| Photoperiod and thermo-sensitive genic male sterile(PTGMS)rice is the basic material of two-lines hybrid rice,and a large amount of research has been carried out on its fertility alteration mechanism.The sterility genes were located and its function and fertility regulation mechanism of photoperiod and temperature were researched.However,the specific relationship between sterile genes and pollen development and fertility determination remains unclear.Lignin metabolites are important components of pollen wall and play an important role in anther development and pollen grain formation.This study take pollen development regulation as the starting point,using PTGMS rice PA2364 S as materials.The role of lignin metabolism in the fertility alteration of PA2364 S was explored through fertility induction of light and temperature treatment,fertility identification,cytological observation,detection of lignin metabolite gene transcription level analysis,enzyme activity determination and mi RNA expression analysis.The main results are as follows:1.In the fertile sensitive period of PA2364 S,artificial climate box was used for different temperature treatment,and different fertility levels of treatment effects were obtained.The results showed that the pollen of PA2364 S was completely sterile under 14 h long day at 30℃.The pollens treated at 21℃ were partially fertile and the iodine staining rate was 39.30%.The results showed that the fertility temperature sensitivity of PA2364 S was obvious.2.The differences of anthers and pollen walls under high and low temperature were observed by fluorescence staining,scanning electron microscopy and transmission electron microscopy.The results of pollen wall staining showed that the staining degree of sterile pollen grains under high temperature treatment was shallower than that under low temperature treatment.The results of scanning and transmission electron microscopy showed the concave of anthers surface of PA2364 S under high temperature treatment and the lack of pollen exine sculpture.The exine of pollen showed a jagged fracture the lack of baculum.The results showed that the accumulation of sporopollenin and lignin metabolites in sterile pollen wall was inhibited under high temperature treatment.3.The results of lignin metabolism detection of PA2364 S spikelet showed that lignin metabolism of fertile spikelet under low temperature treatment was more active and lignin metabolic flux was larger.Moreover,the contents of 4-coumaric acid,ferulic acid and G monomer in lignin metabolites were increased,which are precursors of pollen sporopollenin,and the difference of G monomer content reached a significant level.4.Transcriptional analysis of 22 genes involved in lignin metabolism and mi R5488 showed that 10 genes of PA2364 S,including 4CL3,HCT1,COMT1,CCo AOMT2,COA1,COA26,CCR19,CCR20,CAD1 and CAD2,were up-regulated in pollen wall synthesis stage 9-11 under low temperature treatment.Three genes,C4H1,CCR1 and CCR21,were down-regulated in stages 9-11.Except PAL and C4 H,the transcription levels of lignin metabolism genes were up-regulated under low temperature treatment.However,there was no significant difference in the expression of F5 H in stages 9-11.The expression pattern of mir5488-4CL3 showed that mi R5488 was highly expressed in high temperature treatment PA2364 S spikelet at 8a-9 stages,which was opposite to the expression pattern of 4CL3.The expression of mi R5488 at 10-12 stages showed no significant difference.Therefore,the results showed that the up-regulated transcription of PA2364 S lignin metabolic genes led to the products flow to G monomer under low temperature treatment,and mi R5488 regulated the early differential expression of 4CL3 and participated in the regulation of lignin metabolism.5.The results of the determination of PAL,C4 H,4CL and CAD enzyme activity of PA2364 S anther showed that the enzyme activity of PAL and C4 H at the 10 stages of low temperature treatment was significantly higher than that at high temperature treatment.The enzyme activity and transcription levels of 4CL and CAD were consistent,and the enzyme activity was higher in the 10-12 stages at low temperature treatment,and the difference of enzyme activity in 10 stages reached a significant level.In this study,the differences of anther and pollen wall of PA2364 S under high and low temperature treatment were observed by staining and electron microscopy,and the abnormal development of anther and pollen wall of PA2364 S under high temperature treatment was confirmed.At the same time,it was found that the expression of PA2364 S lignin metabolism gene was up-regulated under low temperature treatment,and the enzyme activity increased,more sporopollenin precursors were produced,which promoted the synthesis of pollen wall and anther development.It was one of the reasons for the fertility recovery of PA2364 S under low temperature.mi R5488 was one of the regulatory factors of this process. |
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