Detection And Analysis Of Viruses Using Small RNAs In Small Yellow Ginger(Zingiber Officinale Rosc.)

Ginger(Zingiber officainale Rosc.)is a monocot perennial herb and an important economic crop in china,which belongs to Zingiber(Zingiberaceae),Zingiber(Zingiber Mill.)in plant taxonomy.Ginger does not bloom or rarely blossoms,and has long been used for asexual reproduction.There are many kinds of bacteria and virus in ginger,and the infection of virus caused the decline of ginger yield and quality.It is significant to detect and analysis the unknown virus of yellow ginger.Hence,the small RNA high throughput sequencing analysis was used to detect the viruses in small yellow giner of the Liupanshui city of Guizhou province.A new ginger virus was detected by assemble and alignment of the sRNA sequence,the virus patial genome was cloned and identified by reverse PCR cloning and fragment sequencing.The main results were summarized as follows:1.This study uses sRNA high-throughput sequencing technology detecation ginger virus,and the ginger collected form zhe Xi country,Liupanshui City,Guizhou Province.Results showed that the virus is a member of badnavirus.This study successfully established the analysis pipelines of bioinformatics to detect plant viruses with small RNA high-throughput sequencing technology.To our knowledge,this virus infection in small yellow ginger is reported for the first time in china.2.New strains of badnavirus or related novel virus may exist in small yellow ginger samples.1)Using the degenerate primers of the Badnavirus amplified 6 genes of RT/RNase H form ginger.These sequences showed less 74% similarity with Badnavirus RT/RNase H sequence.According to the current international on Taxonomy of viruses Committee report,the similarity of nucleotide sequence of RT and RNase H in Badnavirus is less than 80%,which can be divided into different virus species and named Zingiber bacilliform virus taporary.Construct the Phylogenetic tree with 21 Badnavirus members showed these isolates exist 3 novel virus.2)The reference genome of ZBV is circular and contained 5564 bp,Using bioinformatics analysis of the cloned sequence found that its exist conserved domain are exist in multiple members of the Badnavirus.ZBV may be deleted a part of fragment in the evolution.In nature,the virus is the fastest rate of variation in all biology,through genome reorganization,deletion or insertion of fragments to obtain a new genome sequence,in order to increase the fitness of the host.The results showed that there was a complex polymorphism in the process of ZBV evolution.3.Using the Perl language script written by ourselves,we analyzed the hot spot distribution of the small interfering RNA in the reference genome of ZBV.The sRNA length mainly are 21,22 and 24 nt in size.In the process of plant defense virus infection,the third kinds of digestive enzymes in plant DNA(RNase III-type)Dicer-like family(DCL enzyme)cleavage the double stranded RNA and product overlap sRNA,the length usually are 21~24nt.In Arabidopsis thaliana,dsRNA digested by DCL2,DCL3 and DCL4,respectively,formed a virus derived small RNA with length of 21,24 and 22 nt,respectively.It is speculated that DCL3 plays a major role in antiviral activity in small yellow ginger.4.Using PCR detecation the badnavirus in Guizhou,Hainan,Yunnan,Hubei and Guangdong 20 yellow gingers,the primer is the report of the badnavirus universal primer BadnaFP/BadnaRP.Results showed that all samples were amplified products.The detection rate was 100%.Using the detoxification of sterile zhexi yellow ginger from our laboratory tested the badnavirus.Results showed that it can get amplified products.Indicating that virus whole genome sequence or patial genome sequences has been integrated into the host plant genome of yellow ginger.Some or all of the viral genome sequences integrated into the host plant genome my be expressed in plant cells and further affect the growth and development of the yellow ginger.