| Male sterility caused by high temperature is a common phenomenon in plants,and it is also one of the main factors that lead to the yield instability of three-lines hy-brid F1 with high temperature sensitivity.Global warming intensifies,making hot heat damage more frequent.Sustained high temperature will lead to insufficient anther cracking and significantly decreased pollen survival rate of plant three-lines hybrid F1,which will eventually lead to reduced pollen fertility and reduced yield.However,the molecular mechanism of male fertility instability of three-lines hybrid Fi caused by high temperature stress is still unclear.In this study,high temperature treatment was carried out on high temperature-resistant and high temperature-sensitive soybean three-lines hybrid F1,transcriptome sequencing was performed on the flower buds of before and after high temperature stress of two soybean three-lines hybrid F1 with differrent sensitivity to high temperature,a large number of differentially expressed genes（DEGs）were screened for the effects of high temperature stress on soybean male fertility.At the same time,functional studies were carried out on the three DEGs（GmHSFA2,GmHSP20 and GmHSP70）in response to high temperature stress,to identify their role in the regulation of male fertility of soybean three-lines hybrids F1 under high temperature stress.The main results are as follows:1.In order to study the effect of high temperature stress on the male fertility of soybean three-lines hybrid Fi,we used a plant incubator to conduct temperature gradient treatment on soybean three-lines hybrids NF1（NJCMS1A×N4608,heat-resistant hybrid F1）and YF1（NJCMS1A×YY6,heat-sensitive hybrid F1）during the flowering period of soybean,and found that the anther powder and pollen fertility of high temperature-sensitive hybrid F1 reduced with the temperature rise.Subsequently,transcriptional sequencing technology was used to analyze the mRNA expression levels of mixed flower buds of different sizes of NF1 and YF1 under normal te-mprature and high temperature stress,and a total of 8,784 differentially expressed genes（DEGs）were identified.Furthermore,GO functional annotation and KEGG pathway analysis were carried out on DEGs,and qRT-PCR was carried out on related DEGs.We speculated that:（1）High temperature stress would lead to the down-regu-lated expression of pectase genes such as PLs,PMEs,PGs,Exo-PGs,etc.,thus affect-ing the degradation of pectin,causing anther wall damage,leading to anther not to cr-ack;（2）High temperature stress affected the normal expression of carbohydrate meta-bolism-related genes such as beta-glucosidase 13,ATP-D6P7,UDP-GAD2,STP11,SWEET5 and SUC8 resulting in insufficient energy supply during pollen developme-nt;（3）High temperature stress interfered with auxin signal transduction,resulting in abnormal auxin metabolism,thus affecting normal anther cracking of high-temperatu-re sensitive F1 hybrids.In addition,transcription factors such as HSFA2 and heat sho-ck proteins such as HSP20 and HSP70 are also responsive to high temperature stress.2.GmHSFA2 was induced by high temperature in NF1 flower buds,but inhibited in YFi flower buds.Sequence analysis showed that GmHSFA2 has the classic structure of plant HSFA family,and has a high homology with tomato,arabidopsis thaliana and other HSFA2 genes.Functional study of GmHSFA2 found that,compared with the receptor control,35S::GmHSFA2 transgenic arabidopsis thaliana had more leaves,smaller leaves,upturned leaves,compact plant type,darker leaves,lower plant height and more branches.High temperature treatment in flowering period showed that leav-es and inflorescence of 35S::GmHSFA2 were less damaged by high temperature stre-ss,and showed stronger high temperature resistance than the receptor control.qRT-PCR analysis of potential downstream genes of GmHSFA2 in arabidopsis thaliana showed that the expression levels of AtsHSP,AtHSP20,AtHSP40,AtGolSl and AtGo-lS2 in 35S::GmHSFA2 were significantly up-regulated compared with the receptor control.These results indicate that GmHSFA2 may enhance the high temperature resi-stance of GmHSFA2 transgenic arabidopsis thaliana through the induction and regulation of HSP and GolS.3.GmHSP20 and GmHSP70 were induced by high temperature in NF1 flower buds,but inhibited by high temperature in YF1 flower buds.Functional study of GmHSP20 and GmHSP70 found that,GUS proteins were largely expressed in the seedlings and anthers of GmHSP20 Pro::GUS and GmHSP70 Pro::GUS.Reactive oxygen species staining showed that,DAB and NBT showed relatively shallow staining of GmHSP20 and GmHSP70 overexpressed transgenic Arabidopsis thaliana seedlings after high temperature stress,indicating that GmHSP20 and GmHSP70 genes had a scavenging effect on reactive oxygen species. |
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