| Tall fescue(Festuca arundinace Schreb.)is a perennial tufted grass of the genus Festuca of Gramineae,which has the advantages of long green period and high nutritional value,so it’s an excellent cold-season turfgrass and forage grass.However,it can not be wildly planted and popularized because of its few tiller number and the difficulty of living through the summer in the south of the Yangtze River.Using new methods to release the full potential of tall fescue genome and improve crop varieties is the focus and difficulty of tall fescue breeding at present.miRNA plays a key role in plant regulation-related gene expression in response to stress.However,the role of tall fescue miRNA in abiotic stress is not clear.In this paper,a new tall fescue material STTM159/160 was created by double mutation of miRNA159/160 in tall fescue by STTM technique,and its high temperature resistance mechanism was preliminarily explored.The main studies are as follows:(1)Silencing miRNA159/160 by STTM technique dwarfed tall fescue plants and increased the number of bugs.Through the comparison between 3-month-old wild-type plants and STTM159/160 plants,it was found that the leaf width and leaf length of STTM159/160 plants were about 3 times smaller than those of WT.Through scanning electron microscope observation,it was found that the leaves of STTM159/160 plants had more hairs,which increased the roughness of the leaves.(2)Silencing miRNA159/160 by STTM technique increased the heat tolerance of tall fescue.WT and STTM159/160 plants were treated with high temperature for 4 days(42℃/ 37℃)and then transferred to the optimum growth temperature(24℃/ 22℃)for 7 days.The wilting degree of STTM159/160 plants was higher than that of WT.Through the determination of relevant physiological indexes,it was found that STTM159/160 transgenic plants could alleviate the damage of plant cell membrane caused by high temperature stress by reducing electrical conductivity and improving photosynthetic efficiency.(3)In order to further analyze the gene changes of STTM159/160 transgenic plants,wild type plants and STTM159/160 plants before and after high temperature treatment(40℃,6 h)were selected for transcriptome sequencing.Through the analysis of transcriptome data,the phenotypic changes of growth and development of STTM159/160 and the regulation mechanism of heat tolerance improvement were further explained.The candidate target genes were found by transcriptome data,and the expression of candidate target genes was analyzed.In the results of T0159-vs-T0k31 comparison group,four Fa ARF candidate target genes were found:ARF11(Gene ID:Unigene9781＿All),ARF15(Gene ID:Unigene8326＿All),ARF16(Gene ID:Unigene36706＿All),ARF21(Gene ID:Unigene16704＿All).There are 5Fa MYB candidate target genes MYB2(Gene ID:CL12285.Contig1＿All),MYB36(Gene ID:CL4587.Contig7＿All),MYB44(Gene ID:CL966.Contig4＿All),MYB64(Gene ID:CL8710.Contig4＿All)and MYB96(Gene ID:CL9271.Contig7＿All).The expression level of these candidate target genes increased in STTM159/160 transgenic plants,indicating that the use of STTM technology can successfully silence miRNA159/160 to increase the expression level of possible candidate target genes.In this study,two miRNA mutations were simultaneously carried out in tall fescue by STTM technique for the first time,which changed its agronomic characters,improved the heat tolerance of tall fescue,and created a new material with multi-tillering,dwarfing and stress resistance. |
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