| Oat(Avena sativa L.)is an annual Gramineae plant,which is used for both grain and feed and has the function of dietotherapy.It has high nutritional value and health care function.β-glucan is one of the most important nutritional components in oats.RNA-seq can not only quantify the expression level of specific differential genes in different periods,but also identify pathways and genes related to physiological processes.Therefore,in this study,high β-glucan content oat variety Bayou 18 and low β-glucan content oat variety Diyan 5 were used to determine the content of β-glucan in grains at different filling stages.the grains at different filling stages were sequenced,the main functional and metabolic pathways were explored,and the differentially expressed genes related to β-glucan synthesis were analyzed and mined.The differentially expressed genes were analyzed by WGCNA association analysis and qRT-PCR verification,in order to reveal the molecular mechanism of oat β-glucan synthesis and accumulation.The main results are as follows:1.The accumulation rate of β-glucan in oat grain showed the characteristics of "slow-fast-slow".The 6d-16 d accumulation rate of grain filling was the fastest,and the accumulation rate of β-glucan slowed down after 16 days of grain filling.The middle filling stage is the key period for the synthesis and accumulation of β-glucan in oat grains.There was a significant difference in the content of β-glucan between the two varieties.The content of β-glucan was 5.21% in Bayou 18 and 2.03% in Diyan 5 at maturity.2.Compared with the first day of grain filling,the number of differential genes in the grains of the two varieties increased continuously,and reached the maximum on the 21 st day of grain filling.The number of differential genes among the comparison groups of Bayou 18 changed more obviously.in the expression pattern,there were more differential genes up-regulated than down-regulated in each comparison group of the two varieties,and there was a great overlap in gene expression at different stages.3.Seven differentially expressed genes related to β-glucan accumulation were identified as novel.2511,novel.2224,TRINITY＿DN34252,TRINITY＿DN15420,Pepsico2＿Contig6346,TRINITY＿DN34723 and Pepsico1＿Contig10341.Except for novel.2511 and novel.2224,the overall expression patterns of these genes among varieties were as follows: the differential expression of β-glucan synthesis related genes of Bayou18 was earlier than that of Diyan 5.In the same period among varieties,the relative expression ratio of up-regulated genes in Diyan 5 was lower than that of Bayou 18,while the relative expression multiple of down-regulated genes was higher than that of Bayou18.The overall expression pattern of these genes among varieties was as follows: the differential expression of β-glucan synthesis related genes of Bayou 18 was earlier than that of Diyan 5,and the relative expression ratio of up-regulated genes in Diyan 5 was lower than that of Bayou 18 in the same period.however,the relative expression multiple of down-regulated genes was higher than that of Bayou 18.4.A total of 25 β-glucan related genes were screened among different varieties(BY4vs DY4),including 18 endonuclease 1,3-β-glucosidase genes,4(1-3)(1-4)-β-D-glucanase genes,1 β-glucuronosyltransferase gene,1 mixed ligated glucan synthase gene and 1 endonuclease 1,4-β-glucosidase gene.5.WGCNA analysis of DEGs in oat grain filling stage showed that 21 DEGs of the 32 differentially expressed genes were located in the blue module,5 in the magenta module,1in the lightcyan module and 1 in the white module.The differentially expressed genes in these modules may play an important role in β-glucan biosynthesis.6.Using barley Actin gene as internal reference gene,the results of qRT-PCR verification of 5 differentially expressed genes in selected varieties were basically consistent with the results of transcriptome sequencing,which proved that the transcriptome data were accurate and reliable. |
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