Study On Molecular Detection Of Major Diseases,WOX Transcription Factor Family,in Situ Hybridization And Shoot-tip Grafting Of Citrus

The bacterial and viral diseases of citrus have caused a huge impact on the citrus industry.By analyzing the molecular detection technology of citrus bacterial and viral diseases,this research carried out molecular detection of diseases on Volingxia orange callus and citrus plants.At the same time,the phylogenetic tree,gene structure,conserved motifs,gene colinearity and expression of 11 WOX transcription factor families identified from the citrus genome were analyzed;probes for Cs H4-1、Cs H4-2、Cs WUS、Cs SAM、HLB、CTV and CYVCV were prepared.The shoot apex meristem of suitable size was cut for citrus shoot apex shoot-tip grafting.1.Analysis of molecular detection of citrus major diseasesThe callus of Volingxia orange was tested by q PCR and conventional PCR for HLB,as well as RT-q PCR detection of CTV,CYVCV,CTLV,CLBV and CPV.Primers A04F/A04 R and HLBas/HLBr correspond to the target gene CT values of 27.84-33.77.The conventional PCR detection found that the primers A04F/A04 R for HLB have non-specific amplification;at the same time,in RT-q PCR detection,the CT values of target genes such as CTV are in the range of30.35-33.37,which further verified the extremely low or even non-toxic properties of callus,which can be used as a negative control for molecular detection of citrus diseases.At the same time,RT-q PCR detection of CTV and nested RT-PCR detection of CYVCV were carried out on leaves of 66 citrus plants of ’Tianyou’,’Juyou’,’Lane Late’,’Maogan’,’Rinan’ and ’Xunwu’.Among them,20 strains were infected with CTV,59 strains were infected with CYVCV,and 20 strains were combined with infection.2.The citrus WOX transcription factor familyEleven members of the WOX transcription factor family were identified from the citrus genome,and it was found that all members of the citrus WOX transcription factor family could be divided into modern clades,intermediate clades,and ancient clades,and contained highly conserved homeodomains,which were similar to conserved clades.The motif motif-1 corresponds,while modern clade members contain the WUS-box domain,which corresponds to the conserved motif motif-3.And found that the gene structure of citrus and Arabidopsis WOX orthologous genes is highly conserved,and there is a collinear relationship,which further indicates the reliability of the results of the two phylogenetic trees.At the same time,the citrus WOX transcription factor family promoter contains a large number of TATA-box,CAAT-box cis-acting elements,as well as ABRE,MYB and other stress-related cis-acting elements.In addition,the expression of citrus WOX transcription factor family was preliminarily analyzed.The expression levels of CTV,CYVCV,and Cs SAM were high in the shoot tips of ’Xunwu’,while the expression of Cs WUS was low in the shoot tips.The high expression levels of petals,stamens and pistils indicated that Cs WOX3 was involved in the development of leaves and floral organs.3.In situ hybridization and shoot-tip grafting analysis of citrusThrough the multi-sequence analysis of the pathogens of HLB,CTV and CYVCV,and the construction of Cs H4-1,Cs H4-2,Cs WUS,Cs SAM gene vectors,the target fragment of the probe amplified by the recombinant plasmid was used as a template for in vitro transcription.Probes for Cs H4-1,Cs H4-2,Cs WUS,Cs SAM,HLB,CTV and CYVCV,optimized the production of citrus shoot apex paraffin sections,and performed citrus shoot apex in situ hybridization experiments.Histone Cs H4-1,Cs H4-2 was highly expressed in citrus tissues,which was used to test the feasibility of the in situ hybridization experimental method,but hybridization signal wasn’t detected,and no hybridization signal wasn’t detected by Cs WUS,Cs SAM,CTV,and CYVCV probes.The 16 S r DNA sequence is designed for HLB in situ hybridization probe.The specificity of the HLB-2 probe is not strong,and the in situ hybridization result may be background or false signal.The specificity of the HLB-1 probe is strong,and the in situ hybridization result may also be background or false signal,which needs to be verified repeatedly.The stem tips of citrus ’Tianyou’ containing 2-3 leaf primordia and growth points were observed by stereo microscope and scanning electron microscope,and the size was about0.18～0.25mm;cut out the leaf primordia and growth points containing about 2-5 shoot tips were grafted on the incision of the yellowing rootstock of Citrus aurantium,and the grafted seedlings were colonized in MS liquid medium for culture,and the temporarily surviving grafted seedlings were transplanted into the matrix in the second week,After that,there were 4 ’Tianyou’,15 ’Juyou’,7 ’ Lane Late’,11 ’Maogan’,2 ’Rinan’ and 1 ’Xunwu’.