Studies On Histological Localization, Gene Expression And Potential Utilization Of Dedifferentiation Of Mature Wheat Embryos

Wheat is one of the most important crops in the world.The utilization of biotechnology in its cultivar improvements,however,is rather retarded comparison with that in other crops.The bottleneck of the problem is the immaturation of wheat tissue culture technique.The immature embryos are current targets in wheat transformation.But acquirement of immature embryos is strictly limited by seasons and consistency of development stages is different to judge which not only increases the labour constraints but also hampers experimental flexibility of wheat genetic transformation heavily.Mature wheat embryos have a lot of advantages in their availability throughout the year and consistency of development stages.Using mature wheat embryos as explants is also not practice at present due to lower regeneration rate in vitro culture.Therefore it is of significance to improve ability of differentiation and regeneration in mature embryo culture and to reveal molecular mechanism at the level of gene expression.Some common wheat cultivars(Triticum aestivum L.cv)in Henan Province were used as experiment material to study the process of mature wheat embryos dedifferentiation from morphology,cell structure to gene expression level in present paper.The potential utilization of mature embryos was also investigated.The main results were as following:1.Where and when about dedifferentiation of mature embryos were confirmed according to the changes of histological structure in this process and four different stages were divided.The observation of micro- and trans-electronic scopy showed that dedifferentiation originally arose from the top of sheath inner cells of the main radicel.The process of dedifferentiation of this part of cells was finished about 24 h. The four stages were divided apparently according to changes of histological structure in dedifferentiation.The first(0～2 h)was the stage of signal transduction(SST)in which the perception and transduction the stimulation of auxin signal were occurred in cells.The second(2～12 h)was stage of signal response(SSR)in which the cells quickly responded the stimulation and a series of biological changes occurred in the cell wall,cell membrane and cytoplasm,so that the cell changed in morphology.The third(12～24 h)was stage of cell remodification(SCR)in which the cell structures were remodeled based on the changes occurred in SSR and prepared for cell division and formation of new cell. The fourth(about 24h)was stage of calli formation(SCF).The main missions of this stage were formation of calli.2.The changes of gene expression were studied by means of gene microarray technique and a number of essential genes in involved in some biological processes were confirmed.Because of the complexity of dedifferentiation in mature wheat embryos,the present paper investigated the changes of gene expression in the process(0,2,6, 12,24 and 72h)of dedifferentiation of mature wheat embryos by means of Affymetrix wheat GeneChip(?),which contain 61127 probe sets representing 55085 genes.The results showed that in dedifferentiation in wheat mature embryo there were 2413 genes expressed significantly,in which there were 1510 genes with known functions involved in different biological processes,such as:receptor protein (CA630632),calmodulin- binding protein(CA613597),and 14-3-3 protein (CD901339)in total 104 genes that related to signal transduction,ion transporting protein(BJ280396),aquaporin(BQ171992,CD904599)and W~+-transporting AYPase gene(CK208579 and AY543630.1)in total 107genes that related to biomembrane and transportation,extensin(AY543544.1,CK167447 and AY543542.1)in total 22 genes that related to cell wall synthesis and degeneration, transcription factor including AP2/EREBP family(44),b ZIP family(74),MADS family(9),MYB family(19)and WRKY family(7)in total 153 genes that related to gene expression,fimbrin-like protein gene(CK156089),myosin-like protein (BJ236428),microtubule-associated protein(CA663231)and actin (CK203076)in total that related to cell skeleton association and disassociation, 153 genes that related to stress response,363 genes that related to metabolism,DNA replication licensing factor(BQ806632)and DNA primase gene(CA595630) in total 46 genes that related to DNA replication and repair,translation initiation factor-like factor gene(BJ230659 and BQ167634),ribosome cycling factor gene (BJ235250)and translation elongation factor gene(CA637708)in total 231 genes that related to transcription and translation,ubiquitin extension protein gene (BJ266750),protein phosphatase(CD871466)and 26S proteasome regulatory particle gene(CA702474)in total 57 genes that related to protein degeneration, molecular chaperone such as HSP70(BJ217967,BJ320338,BJ272656 and BJ283294)in total 8 genes that related to protein assembling or folding folding, BQ166624,CA500212and BJ213871in total 55 genes that related to histone modification and chromatin remodeling,CA727622,CA726369 and BJ231319 in total 7 genes that related to cell division,and some unknown functional genes. Expression fold changes of these genes ranged 0-1500 times.There were also 633 genes with unknown functions or difficulty to sort them into relevant biological processes.3.The full-length of PM H~+-ATPase gene was cloned by RT-PCR and TAIL-PCR.The molecular structure of the gene and its protein was analyzed by means of bioinformatics methods.Because PM H~+-ATPase was an important enzyme in dedifferentiation,a full-length DNA sequence of PM H~+-ATPase gene was cloned by RT-PCR and Tail-PCR method for the first time according to Accession Number AY543630(PM H+-ATPase gene sequence)registered in GenBank.Sequencing result showed that it was of 6125 bp nucleotide with 15 exons and 14 introns.Bioinformatics analysis indicted that it encoded a protein of 951 amino acid residues.Structure prediction of the protein showed it contains 10 trans-membrane domains and three function domains,including Cation_ATPase domain in 5-86 at the N-terminus, E1-E2-ATPase domain in 98-319 and hydrolyase domain in 323-609.There were also 44 functional sites in different amino acid residues.The results of alignment of 9 deduced protein sequences from other plants indicated that PM H~+-ATPase in wheat is homologous to that in cotton(84.4%)and rice(71.1%).These suggested that PM H~+ ATPase from different plants are extremely conservative in primary structure.The complexity of structure determined the importance of PM H~+-ATPase function in wheat.4.Study on potential utilization of mature embryos in wheat tissue cultureResults in the present paper showed that when embryos were cultured under endosperm supported condition(endosperm supported culture,ES),the percentage of callus induction was significantly lower than that embryos were cultured in the absence of endosperm(non endosperm supported culture,NES),but ability of regeneration of plantlet was apparently improved in ES culture(up to 65.8%), showing potential utilization of mature embryos in wheat tissue culture.