Optimization Of Oncidium In Vitro Culture System And Transformation Ananlysis Of Ferredoxin Genes

Onchidium belongs to the Orchidaceae,mainly used for potted and cut-flowers.It is considered as the highest economic ornamental value in the world.Onchidium is very susceptible to disease during cultivation,especially soft rot caused by Erwinia spp which is the devastating disease of the Onchidium industry.The disease can occur every developmental stages of Onchidium,but it has no effective prevention and treatment methods,so selection of resistant varieties of soft rot Onchidium is an important route of Onchidium breeding.Ferredoxin(Fd)is a large gene family that has generally found in plants and is closely related to the accumulation of ROS in plants.Fd has been confirmed that it can contribute in broad-spectrum resistance of some plants,such as rice,tobacco,Arabidopsis and Onchidium.In this study,we used Onchidium "little cherry" as material and base on orchid genome data.The reverse transcriptome-PCR(RT-PCR)combining with rapid-amplification of cDNA(RACE)method was used to clone the complete cDNA sequences of OnFd and OnFNR which are resistance to soft rot related genes.Subsequently,through real-time quantitative PCR and protocorm-like bodies transformation of Onchidium were used to study the function of the two genes,in order to provide a scientific basis about ferredoxin resistance to soft rot in Onchidium.1 Optimization of in vitro culture of OnchidiumOnchidium "little cherry" as material was used to study propagation of protocorm-like bodies of Onchidium.Protocorm-like bodies were placed under different amount of bananas mediums.The result illustrated that the proliferation rate was highest when 50 g/L banana puree was added to 1/2MS plus 1g/L activated carbon.The average growth rate was 5.41 in which the PLBs showed bright green color,growing closely and without differentiation.Moreover,the effects of different hormone types and concentration,natural additives and amount of inoculation on differentiation of protocorms proved that 1/2 MS + 50g/L apple mud inoculated with 8 explants per bottle gave the best differentiation rate of 100%.It can be concluded that the organic addition has effect on the differentiation of PLBs and the proliferation of the cells on Onchidium.2 Cloning and bioinformatics analysis of OnFd and OnFNR in OnchidiumFerredoxin has been proved to aid many plants to obtain broad-spectrum resistance.Based on the orchid gene website,we obtained seven gene sequences which were closely genetic relationship with Fd gene.The length of these seven genes were 244,505,452,616,933,209 and 270 bp,the length of 505,452,616,and 933 have complete ORF sequence.The study used Onchidium "little cherry"health and infection of soft rot of the plant as a material.When screen these seven genes through the qPCR we found a significant increase and decrease in the relative expression of two genes in infected soft rot.So the reverse RT-PCR combining with RACE of cDNA method was used to clone the complete cDNA sequences for them,which were named as OnFd(GenBank accession number KX461907)and OnFNR(GenBank accession number KX461908).Sequence alignment showed that the similarity of these two genes was low,their nucleotide sequence and the length of the encoded protein were quite different which indicated that there was difference in the evolutionary level between the ferredoxin families.Bioinformatic analysis suggested OnFd contains a typical[2Fe-2S]domain,whereas the OnFNR belongs to the FNR-like superfamily which contains the FAD binding domain and the NADP +binding domain.herefore,presumed they have similar functional,though there are some differences in specific parameters,especially physicochemical Nature,transmembrane structure and curly spiraletc that revealed functional difference in different members.3 Determining the expression levels of OnFd and OnFNR in OncidiumThe expression of OnFd and OnFNR genes in different plant tissues and organs of Oncidium through qPCR.The results showed that OnFd and OnFNR were expressed in roots,pseudobulbs,leaves and flowers.The expression of OnFd was the highest in the flower,indicating that OnFd may be involved in the flowering.The expression level of OnFNR was the lowest in root and the highest in the upper and lower