Cloning,Localization And Expression Analysis Of TaEPFL1 Gene In The Pistillody Line Of Common Wheat HTS-1

The stamen is an important reproductive organ in plants,whose growth and development directly affects crop yield and quality.Male sterility in plant is a common phenomenon that the abnormal development of stamen can't produce fertile offspring.Male sterile lines play a slightly important role in hybrid breeding,the improvement of crop yield and quality,and the enhancement of plant resistance and adaptability and so forth.Wheat(Triticum aestivum L.)is a self-pollinating crop,in the process of cross-breeding,whose male sterile line is usually enabled to use as an excellent female parent that can produces hybrid progeny,which is superior to those of its parents in terms of viability,reproduction rate,stress resistance and yield.The process of cross-breeding is simple,which can eliminate the difficulty of artificial pollination and improve the seed purity of the progeny.Therefore,studying the development of wheat floral organs is substantially important for improving crop yield and quality by genetic technology.HTS-1 is a novel pistillody mutant in common wheat,first accidentally discovered by Professor Peng from near-isogenic line Chinese spring TP(CSTP)with Pis gene derived from TP mutant on Chinese spring(CS)background,which belongs to the variant plant of CSTP.HTS-1 mutant has some properties,including that less than 3 stamens and more than 3 pistils per floret,transforms all or part of the stamens into pistil,the abnormal stamens that cannot produce normal fertile pollen.Therefore,the stamen is completely or partially sterile.In addition,the HTS-1 mutant does not contain exogenous cytoplasm,and the pistillody trait is controlled by the interaction of two recessive karyogenes hts1 and hts2,which is completely different from that of male sterile wheat with early nucleo-cytoplasmic interaction.It is helpful to study the phenomenon of the nucleotide-controlled stamen homology transforming into pistil alone.Therefore,HTS-1 mutants are excellent materials for the study of stamen development.In order to further investigated the molecular mechanism of pistil-like traits present in the HTS-1 mutant,we compared the transcriptional profile of the Pistillody Stamen(PS)and pistil(P)of HTS-1 and the stamen(S)of CSTP previously.An unknown gene encoding cysteine-rich polypeptide(CRPs)was isolated from HTS-1 in our RNA-Seq analysis.In this study,we will further study the structure and function of the candidate gene,and identify whether it can regulate the development of floral organs during the development of wheat flower or not.The cDNA sequence and DNA sequence of the target gene were amplified by nucleic acid extraction and gene cloning and chromosome mapping,respectively.The sequence alignment,gene location,phylogenetic tree construction and other bioinformatics analysis were carried out.Subsequently,the expression pattern of the target gene in floral or gans of wheat was analyzed by qRT-PCR,of which the function was further analyzed by the overexpression in Arabidopsis thaliana.The main results are as follows:1.After cloning and sequencing,we found that the target gene contains three homologous genes,and the identity of these three homologous genes is as high as 98.18%,whose amino acid sequences are 100%,95.83% and 95.83% similar to AeEPFL1 of Aegilops tauschii,respectively.Therefore,these three homologous genes were tentatively designated TaEPFL1.1,TaEPFL1.2 and TaEPFL1.3(Gene Bank No.MH638998.1,MH638999.1,MH639000.1,respectively).The open reading frame(ORF)of three homologous genes is 363 bp in length,encoding 120 amino acids.Among these genes,the ORF of TaEPFL1.2 and TaEPFL1.3 are identical totally.Gene structure analysis shows that the DNA sequence length of these three homologous genes,TaEPFL1.1,TaEPFL1.2 and TaEPFL1.3,is 728 bp,713bp and 753 bp,respectively,they are all composed of two exons and one intron,and there is no difference in the length of their corresponding exons.2.Chromosome mapping analysis using wheat nulli-tetrasome(NT)as material showed that TaEPFL1.1,TaEPFL1.2 and TaEPFL1.3 are located on chromosome 6D,6B and 6A,respectively.3.Phylogenetic tree analysis showed that these two homologous sequences,TaEPFL1.1 and TaEPFL1.2,are clustered together with ObEPFL1,BdEPFL1 and AeEPFL1,which belonged to EPFL1 protein,and had the closest relationship with Aegilops tauschii.4.Real-time PCR results showed that the expression level of TaEPFL1 in pistillody stamen(PS)of HTS-1 was significantly higher than that in wheat pistil(P)and stamen(S).5.By comparing wild-type and transgenic in Arabidopsis thaliana,the results showed that the six filaments per flower of Col-0 were as long as or longer than the stigma,while the filaments of transgenic plants were significantly shorter than the stigma,and the ratio of filaments to stigma ranged from 0.35 to 0.79 at the exact-pollination stage.The number of filaments per flower in transgenic lines varied from 2 to 6,compared with Col-0 which consistently contained six filaments per flower.At the end of reproductive growth,most of the siliques of transgenic plants were short,empty,unable to produce seeds,and some had only residual pedicels without siliques.The expression level of three key speed-limiting enzymes genes in ethylene synthesis pathway was analyzed.The results showed that the overexpression of TaEPFL1 could reduce the expression level of AtACO2 gene.This study characterized and analyzed the structure and function of TaEPFL1 gene,which confirmed that TaEPFL1 gene could regulate the development of stamens in wheat by analyzing the expression of TaEPFL1 in transgenic Arabidopsis thaliana and might negatively regulate ethylene biosynthesis.These data provided a scientific basis for further elucidating the molecular mechanism of the homologous transformation of stamens into pistils in wheat.