Germplasm Materials Obtainment Of Non-host Resistance Gene Mediated Wheat Resistance To Fungal Disease And The Study Of Protoplast Preparation And Regeneration Of Rhizoctonia Cerealis

Wheat?Triticum aestivum?is one of the three most widely distributed food crops in the world,which is also very important in C hina.During the cultivation of wheat,Fungal disease is an important factor that affects the yield and quality.wheat sharp eyespot and root rot of wheat are two important soil spread fungal diseases of wheat root,especially in the main producing areas of winter wheat in China that are increasing year by year,which can accumulate in the soil over the years and infect wheat from seedling to adult plant stage,causing root-rot of wheat and serious impact.So far,there is no efficient chemical agent to control such kind of soil spread fungal diseases.Breeding and applicating new wheat variety is undoubtedly the most economical and effective way to control the two diseases.Attribute to less understanding of genetic mechanism and lack of resistance gene resource,It's necessary to consider using new resistance genes to create a broad spectrum and stable new germplasm.In this study,A cotransformation strategy of“Avr/R transgene combinations”was applied to connect pathogen inducible promoter with improved avrRxo1gene fragment cloned from Xanthomonas oryzae pv.Oryzicola,and Co-transformate the connected fragment to wheat varieties Bobwhite with no n-host resistance gene Rxo1 cloned from maize.The molecular mechanism was explored to comprehend the interaction between non-host resistance gene Rxo1 and avrRxo1 gene fragment against fungal diseases such as root rot of wheat.The results are shown below:The pathogen inducible expression vectors were constructed and transformed to wheat by means of gene gun.By Agrobacterium mediated transient expression system,the function of pathogen inducible promoter 35S-mini was identified that it can start the expression of GFP from 12 h after inoculation with Bipolaris sorokiniana and Rhizoctonia cerealis.The expression vector including the pathogen inducible promoter connected with the improved avrRxo1 gene fragment were constructed,and was co-transformed with Rxo1 gene to the wheat variety Bobwhite by gene gun mediated method.132 transgenic regeneration plants were obtained through the screening of herbicide stress during the prosses of regeneration and acclimatization.The positive seedlings of transgenic wheat were screened and verified by molecular detection.Molecular detection of PCR was carried out on the regenerated seedlings of transgenic wheat,and 12 positive plants containing both Rxo1 and avrRxo1 genes simultaneously were obtained.RT-PCR detection was carried out 48 h after inoculating Bipolaris sorokiniana on the leaves of positive transgenic wheat,line KCA271,CA411,KCA352,the results showed that avrRxo1 and Rxo1 gene expression were both detected in the three transgenic lines.The disease resistance of the T₂ transgenic wheat was analysised.The pathogenic fungi of Rhizoctonia cerealis and Bipolaris sorokiniana was inoculated separately to T₂ generation lines KCA271,KCA348,KCA352 and CA411.and the results showed that,compared with wild type controls,these four lines of T₂ generation transgenic showed varying degrees of resistance to wheat sharp eyespot and root rot,and line CA411 which contains pathogen inducible promoter 35S-mini showed the strongest disease resistance.By inoculating B.sorokiniana on the leaves of CA411 strains of T₂ transgenic wheat,the disease resistant mechanisms of transgenic wheat were preliminary explored through cytological observation,determination of active oxygen content and protective enzyme system activity,and defense gene expression respectively.?1?Deposition of lignin,callose,and phenols were enhanced in the T₂ transgenic wheat lines after inoculation with Bipolaris sorokiniana on the leaves.?2?A significant accumulation of reactive oxygen species was tested in the transgenic leaves 24 h after inoculation,and the second peaks appeared after 96 h.Activity of SOD was significantly increased in 24 h,and the upward trend continued until 48h after inoculation;Compared with wild type plants,the C AT activity of CA411 lines was relatively low in the early stage of pathogen inoculation.?3?Real time RT-PCR analysis showed significant expression increasing of avr Rxo1 and Rxo1 at 24 h after inoculation,which indicated that the the expression of exo genous gene were induced by pathogen inoculation.TaPR1,TaPR2,TaPAL,TaSOD and TaAPX genes in CA411 lines were up-regulated during 24⁷2 h after inoculation.The highest expression level of TaPAL and TaAPX was detected at 24 h after inoculation,while TaPR1 and TaSOD at 48 h.there was no higher level of expression for TaCAT than the wild type plants in the process of pathogen infection.R.cerealis is the main pathogen of wheat sharp eyespot in our country.In order to obtain high quality protoplasts fo r the transformation of R.cerealis,the effect factors on protoplast preparation and regeneration of R.cerealis were studied.The results showed that the suitable conditions of protoplast preparation were 6 d mycelium,in the mixture enzymes of 15 mg/mL Lywallayme and 10 mg/mL Snailase,digesting at 30?for 4 h,the protoplast yield reached3.0×10⁶ cell/mL.The optimal regeneration conditions of R.cerealis protoplast were using SuTC as osmotic stabilizer,adopting TB3 medium for protoplast regeneration,inoculating the protoplasts in the monolayer regeneration medium by mixing the protoplasts with the unsolidified medium,protoplast regeneration rate can reach 58.6%.In summary,it is for the first time in this study to transfer the non-host resistance gene Rxo1cloned from mize into wheat.And a cotransformation strategy of“Avr/R transgene combinations”was applied in order to induce the expression of disease resistance.The transgenic wheat lines showed higher resistance to wheat sharp eyespot and root rot.This experiment provides a theoretical basis for the the wheat resistance gene engineering to introduction non-host resistance genes from other species to create a new broad spectrum,sustained and stable disease resistant wheat germplasm.