Cloning Of Two MBD Genes, Construction Of Their Plant Expression Vectors And Genetic Transformation In Wheat

DNA Methylation plays important regulation roles in gene expression, cellular differentiation and phylogenetic development. MBD is a kind of trans-acting factor which binding with DNA methylation, and play a considerable function druing the growth and development in plant. The open reading frame (ORF) of two MBD were isolated by in silico cloning and RT-PCR method, Overexpression and RNAi expression vectors for them were constructed, and were transformed with wheat by the pollen tube mediated transformation methods. Some transgentic plantlets were confirmed by preliminary identification.The main results are as follows:1. Two genes which encode methyl-CpG binding protein were cloned, designated TaMBD1 and TaMBD2. Sequence analysis showed that the cDNA of TaMBD1 is 668 bp in length and the coding region 579 bp, which contains 36 bp in 5' UTR and 53 bp in 3' UTR.The genome structure analysis indicate that a 1386bp intron was included in TaMBD1.The cDNA of TaMBD2 is 1427 bp in length and the coding region 942 bp ,which contains 131 bp in 5' UTR and 354 bp in 3' UTR .2. Sequence analysis and BLASTX results revealed that TaMBD1 and TaMBD2 had high similarity to a group of MBD protein genes of different species. TaMBD1 has higher amino acid sequence similarity with MBD in rice (Accession number: CT831688), maize MBD101 (Accession number: EU976548) and Arabidopsis AtMBD1 (Accession number: NM₁18401.5) were 76%, 74% and 49%, respectively. TaMBD2 has similarity amino acids with Zea mays MBD108 (Accession number: AAK40309), Arabidopsis AtMBD2 (Accession number: NP₉74850) were 73%, 60%, respectively.The interPro analysis showed that the deduced amino acid of cloned genes contains typical MBD conservative region. Phylogenetic analysis suggested that the two genes belonged to different subclasses, the TaMBD1 was included in II subclass with AtMBD1 and AtMBD4, and TaMBD2 was belonged to III subclass with AtMBD2, which implied that two new MBD genes were islated in wheat.3. The overexpression and repression expression vectors of TaMBD1 and TaMBD2 were constructed, respectively. In order to enhance capability of gene silence, the forward, intron and reward orientation of cloned gene were included in vector named RNAi. Constructed vectors were transformed in wheat cultivar "YuMai34" by the pollen tube mediated transformation methods, respectively. Some transgentic plantlets were detected by PCR, the result showed that 2 transgenic wheat plants with pCAM-M1-RNAi, 1 transgenic wheat plants with pCAM-M2-over and 10 transgenic wheat plants with pCAM-M2-RNAi were confirmed by preliminary identification. The abnormal phenotype of transgenic pCAM-M2-RNAi plantlet were observed.