| MicroRNAs(miRNAs)are a kind of endogenous single-stranded small molecule RNA with 20~24 nucleotides,mainly exist in eukaryotes.They could regulate gene expression through post-transcriptional level,widely involved in plant growth and development.Previous studies have shown that some specific miRNA participate in the process of anther development in plants,which is one of the important reasons for anther development abnormality and male sterility.At present,the research reports on miRNA in plant anther development are mainly on rice,maize,cotton,rape and other crops,while in wheat research has been few.So it is important to study the role of specific miRNA in wheat anther development for male sterility and heterosis utilization in wheat(Triticum aestivum L.).In our previous research,we used anthers of wheat photo-thermo-sensitive male sterile line 337S under short day low temperature sterility and normal fertility condition as materials identified some differentially expressed miRNAs by RNA sequencing analysis.In this study,we choosed miR1127 b,miR9652,miR2275 as candidate genes to construct the over-expression vector for genetic transformation in different wheat callus.Since no target genes of miR1127 b and miR9652 were identified in degradome sequencing,the target gene CAF1 was only found for miR2275,so we further cloned the CAF1 gene and carried out genetic transformation of miR2275 gene in order to understand the role of miR2275 and the target gene CAF1 in wheat reproductive development.The main results in this research were as follows:1.Genetic transformation of miR2275,miR9652,miR1127 b gene: The over-expression vector of miR2275,miR9652,miR1127 b were constructed by means of Gateway technology and transformed into different wheat varieties by Agrobacterium tumefaciens and gene gun method.According to preliminary PCR detection in transgenic plant,a total of 18 positive seedlings were obtained from miR2275 gene transformation in the genetic transformation experiment of wheat immature embryos mediated by gene gun,10 of them from Huamai 2566,7 plants from Bobwhite,and 1 from 337S.The positive rate was 1.94%,1.51% and 0.23%,respectively.In the transformation ofimmature embryos mediated by Agrobacterium tumefaciens,only one positive seedling was obtained from Huamai 2566 in miR1127 b gene transformation,with a positive rate of 0.19%;the transformation of miR9652 gene also had one positive seedling in Bobwhite,the positive rate was 0.19%;the transformation of miR2275 got three positive seedlings from Huamai 2566,and the positive rate was 0.99%.2.Homologous cloning of miR2275 target gene CAF1: In the previous study,the anthers of 337S were sequenced and the target gene CAF1 of miR2275 was found.Six homologous genes of CAF1 were cloned by homologous cloning method and named CAF1-3A-1,CAF1-3A-2,CAF1-3B-1,CAF1-3B-2,CAF1-3D-1,CAF1-3D-2,respectively.Bioinformatics analysis showed that all the genes contained highly conserved domains of CAF1 family except CAF1-3B-2.There was a high homology between CAF1-3A-1 and CAF1-3A-2 genes,and the deletion of 34 bp in CAF1-3B-2 resulted in the termination codon,which caused the early termination of translation.Only CAF1-3D-1 protein had a transmembrane region.Besides,CAF1-3B-1 and CAF1-3D-2 were hydrophobic proteins,and the rest were hydrophilic proteins.3.Subcellular localization and expression of the target gene CAF1: The subcellular localization vectors of GFP fused with the N and C terminal of CAF1-3A-1,CAF1-3B-1,CAF1-3D-1,CAF1-3D-2 were constructed respectively,and transiently expressed in Benn’s tobacco.The results showed that only the GFP fused with the N-terminal of CAF1 all showed fluorescence in nucleus,cell membrane or cell wall of tobacco,it was proved that the 4 proteins were expressed in the cell nucleus,cell membrane or cell wall.Besides,the prokaryotic expression vector of CAF1-3A-1,CAF1-3D-1,CAF1-3D-2 were constructed and the suitable expression conditions were determined in Escherichia coli strain BL21(DE3).The expression of these three proteins determined to be 18℃,0.5mM IPTG induced for 12 h.4.CAF1 promoter cloning: Promoters of six CAF1 genes were cloned,and GUS fusion protein expression vector driven by promoters were constructed and transformed into wild type Arabidopsis thaliana by inflorescence infection mediated by Agrobacterium tumefaciens and obtained corresponding transgenic plants.5.The phenotypic observation of transgenic positive seedling in wheat: Compared with wild type Huamai 2566,Huamai 2152,Bobwhite and 337S,the positive seedlings of transgenic miR2275 were weak.One of the positive plant from Bobwhite was short and not fruited,another positive seedling from the 337S showed a large glume opening angle from the middle of the ear to the top,with the pistil exposed and only a fewer seeds at the base of ear than the wild-type plant.This further showed that miR2275 is one of the important factors determining the sterility of 337S. |
