Cloning And Functional Analysis Of Disease Resistance Related Gene TwRLR1 In Triticale

Triticale(× Triticosecale Wittmack)is a generic hybrid derived from a cross of wheat(Triticum)and rye(Secale),which possesses strong heterosis and can adapt to alpine climate conditions.It is an excellent forage for feed production in high altitude areas.Triticale,on the other hand,is exposed to a lot of biological stress in the environment during cultivation,especially yellow rust,which pathogen prefers humid,cool habitats,it can lead to huge losses in the yield and quality of triticale.Yellow rust caused by Puccinia striiformis f.sp.tritici(Pst),the pathogen is virulent to triticale genotypes and frequently variation.A large number of studies and practices have shown that the most effective approach to control the yellow rust epidemic is worthy of breeding excellent resistant cultivars.In this study,using transcriptome sequencing technology,the signaling pathways of interaction between triticale and Pst,and differentially expressed genes were explored and analyzed at the level of transcriptome,along with screen and identify of the yellow rust resistance related candidate genes,is of greatly importance to elucidate the molecular mechanisms of triticale resistance to Pst infection,it is of great significance to genetic improvement and appropriate deployment of resistant cultivars to achieve sustainable control of the disease.Therefore,in this study,seedlings of two triticale cultivars with significant differences in yellow rust resistance,highly resistant Gannong No.2 and susceptible Shida No.1,were inoculated with highly pathogenic Pst race CYR34.Transcriptome sequencing(RNA-seq)was then used to investigate their transcriptional responses against pathogen infection.The molecular mechanism of triticale-Pst interactions was preliminarily analyzed,and the key candidate genes related to yellow rust resistance were screened and identified.Further study cloned candidate gene,RPM1-like resistance gene(Tw RLR1)in triticale.Besides gene expression and protein structure study,Tw RLR1 gene function was preliminarily analyzed by virus-induced gene silencing(BSMV-VIGS).In addition,the regeneration system of mature embryos of triticale was optimized to lay a foundation for genetic transformation of triticale callus and functional analysis of triticale genes.The main findings are as follows:(1)Seedlings of highly resistant cultivar(Gannong No.2)and highly susceptible cultivar(Shida No.1)were inoculated with Pst race CYR34,leaves of two cultivars seedlings were collected both 10 and 20 days after inoculation,and RNA-seq was then performed.The results showed that the number of up-(3,814)and down-(2,581)regulated differentially expressed genes(DEGs)in the resistant cultivar was greater than in the susceptible cultivar(3,335 up-regulated and 1,578 down-regulated).A total of 5,869 DEGs(2,655 up-regulated and 3,214 down-regulated)were identified on the10 days after inoculation,and 5,685 DEGs(2,561 up-regulated and 3,124down-regulated)were uncovered on the 20 days after inoculation,there was no significant difference between these DEGs that up-regulated and down-regulated on the 10 days and 20 days.According to a venn diagram analysis,a total of 2,560 DEGs commonly expressed in the two cultivars on two sampling dates were subjected to pathway analysis,which revealed that most DEGs were closely associated with defense and metabolic activities,such as phenylpropanoid biosynthesis,biosynthesis of amino acids,carbon metabolism,plant-pathogen interaction,plant hormone signal transduction.NAC,WRKY and other transcription factors were involved in the expression regulation.242 DEGs were defined as plant R protein expression genes,of which 120 were annotated with specific domains.(2)A full ORF length of Tw RLR1 gene was 3075 bp,encoding 1024 amino acids was cloned from triticale.Bioinformatics analysis showed that the deduced amino acid contained a Rx-CC＿like and a NB-ARC domain in N terminal;The homology of Tw RLR1 with wheat RPM1-like was 100%;It was localizated on the nucleus and membrane by transient expression in tobacco;The expression pattern of Tw RLR1 gene was analyzed by real-time quantitative PCR(q RT-PCR),the results showed that Tw RLR1 was upregulated in both resistant and susceptible cultivars at different treatment time after inoculation(0 h、24 h、48 h、72 h、96 h、120 h、240h、480 h),the expression of Tw RLR1 gene in Gannong No.2(resistant cultivar)was significantly higher than that of Shida No.1(susceptible cultivar),and expression level was highest at 72 h after inoculation;In addition,Tw RLR1 was expressed in triticale roots,stems and leaves,with the highest expression level in roots,followed by leaves and very low expression level in stems.(3)The expression level of Tw RLR1 in Gannong No.2 was inoculated with BSMV:Tw RLR1 decreased significantly compared with that in the control(Mock and BSMV:00)by BSMV-VIGS technique;A little uredinium appeared on the leaves of triticale that had silenced Tw RLR1 after inoculation with Pst race CYR34,and the infection types of response to Pst also changed,however,the control seedlings infected with CYR34 exhibited a few necrotic stripes,but no sporulation.The above indicated that transient silencing of Tw RLR1 was significantly reduced triticale resistance indicating that Tw RLR1 plays a positive role in triticale resistance against Pst.(4)Mature embryo of susceptible triticale Shida No.1 was used as explants to study the induction and differentiation of callus,and the effect of different concentrations of 2,4-D and Picloram(1.0、1.5、2.0、2.5、3.0 mg/L),different cutting ways to mature embryos(The intact embryo,longitudinal cut,cross cut),and different differentiation media(including three ratios of hormone,6-BA、KT、NAA)on mature embryo in vitro culture.The Agrobacterium-mediated genetic transformation of triticale was also studied.The results showed that there was no significant difference in the ability to induced calli on two hormones at their appropriate concentrations,but the quality of callus induced by picloram was better;The induction rate and quality of callus was promoted by longitudinal cut of mature embryo,and the frequency of embryonic callus rate was significantly higher than that of inoculating the intact embryo and inoculating 1/4 embryos by cross cutting;The addition of 2 mg/L KT,3mg/L 6-BA and 0.2 mg/L NAA in the differentiation medium resulted in a higher differentiation rate of Shida No.1 callus.The Agrobacterium strain GV3101 harboring binary vector p CAMBIA2300-Tw RLR1-e GFP which contained npt II as the reporter gene,transformed into embryogenic callus for optimizing transformation conditions.The results showed that the optimal concentration of kanamycin was less than 90mg/L,on this basis,the Agrobacterium-mediated genetic transformation system of triticale needs further research and discussion.