leaves which expression level was about 100 times than root.OnFNR belonged to LFNR of photosynthetic type.The use of different hormone treatment and biological stress in Onchidium healthy plants showed that OnFd and OnFNR responded to IAA,ABA,GA,SA,NaCl and PEG.The effects of different hormones on the expression of genes were different and there was certain difference in expression between the two genes under the same hormone treatment.OnFNR expression showed upward trend under AA,GA,SA,ABA and NaCl.In contrast,only PEG treatment showed the downward trend.OnFd showed a downward trend under IAA,SA,ABA and NaCl treatment,and under SA,ABA and NaCl treatment showed an upward trend.These results suggested that both exogenous hormones and biological stresses affect the expression of OnFd and OnFNR.This effect is closely related to the accumulation of ROS.4 Determining the expression levels of OnFd and OnFNR in different stage of disease of OncidiumThe soft rot pathogen was isolated from soft rotted leaves of Oncidium "Grower",the DNA sequence of pathogens with a similarity of 99%of Erwinia carotovora pectinase gene(J03673.1)was obtained by primer Y1/Y2.Indicating that the main pathogens of soft rot in the study of Oncidium was Erwinia.The roots,pseudobulbs,upper and bottom leaves form Oncidium which has infected after Id,3d,6d,9d,12d and health plants as material.The qPCR result showed that OnFd relative expression was upward trend in all parts of the plant.The Pseudobulb expression levels showed significant increases(P<0.01)after pathogen inoculation on the 1st day and the expression level in the five susceptible stages were 2.83?3.98 times than healthy plants.OnFNR gene upper and bottom leaves,and bulbs of the vaccination site expression showed downward trend,the expression showed significantly reduced(P<0.01)after pathogen inoculation.However,the root demonstrated a significant upward trend,and reached the highest value on the 12th day which the expression was about 4.5 times than the control group.Therefore,both the OnFd gene and the OnFNR gene had a significant response to the soft rot bacteria,indicating that the two genes may play an important role in the process of response to soft rot in Oncidium.5 The subcellular localization analysis of OnFd and OnFNR proteinsAfter obtaining the full length cDNA sequences of OnFd and OnFNR,we made constructs of these genes tagged with green fluorescent protein(GFP)subcellular localization vector and ectopic expression in tobacco through Agrobacterium-mediated method and observed the protein localization.The results showed that OnFd and OnFNR were all located in chloroplast6 OnFd and OnFNR gene was transferred to OncidiumConstruction of 35S:OnFd and 35S:OnFNR as overexpressing vector,Construction of OnFd RNAi and OnFNR RNAi as inhibition vector which was used pJM007 as the intermediate vector.Agrobacterium tumefaciens-mediated method was used to transfer them into PLBs of Oncidium.The results showed that transgenic PLBs did not grow well in the resistance culture and the color was yellowish,but the color changed to green after entering the differentiation culture and the volume of PLBs were larger than non-transgenic.The expression of OnFd and OnFNR in PLBs were highly increased.The expression levels of OnFd and OnFNR in PLBs that inhibited OnFd expression were significantly decreased.The expression of OnFNR in PLBs that inhibited the expression of OnFNR was significantly decreased,whereas,the expression of OnFd was significantly increased.When soft rot pathogens infected to PLBs,we found overexpression of OnFd and OnFNR in PLBs were more resistant to soft rot than inhibited on OnFd and OnFNR expression.SOD and ferredoxin have a close relationship,so in this study the transgenic PLBs was used as the material.In qPCR we found overexpression OnFd and OnFNR on PLBs.The expression levels of Cu/ZnSOD and FeSOD were significantly down-regulated.When the expression of OnFd and OnFNR was inhibited.The expression of Cu/ZnSOD was significantly up-regulated but the expression of FeSOD was down-regulated.Only the expression of MnSOD showed a significant increase in OnFd and OnFNR overexpression or inhibition of expression.The changes of SOD-related gene expression may be related to the redox reaction of OnFd and OnFNR in plants.