| PTGMS rice as an important germplasm resource.Light and temperature regulation mechanism of fertility is the theoretical and technical basis for the application of two-line hybrid rice.Although our scholars have obtained abundant results in the basic research of rice fertility regulation,the internal relationship between light,temperature and fertility is still unclear.In this study,high-throughput sequencing technology was used to analyze the small RNA of different types of photo-thermo-sensitive sterile material under different fertility conditions,qPCR technology was used to detect the expression differences of screened microRNAs and their target genes,we tried to obtain some intrinsic physiological indicators related to fertility differences.The main results are as follows:1.Temperature treatment under the same illumination can effectively induce pollen fertility conversion of Pei’ai 64 S.Pollen fertility of D52 S and Nongken 58 S can be effectively induced by photoperiod treatment at the same temperature,and the fertility of the two materials was opposite under the same photoperiod.2.Small RNA was detected by Illumina HiSeqTM2500 system at stage 3,6,7 of rice panicle materials with different fertility.The number of reads in all replicates is over 14 million,with an average length of 22-23 nt.The correct rate was above 99%,482 known miRNAs were detected,and 48 new miRNAs were detected.3.Using natural temperature treatment as the control,93 differentially expressed miRNAs were found at stages 6,of which 46 were up-regulated and 47 were down-regulated;103 differential miRNAs were found at stage 7,of which 61 were up-regulated and 42 were down-regulated.In addition,39 differential miRNAs were appeared at both the stage 6 and stage 7,including 29 known miRNAs and 10 novel miRNAs.4.The reliability of sequencing results was verified,and some differentially expressed microRNAs were screened by KEGG and GO enrichment.According to the function of their predicted target genes,they were divided into three categories:glycosyltransferase-regulated miRNAs(miR1436,miR818 a and miR5494),Lipidtransporter-related microRNAs(micro1862d,micro171 g and micro171b)and methyltransferase-related microRNAs(micro1860-5p,micro2863 a,micro5504,micro5794,micro6251 and micro2876-3p).5.The expression of target gene in Pei’ai 64 S under different fertility in different stages was detected by q-PCR.miR818 a,miR5494,miR860-5p,miR2863 a,miR5504,miR2876-3p were contrary to the target gene expression trend;miR1436,miR1862 d were same trend as target gene expression.6.Two target genes with glycosyltransferase activity have the same expression trend at stage 6 of fertility conversion,stage 6 is a important stage for fertility conversion,and all are up-regulated under low temperature treatment;Both miR171 b and miR171 g are up-regulated under low temperature induction,and we believe that these two miRNAs may be involved in temperature response;Four target genes with methyltransferase activity were down-regulated by low temperature induction at stage 6.The other three target genes except Os02g0237100 were up-regulated at stage7,indicating that the fertility conversion of Pei’ai 64 S may be related to methylation.7.Analysis of the expression of screened microRNAs and their target genes in D52 S and Nongken 58 S.The same of the expression trend is defined as participation in photoperiod response including: miR1860-5p,miR5504,miR2876-3p;The opposite of the expression trend is defined as participation in photoperiod regulation including: miR1436,miR171 g,miR6251.The specific mechanisms by which these miRNAs participate in photoperiod response and regulation still require in-depth research.In this study,we obtained a lot of valuable information by using small RNA sequencing technology.It is clear that small RNA plays an important role in fertility transformation of photo-thermo-sensitive male sterile rice.Some of the selected microRNAs that may be involved in fertility regulation can be used as molecular indicators for fertility expression level detection and the basis of metabolic process research after further validation. |
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