| Poplar is one of the most important worldwide tree species and it is usually clonal propagated.Thus,the ability for forming adventitious root(AR)is the key factor for its growing.In this study,six genes were identified as candidate regulators for AR formation and development by using RNA-Seq analysis.The over-expression(OE)transgenic lines for these 6 genes were generated.The traits related to AR formation were observed and recorded.Two genes,names as WZ4(a CML protein)and XZ6(a NAC transcription factor),showed increased ability for AR development.The WZ4-OE transgenic lines were selected for carefully characterization.The main research progresses are as follows:1.By observing the adventitious root dynamics of the tissue-cultured seedlings of NL895 poplar(P.deltoids× P.euramericana)cultured in WPM medium,making paraffin section of stem segments of tissue-cultured seedlings,the incidence of adventitious root primordia on day 0-8 after cutting into WPM medium was counted,and the main time nodes of adventitious root formation of poplar were determined.The RNA at the base of Poplar cuttings was extracted for transcriptomic analysis.According to the certain thresholds and identification scheme,six genes which may participate in adventitious root formation and development of poplar were selected.These genes correspond to the reference genome numbers of Populus trichocarpa: Potri.017G126200,Potri.002G065600,Potri.014G103000,Potri.004G042600,Potri.004G033100,Potri.001G404100.For the convenience of recording,it is renamed as WZ4,XZ2,XZ3,XZ4,XZ5,XZ6.The genes were cloned and the GFP fusion expression vectors were constructed.Subcellular localization was carried out in tobacco epidermal cells.It was found that the transcription factor proteins of these genes were located in the nucleus,indicating that all the six genes may have the function of transcription factors.At the same time,the fusion protein of WZ4 gene is also located on the cell membrane,which indicates that the expression product of WZ4 gene may not only act as a transcription factor,but also participate in the activity of cell membrane as a structural protein.2.The over-expression vectors of the six genes were constructed and transformed into NL895 poplar by Agrobacterium tumefaciens mediated transformation and leaf disc transformation.The positive transgenic lines were obtained by three levels of DNA,RNA and Protein,and the positive lines were amplified and propagated to obtain enough individual plants.3.With wild-type NL895 poplar as a control,the adventitious root characters of the positive lines were observed and compared under tissue culture conditions.The transgenic lines with obvious differences were identified more accurately under water culture and soil culture conditions.At present,it has been found that the length of adventitious roots of transgenic lines with WZ4 gene is significantly longer than that of wild-type NL895 poplar under three different conditions.Based on this result,we analyzed the function of WZ4 gene in detail.4.By homologous analysis of WZ4 gene sequence,we preliminarily concluded that WZ4 is a calmodulin like protein regulatory gene(CML23-1).At the same time,tissue-specific expression analysis showed that the expression level of WZ4 gene in adventitious root tissue was the highest,which was significantly higher than that in stem,petiole and terminal bud tissue,indicating that the main part of WZ4 gene in poplar was adventitious root tissue.The transcriptome analysis of WZ4 over-expression transgenic lines showed that the expression of a number of regulatory genes was significantly increased.Among them,Potri.001G011300,which was annotated as hydrogen peroxide synthase,showed the highest expression level.It is speculated that WZ4 gene may indirectly participate in the regulation of hydrogen peroxide synthesis,thus regulating the development of adventitious roots.5.The promoter sequence of WZ4 gene was cloned to construct yeast single hybrid vector and GUS expression vector(a vector for analyzing the tissue specificity of gene expression).The constructed yeast single hybrid vector transformed yeast strain Y1H-Gold,which was inoculated on yeast defective medium containing antibiotic Ab A.The results showed that Y1 H containing WZ4 promoter could still grow normally in high concentration Ab A,indicating that the WZ4 yeast strain appeared self-activation.The positive transgenic plants obtained by transforming GUS expression vectors were stained with GUS histochemistry,and the leaves,root tips and shoot tips were stained blue,which was basically consistent with the results of tissue-specific expression analysis.Through the development of this study,we have successfully obtained a batch of genes involved in the regulation of adventitious root formation and development in poplar,and identified some of them for adventitious root traits as well as systematically studied the function of WZ4 gene.Based on this study,we have deepened our understanding of adventitious root traits of poplar,obtained a number of gene resources related to adventitious roots of poplar and a gene with the potential to improve the development ofadventitious roots of poplar,which laid an important theoretical foundation and genetic resources for the creation of new varieties of resources. |
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