Study On MRNA Exchange And Regulatory Network Of Populus Szechuanica Var.tibetica And Populus Tomentosa Heterografts

Plant grafting,an ancient asexual propogation techniques,has been widely used in agriculture and horticulture for its advantages in easy operation,rapid breeding and trait improvement.Researchers have been working on the mechanism of interaction between rootstocks and scions.In recent years,rapid development of molecular technology made them focus on genetic materials exchange across the graft union.In this study,tissue culture materials of P.szechuanica var.tibetica and P.tomentosa were grafted,and expression data were obtained by transcriptome sequencing to analyze mRNA exchange between rootstocks and scions.The main research contents and results are as follows:1.Micrografting method was chosed to reduce the influence of external environment,and tissue culture materials were established.I designed different concentration gradient of hormones and successfully established tissue culture materials of P.szechuanica var.tibetica.Age effects of P.tomentosa materials existed in our lab were eliminated by induction of callus redifferentiation.Then P.szechuanica var.tibetica was micrografted with P.tomentosa and vice versa,and autograft of this two poplars were used as control.I found the two poplars had good graft compatibility,and taking P.szechuanica var.tibetica as rootstock was the best option.The time of vascular reconstruction between scion and rootstock was 5-6 d after grafting which was detected by 6-CFDA.2.Stems of scions and rootstocks were collected at 5 d,6 d,8 d,12 d,18 d,26 d,36 d and 48 d after grafting,and RNA of each sample was extracted for transcriptome sequencing.Trinity was applied to assemble clean reads of control group for P.szechuanica var.tibetica and P.tomentosa,respectively.After asscmbling,unigenes were sort out and reassembled by GICL.Finally,83,222 contigs for P.szechuanica var.tibetica and 106,402 for P.tomentosa were obtained.RSEM was used for quantitative analysis of gene expression,and correlation detection after eliminating batch effect indicated reliability and accuracy of the results.3.Transcriptome of the two poplars were blasted to obtain specific sequences for each poplar,and then I detected expression of P.szechuanica var.tibetica specific sequences in P.tomentosa samples which had grafted with P.szechuanica var.tibetica and vice versa to identify transcripts exchange between the two poplars.273 transcripts were found move from P.szechuanica var.tibetica to P.tomentosa,and 1678 transcripts move from P.tomentosa to P.szechuanica var.tibetica.Results of functional annotations and classification demonstrated these exchanged genes participated in metabolism,response to stimulus and regulation of biological process.4.ImpulseDE2 was applied to analyze temporal differential expression of exchanged genes between P.szechuanica var.tibetica and P.tomentosa in each recipients,and the possible function and fate of these genes were also predicted.5.Regulatory network of the exchanged genes were constructed by combining high-dimensional differential equations with evolutionary game theory,and some pivotal genes were identified,which give theoretical direction for subsequent studies in validating factors playing important roles in rootstock and scion interaction.