| The pollen fertility of PTGMS rice is regulated by the photoperiod and temperature during the ear development period.After a lot of research,the mechanism of the regulation of photo-fertility is still unclear.Pei'ai 64 S is an indica photo-temperature-sensitive nuclear sterile line derived from Nongken 58 S as a sterile gene source.It is a widely used female parent in a two-line hybrid rice combination.However,it is easy to be used in actual production due to its sterility.Occasionally,seed production losses are affected by low temperatures.Studying the regulation mechanism of temperature on fertility has the theoretical significance of reproductive development regulation and the guiding role of the cultivation of temperature-stable sterility materials.In the study of the regulation mechanism of light-sensitive sterility genes,it has been found that mi RNAs participate in the process of switching regulation of fertility in light-sensitive sterility rice.This study attempts to explore whether mi RNAs are also involved in the process of temperature-controlled fertility.Mi RNA related to sexual temperature regulation analyzes its regulation mechanism.The study selected Pei'ai 64 S near isogenic lines(PA2364S,PA2864S)that responded differently to fertility temperature.The young ears under different temperature conditions were subjected to degradation group sequencing,combined with transcriptome sequencing and Small RNA sequencing combined with previous studies.Screening the expression differences of specific mi RNAs and their target genes further analyzes the mechanism of fertility temperature stability,and provides basic information for the selection and identification of temperature-stable sterile lines.The following main results were obtained:1.The fertility of previous male sterile line materials was verified,microscopic examination of pollen iodine staining showed low temperature treatment can effectively restore the pollen fertility of Pei'ai 64 S.Near-isogenic lines PA2364 S and PA2864 S show significant differences in fertility temperature response.Under 14.0h long light,both were fertile at 21?;PA2364S was sterile at 25?,and PA2864 S was fertile;both were sterile at natural high temperature.2.Using the PA2364 S ears under fertile(21 ? treatment)and sterile(high temperature treatment)conditions to construct a degradation group library,a total of 111,356 degraded fragments were obtained,which were compared with the Japanese c DNA library and combined with target gene prediction software 342 mi RNAs cut 1799 target genes,of which 742 were newly discovered.3.The degradation group sequencing combined with the existing small RNA sequencing and transcriptome sequencing in the early stage of the laboratory revealed that there were 68 mi RNA-target gene regulation pairs in the panicle differentiation stage VI,of which 37 pairs had negative regulation relationships;There were 75 mi RNA-target gene regulation relationship pairs in Phase VII,of which 38 pairs were negatively regulated.Concentration analysis of the significantly different target genes GO and KEGG in the joint analysis found that differentially expressed mi RNA-target gene pairs related to fertility were divided into three categories according to the function of the target genes: Target genes of 12 mi RNAs,such as mi R156 a,mi R319a-3p.2-3p,mi R396a-5p,mi R5809,are related to cell division and differentiation;target genes of 11 mi RNAs,including mi R397 a,mi R399 a,mi R5488,are related to secondary metabolism;The target genes of 16 mi RNAs such as mi R164 a and mi R171i-3p are related to plant hormones.4.The expression difference of PA2364 S and PA2864 S under different temperature treatment conditions was verified by q PCR.Under 21? treatment and high temperature treatment,the expression of opposite fertility in PA2364 S and PA2864 S lines was up and down The results showed consistency,and finally verified that the expression difference between PA2364 S and PA2864 S strains was consistent at 25?.It is speculated that mi R156 a,mi R319a-3p.2-3p;mi R5488;mi R159 a.1 these mi RNAs regulate the expression of the corresponding target genes May be related to the difference in fertility temperature sensitivity.In this study,through the joint analysis of multi-omics data and verification between near isogenic lines with large differences in infertility critical temperature,preliminary selection of mi RNAs that may be related to the difference in fertility sensitivity was combined with the function of corresponding target genes to construct temperature-influencing cells.Division and differentiation,accumulation and transport of secondary metabolites,and plant hormone-mediated fertility regulation network provide some references for further exploring the mechanism of temperature regulation of fertility.This study initially screened out a batch of mi RNAs that are related to differences in fertility sensitivity.It also needs a lot of verification to verify its feasibility as a molecular characteristic indicator of temperature stability in sterile lines before it has application value. |
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