Small RNA Sequencing Analysis Of Three-line Hybrid F₁ In Response To High Temperature Stress And Functional Study Of Related MiRNAs In Soybean

Soybean is the main cash crop in our country,in recent years,high temperature weather has occurred frequently,and the fertility of some soybean three-line hybrids F1 has been severely reduced after suffering high temperature stress.The male sterility caused by high temperature is mainly manifested as anther powder and microspore abortion.Prior to this,there was little research on the molecular mechanism of male sterility caused by high temperature.In this study,the small RNA sequencing method was used to analyze the three-line hybrid NF1（NJCMS1A×N4608,high temperature resistant hybrid F1）and YF1（NJCMS1A×YY6,high temperature sensitive hybrid F1）of the mixed flower buds before and after high temperature stess treatment in soybean,excavated 1326 differential miRNAs in response to high temperature stress,and conducted a more in-depth functional study of the differential miRNAs gma-miR156b and gma-miR397a to provide research directions for the regulatory mechanism of YF1 male sterility caused by high temperature.The main results were as follows:1.Small RNA sequencing was performed on mixed flower buds before and after high temperature stress treatment of NF1 and YF1,the results showed that the length of small RNA fragments was mainly concentrated on 21nt and 24nt,differential expression of miRNA analysis showed 1326 differential miRNAs response to high temperature stress,8 differential miRNAs were further selected for qRT-PCR verification,and the results of qRT-PCR were found to be basically consistent with the results of small RNA sequencing.Target gene prediction found that one miRNA can target multiple target genes;multiple miRNAs target one target gene,small RNA sequencing and transcriptome joint analysis results show that some miRNAs and their target genes have a negative regulatory relationship under high temperature stress.Combining the sequencing results and target gene prediction results,two differential miRNAs whose expression was down-regulated in response to high temperature stress were screened:gma-miR156b and gma-miR397a,which supported the database for further researches about miRNAs’ function in male sterility in response to high temperature stress.2.Based on the research of gma-miR156b overexpressing transgenic Arabidopsis in our laboratory,functional study showed that the length of the open flowers of gma-miR156b overexpressing transgenic Arabidopsis became smaller,and the anthers became smaller and the pollen number decreased,the seed setting rate decreased but the seeds increased,and the 100-kernel weight increased.Further research found that gma-miR156b participates in the growth and development of plants by inhibiting the expression of the downstream genes LBD and AGL of its target gene SPL.Small RNA and transcriptome joint analysis and qRT-PCR analysis results showed that gma-miR156b and its target gene GmSPL2b responded to high temperature stress and had a negative regulatory relationship.In order to verify that gma-miR156b is involved in the male reproductive development of plants in response to high temperature stress,this study conducted a high temperature stress treatment experiment on gma-miR156b overexpressing transgenic Arabidopsis and soybean at flowering stage.The study found that under high temperature stress,gma-miR156b overexpressed transgenic Arabidopsis thaliana and soybean both showed anther non-cracking and pollen abortion,while the control group had normal anther cracking.Among them,gma-miR156b overexpressed transgenic soybean showed complete abortion of pollen on the third day of high temperature stress treatment,while the control group showed complete abortion of pollen on the seventh day;further measurement and observation revealed that the water loss rate,DAB and NBT reactive oxygen species dyeing of leaves decreased after high temperature stress in gma-miR156b overexpressed transgenic soybean.The above results indicate that gma-miR156b may play an important role in responding to high temperature stress and regulating male sterility in soybean.3.Small RNA sequencing analysis showed that although high temperature stress suppressed the expression of gma-miR397a,its expression level in YF1 was higher than that in NF1.Functional study showed that the plants of gma-miR397a overexpressing transgenic soybeans were weak and reduced in plant height.After high temperature stress treatment,the results showed that the anthers of gma-miR397a overexpressing transgenic soybean and the control group normal crack on normally before treatment,the anthers of gma-miR397a overexpressing transgenic soybeans did not crack on the 5th day of treatment,while the anthers of the control group were normal,and the anthers of the control group also did not crack on the 7th day.Further measurement and observation revealed that the water loss rate,DAB and NBT reactive oxygen species dyeing of leaves decreased after high temperature stress in gma-miR397a overexpressed transgenic soybean.The above results indicate that gma-miR397a may play an important role in responding to high temperature stress and regulating male sterility in soybean.