Functional Analysis Of TaPK3A And TaTT12 In Wheat And Molecular Characterization Of Transgenic Wheat Expressing DmAMP1W

The stable and high yield of wheat is very important for ensuring global food security.Wheat sharp eyespot is a soil borne disease and it mainly caused by the Rhizoctonia cerealis,which has become an important factor in limiting the production of wheat in China for the past several years.However,it is difficult to make great progress on disease-resistant breeding since the difficulty in identification of wheat sharp eyespot and the lack of breeding materials with high resistance to wheat sharp eyespot,and the key gene for wheat resistance to sharp eyespot has not yet been isolated.Systematically studying the molecular basis of wheat defense response and developing effective resistance genes are the prerequisites for the purpose of the breakthrough in wheat resistance breeding.In this study,the expression profiles of wheat genes TaPK3A and TaTT12 were analyzedand their functions in the disease resistance response to sharp eyespot in wheat were preliminary studied.Based on wheat codon usage bias,an antibacterial peptide gene DmAMP1W was synthesized.Its antifungal activity was tested in vitro,and its molecular detection and inoculation resistance identification of transgenic wheat were carried out.In addition,the disease resistance function was further studied.The main results are as follows:In this study,a wheat receptor-like kinase?RLK?gene,named TaPK3A,was cloned from sharp eyespot-resistant wheat cultivar CI12633.The expression and function of TaPK3A were analyzed.TaPK3A contains an open reading frame with 1983 bp length.It encodes a protein kinase that is consisted of 660 amino acids.RT-qPCR analysis showed that the expression of TaPK3A in sharp eyespot-resistant wheat cultivar CI12633 was significantly induced by the pathogen of sharp eyespot.The TaPK3A gene was expressed in all the tissues,and the expression level in the leaves was the highest.The expression of TaPK3A was significantly up-regulated after salicylic acid treatment.By means of barley stripe mosaic virus?BSMV?based virus-induced gene-silencing?VIGS?,TaPK3A was silenced in CI12633 plants.After Rhizoctonia cerealis?WK207?inoculation,TaPK3A-silenced CI12633 plants displayed significantly decreased resistance to Rhizoctonia cerealis infection compared with BSMV:GFP-infected CI12633 plants?control?.These results suggested that TaPK3A was required for wheat defense response to sharp eyespot.In this study,the expression profiles of multidrug and toxic compound extrusion family?MATE family?gene TaTT12 and its functions in the disease resistance response to sharp eyespot in wheat were preliminary studied.The expression of TaTT12 was significantly up-regulated when Rhizoctonia cerealis invaded the disease resistant varieties CI12633,Shanhongmai and susceptible cultivar Wenmai6.In addition,the expression of TaTT12 in the resistant varieties was higher than that of the susceptible varietiy.The expression of TaTT12 was most significant up-regulated after jasmonic acid treatment among the CI12633 treated with four hormones.The BSMV-VIGS experiment results showed that TaTT12-silenced CI12633 plants significantly decreased resistance to Rhizoctonia cerealis infection,and the transcription levels of of defensin and PR12 was down-regulated,which suggested that TaTT12was is positively involved in wheat resistance to sharp eyespot by regulating the expression of defensin and PR12.Antibiotic experiment was carried out in vitro to determine the anti-fungal ability of Dm AMP1W,which was artificial synthesized according to the gene sequence of an antimicrobial peptides Dm AMP1isolated from dahlia.The results showed that DmAMP1W could inhibit the mycelial growth of wheat sharp eyespot,common root rot and take-all pathogen.Also,DmAMP1W had certain ability to inhibit the mycelial growth of wheat stem rot pathogen.Meanwhile,a transgenic vector pWMB122-DmAMP1W was constructed,and Dm AMP1W transgenic wheat Zhoumai18 was obtained by Agrobacterium-mediated transformation.The 3 generation molecular characteristics of transgenic wheat were analyzed by PCR,semi quantitative RT-PCR and Western blot.The results showed that DmAMP1W could be expressed in T₁-T₂ generation transgenic wheat.The average sharp eyespot disease rating of T₁-T₂ DmAMP1W transgenic lines and Zhoumai18 showed that five transgenic lines?AMP166,AMP163,AMP169,AMP544 and AMP552?enhanced 0.69¹.54 and 1.18¹.88 disease resistance levels compared with the acceptor Zhoumai18,respectively.The average common root rot disease rating of Zhoumai18 and four transgenic lines?AMP162,AMP163,AMP169 and AMP543?were 2.41 and 1.42¹.82,respectively.The results above indicated that DmAMP1W expression significantly enhanced resistance to sharp eyespot and common root rot.