthesis.Over expression of NnMYB4 analysis showed that the key enzyme gene(including At4CL1,AtC3H,AtF5H,AtCOMT1,AtCesA1,AtCesA7,AtCesA8,AtIRX8)of the lignin biosynthesis pathway was significantly reduced in transgenic lines.These results shows that NnMYB4 can negatively regulate the key enzyme genes of lignin and cellulose in plants secondary cell wall metabolism,NnMYB4 may be a negative regulator.