Genetic Variation Of RBSDV S6 And Regulation Pathogenic Mechanism Of Maize Rough Dwarf Disease

Maize rough dwarf disease(MRDD)is one of the important diseases effecting maize production in China and even the world.Rice black streaked dwarf virus(RBSDV)is the main virus causing maize rough dwarf disease in China.RBSDV is a member of Fijivirus of Reoviridae,which contains 10 ORFs and encodes 13 proteins.At present,the pathogenic characteristics,gene structure and the function of some of 13 ORF encoded proteins of RBSDV have been studied.However,how the silencing inhibitor RBSDV S6 participates in the pathogenic process and mechanism of maize rough dwarf disease is not clear.Therefore,we analyzed the variation,evolution and molecular characteristics of 26 S6 sequences from maize and rice by bioinformatics,and analyzed the pathogenic mechanism of P6 involved in maize rough dwarf disease by molecular biological approaches such as transgenic and yeast two hybrid screening library.The main results are as follows:1.Using bioinformatics software such as MEGE X64 and DNA SP to analyze the sequence variation and evolution of 26 S6 sequences of maize and rice from 13 locations.According to the hosts rice and maize,the 26 sequences are divided into three subgroups for analysis,namely M18(maize),R8(rice)and T26(maize and rice).(1)There were significant differences in nucleotide sequence diversity among M18,R8 and T26 populations(P ≤ 0.05),indicating that there was variation and recombination between S6 sequences.(2)By analyzing the amino acids encoded by S6 and codon preference,it was found that there was no significant difference in codon preference among S6 subgroups.ORF6 had other codon preference except GC3 s,and the codon use of the three groups was under the pressure of natural selection.(3)Based on genetic recombination analysis,there were two recombination events(S6-M-13ⅤM-2 and S6-M-13ⅤM-3)in 26 sequences,all of which came from M18.Based on phylogenetic analysis,S6 sequence can be divided into four groups.(4)The analysis of S6 sequence population by population neutralization test and selection pressure showed that ORF6 region was subjected to negative selection and had purification effect to maintain the functional conservation of the gene.(5)By analyzing the genetic differentiation and gene flow of S6 population,the|FST| between populations(T26-M18,T26-R8 and M18-R8)is less than 0.33 and the absolute value of |Nm| is greater than 1,indicating that there is frequent gene flow among populations.The RBSDV population based on S6 segment is showing an expansion trend.2.Bioinformatics analysis predicted that P6 protein was non membrane protein and non-secretory protein.The subcellular localization of P6 based on tobacco system showed that P6 was located in the plasma membrane.q RT-PCR showed that the transcription level of S6 increased firstly and then decreased after inoculation with RBSDV.The plant height of S6 transgenic lines was significantly lower than that of receptor B104 and Nipponbare(P ≤ 0.01).3.The potential interacting proteins of P6 were screened from maize c DNA library by yeast two hybrid system.The interaction between blue light receptor Zm PHR2 and rice homologous protein Os PHR2 and P6 was demonstrated by yeast two hybrid and luciferase complementation tests.Further identification of the phenotype of Zm PHR2 mutant showed that the plant height of mutant zmphr2 was significantly lower than that of wild-type B73.