| Sunflowers are widely planted in Northeast China,Inner Mongolia,Xinjiang and other places,in the process of sunflower production,the sunflower rust caused by Puccinia helianthi Schw.seriously affects the production and planting of sunflower,which resulted in the decrease of resistance and the emergence of new races.In this study,Illumina HiSeq 2500 sequencing platform was used to carry out high-throughput sequencing of the transcriptome of sunflower-rust interaction,to systematically understand the transcriptional characteristics of the interaction between pathogen and host,and to screen and excavate the relevant resistant genes.The main findings of the study are as follows:1.In this study,mRNA was isolated from total RNA by RNA-Seq technique and then reversely transcribed into cDNAs,and then high-throughput sequencing was carried out on the cDNA library.In the 9 RNA samples obtained,the A260 / 280 value was approximately 1.92~1.97,and the samples had obvious peak values at 2000nt-4000 nt.Agilent 2100 analyzer was used to sequence and compare the RNA-seq sequences of three non-compatible combinations of sunflower leaves(B-1,B-2,B-3),Affinity combination(C-1,C-2,C-3)and control group(A-1,A-2,A-3)respectively.The results of RNA-seq sequencing and comparison showed that the percentage of Q20 was higher than 97% Q30 and the percentage of reads compared with reference genome sequence was higher than 76.64% of Total reads,which met the need of analysis.2.Based on the results of high quality sequencing,the obtained clean reads was assembled from de novo.The length range of the assembled sequence was between 100~3000bp and the unigene was classified according to the length of the sequence,and the number of sequences with the length of >=3000bp was the highest.Secondly,the sequence length is in the range of 600~699bp,and the least proportion is the length range of 100~199bp.3.The differentially expressed genes were screened by pairwise comparison of three groups of samples or by comparison of three groups of samples.There were 1799 differentially expressed genes between sample A and sample B,2078 differentially expressed genes between sample B and sample C,and 2611 differentially expressed genes between sample C and sample A.In addition,585 differentially expressed genes were obtained by comparing the three groups of C-B-A samples according to the principle of not considering up-regulation or down-regulation,so long as there were differences.4.Carrying out functional classification on the differential expression gene by GO,the results showed that "cilium" and "motile cilium" were the most significant enrichment of GO terms "chitin binding" and "purine nucleoside binding","purine ribonucleoside binding" is the most significant enrichment of GO terms in molecular function,while "response to stress","recognition of pollen" and "cell recognition" are the most significant enrichment of GO terms in biological process.5.The KEGG pathway annotation of differentially expressed genes showed that,in B-VS-A,B-VS-C,C-VS-A,389,588,634 unigene were involved in six biochemical metabolic pathways,including substance Metabolism,Organismal Systems,Genetic Information Processing,Environmental Information Processing,Human Diseases and Cellular Processes.In the results of significant enrichment analysis of KEGG Pathway,there are 606 differentially expressed genes in B-VS-A annotated to 193 pathways.A total of 1047 differentially expressed genes in B-VS-C were annotated to 215 pathways.A total of 1051 differentially expressed genes in C-VS-A were annotated to 211 pathways.The paths of significant enrichment are sesquiterpenoid and triterpenoid biosynthesis,phenylpropanoid biosynthesis,phenylalanine metabolism,estrogen signaling pathway,drug metabolism-cytochrome P450,MAPK signaling pathway,antigen processing and presentation,etc.These results suggest that pathway enrichment is related to the biosynthesis,metabolism,transport,processing and biotransformation of substances.6.From the analysis of the above digital gene expression profile,the pathogenesis-related proteins,protein kinases,transcription factors,photosynthesis and glutathione S-transferase play an important role in the plant immune response.Therefore,they may be closely related to the rust resistance of sunflowers.In addition,plant-pathogen interaction and phenylpropanoid biosynthesis may also be involved in the disease resistant response.The resistant mechanism of sunflower can be further inferred by the expression of rust resistant genes in sunflower,which lays a theoretical foundation for future utilization of plant resistance genes. |
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