Research Of The Gene Expressions Of Paulownia Witches' Broom Under Methyl Methane Sulfonate Treatment

Paulownia witches' broom(Pa WB)is an infectious disease,caused by phytoplasma,results in billions of dollars in economic losses to the industrialization of Paulownia production,is one of the problems needs to be solved in the forestry production in China.In the past forty years,the researches focus on two aspects(phytoplasma and Paulownia)on the interaction of phytoplasma-Paulownia.Researches on Paulownia,a set of physiological and biochemical variations,gene expression of paulownia were obtained after phytoplasma infection.For Pa WB phytoplasma,progress referred to the way of the infection,classification,and modes of reproduction of the phytoplasma has been obtained.However,the mechanism of Pa WB is still poorly understood.In this study,the modern biological technologies were used to analysis the changes of the DNA sequence,DNA methylation and the gene expression related to Pa WB in P.fortunei,P.tomentosa × P.fortunei and P.tomentosaa at morphological level,DNA level,RNA level and epigenetic level,respectively.The results as follows:1.The phytoplasma can be removed in the Pa WB seedlings treated with suitable concentration of MMS,and the three kind of Pa WB seedlings recovered into healthy morphology.Due to the difference in the three species of Paulownia,they were different sensitivity to the MMS,the suitable MMS concentration that can remove the phytoplasma of P.tomentosa × P.fortunei was moe than 30 mg·L-1,and the phytoplasma 16 Sr DNA could not amplified by Nest-PCR in these seedlings,while the results will opposite when the MMS concentration less than 30 mg·L-1;For P.tomentosa and P.fortunei,there need 40 mg·L-1MMS can remove the phytoplasma,while the results will opposite when the MMS concentration less than 40 mg·L-1.2.The phytoplasma cannot change the DNA sequence at AFLP level.In this study,the AFLP approach was performed to analysis the changes of the DNA sequence in the three kind of Paulownia witches' broom(Pa WB)seedlings and the ones with MMS treatment and the healthy seedlings,the results showed that phytoplasma infection had not changed the DNA sequence of Paulownia at AFLP level.3.Phytoplasma infection changed the Paulownia DNA methylation levels and patterns.In this study,the MSAP appooach was used to analysis the DNA methylation levels and patterns of the three kind of Paulownia in their process of the morphological changes,the observations showed that the DNA methylation levels of three kinds of Paulownia Pa WB seedlings lower than the related healthy seedlings,and the DNA methylation level increased after MMS treatments;while the DNA methylation pattern were also changed,there exists DNA methylation pattern and demethylation pattern in the process of the three kinds of Paulownia Pa WB seedlingsmorphological changes,but given priority to DNA methylation patterns.4.The expressions of the genes related to Pa WB were changed after phytoplasma infection.In this study,the different genes were obtained after comparing analysis the DNA methylation patterns through MSAP approach,and obtained 81 difference genes in the P.fortunei,72 difference genes in the P.tomentosa × P.fortunei,and 60 difference genes in the P.tomentosa,at last,we found two genes closely related to Pa WB after comparing the length and the functions of the aboved difference genes.The result illuminated that the two genes might play important roles in the occurrence of Pa WB.The transcriptome sequencing technique was used to analysis the difference expressed genes in P.tomentosa × P.fortunei and P.tomentosa(?)and(?),and 74,2540 and 902 DEGs were obtained,Among these DEGs,we found the genes related to secondary metabolism,such as folate and flavonoid metabolic pathway,genes related to fatty acid metabolic pathway,genes related to clycolysis/gluconeogenesis metabolic pathway,genes related epigenetic modifications,genes related to photosynthesis and carbohydrate metabolism,genes related to Pa WB symptom expression,genes related to plant defense involved in Pa WB.After comparing the sequences and the functions of these DEGs,9 genes were obtained,at the same times,42 Pa WB-related genes were randomly selected from above DEGs for q RT-PCR assays.The results showed that the results of q RT-PCR were agreement with the data generated from RNA-Seq,these results affirmed the validity and reliability of the transcriptome sequencing technique,and provide a new perspective about the research on the interaction of the phytoplasma–Paulownia.