Cloning And Genetic Transformation Of Two Poplar MYBs Transcription Factors

Lignins play an important roles in composing materials of secondary structure of cell wall in the plants.It makes up the third largest component of cell wall,followed by the cellulose and hem-cellulose.Lignin always cross-linked with other components of cell wall comprised the tricked cell wall-secondary cell wall,play greatest important roles in the process of resisting pest and disease damage,bacterium and a variety of nutrition(water,mineral nutrition,toxic heavy-metal elements).Many documents have reported that lignin response to all kinds of stresses so quickly that lead to the accumulation under stress conditions.Boron is one of essential mineral elements during the life process of plants and there is a phenomenon that the soil boron content is deficient of most poplar culture regions in South China.Early research results of our research group showed that lignin content accumulated significantly in the roots of Nanlin 895 under boron deficiency stress.Profound research by digital gene expression profile(DGEs)proved that there are amount of genes differently expressed under boron deficiency and control conditions.Based on the early DGEs,genes PtrMYB192,PtrMYB199 which were screened from digital gene expression profile were blast with lignin-related AtMYBs in this research.Bio-information analysis were used to predict their primary composition and structure,protein PtrMYB192 and PtrMYB199.Over expression and RNAi conductor were constructed,and then these conductors were transferred into poplar plants by GV3101.The main results were as follows:Amino acid sequences of lignin-related genes,AtMYB58,AtMYB85,AtMYB43,AtMYB63 and that of PtrMYBs differently expressed in DGEs were down loaded from photozome and used to construct phylogeny tree with bootstrap value>60.Results showed that AtMYB58,AtMYB63 and PtrMYB192;AtMYB85,AtMYB43,and PtrMYB199 are in the same subgroup and have closer relatives to each other.Gene structure analysis showed that AtMYB58.AtMYB63 and PtrMYB192;AtMYB85,AtMYB43 and PtrMYB199 are conservative in the size and quantity of exons and introns.Protein primary structure analysis showed that protein PtrMYB192,PtrMYB199 were stabilizing and hydrophilic proteins.Analysis of signal peptide and trans-membrane structure of protein PtrMYB192 and PtrMYB199 showed that they all contain neither signal peptide nor trans-membrane structure.Analysis of domains and function of protein PtrMYB192 and PtrMYB199 showed that they all characterized with 2 domains which contain in MYB transcription factors,function in catalyzing DNA transcript into RNA,but both can not function as enzyme.Thus we concluded that they all members of R2R3 MYB subgroup.Predict analysis of secondary structure showed that the proportion of each kind of secondary structures list from most to least as random coil,a-helix,extended strand,?-turn.Analysis of tertiary structure of protein PtrMYB192,PtrMYB199 showed that AtMYB58,PtrMYB192;AtMYB43 and PtrMYB199 contain similar domains which could be deduced that they shared the similar functions.In order to illuminate the function of genes PtrMYB192,PtrMYB199 in the process of lignin bio-synthesis,the CDS regions were cloned into PCAMBIA1302 named as PCAMBIA1302+MYB192,PCAMBIA1302?MYB199.In these over expression vectors,hygromycin B was selected as the marker gene and CDS region were inserted into the multiple cloning sites between Bgl? and BstP?.Results of enriched PCR detection and double enzyme digestion indicated that the target gene was inserted into expression cassettes,could be used for latering poplar transformation.And reverse fragment of gene PtrMYB192,PtrMYB199 into two ends of intron of intermediate cloning vector psk-int.Constructed hairpin motifs were then linked into mutipule cloning sites of NCOI and BstP? of PCAMBIA1302 contains Hairpin motifs named as RNAiPCAMBIA1302+MYB192,RNAiPCAMBIA1302+MYB199.Results of PCR detection and double enzyme digestion indicated that the the Hairpin motif was inserted into expression cassettes,could be used for latering poplar transformation.In this paper we also constructed RNAi expression vectors RNAiPCAMBIA1302+MYB192,RNAiPCAMBIA1302+MYB199.Hairpin motify were constructed by inserting foreword.In this paper genetic transformation of poplar Nanlin 895 were mediated by Agrobacterium tumefaciens GV3101.Constructed expression vectors were introducted into Agrobacterium tumefaciens GV3101 by eletroporation.Leaves of Nanlin 895 precultured for 72 h,were infected by Agrobacterium tumefaciens GV3101,which contains expression vectors.Then these leaves were transferred on the co-culture mediums contained Cef and AS and lasted for 72 h under dark condition.Then leaves were transferred on the selective medium till produce resistant buds.After a period of time,these resistant buds were cutted and transferred on the medium for elongating these bus.Finally,elongating buds with strong stems were cultured in taking rooting culture medium.Rooted resistant seedings were used for PCR detection,and results showed that we obtain 6 positive plants were obtained,which over express PtrMYBs,containing 4 PtrMYB192 plants and 2 PtrMYB199 plants.we also obtained 1 RNAiPtrMYB199 plants.These results not only clarified the evolution structure of target genes,various structural composition and properties of their translating proteins,providing theoretical basis for their further study,but also laying a solid foundation for revealing their important roles in the mediation of lignin bio-synthesis from the point of Physiology by constructing over expression and RNAi vectors and transformation plants.