Preliminary Study Of The Function Of CENH3 In The Chromosome Elimination Of Wheat-Maize Hybrid

The maize chromosomes in the wheat-maize hybrid embryos are eliminated gradually and form micronucleis due to their centromeres fail to attach to the spindle fibers and hence cannot move to two polar poles during anaphase.Chromosome elimination in wheat-maize hybrid has been widely used in the production of double haploid of wheat.However,the mechanism of this phenomenon is still poorly understood.Several hypotheses have been supposed to explain the chromosome elimination in cross combinations between different plant species,one of which is uniparental centromere inactivity.There have been several research groups reported the important roles of CENH3,which is the fundamental characteristic of active centromere,in chromosome elimination.Sanei et al.(2011)found that missegregated and eliminated chromosomes in the cross between Hordeum vulgare and H.bulbosum were due to centromere inactivity resulted from loss of centromeric CENH3 protein,providing direct evidence for the association of CENH3 with chromosome elimination.To test whether parent-specific CENH3 is involved in the mitosis-dependent process of chromosome elimination of wheat-maize hybrid,in the present research,the maize(Zea mays L.)CENH3 gene was transformed into wheat.Our purpose is to study whether the transformed ZmCENH3 can be expressed and translated in wheat background,and the ZmCENH3 can be allocated to the centromeric region.We also used the transgenic wheat to produce wheat-maize hybrids to study the possibility of suppressing the maize chromosome elimination by overexpressing Zm CENH3.1.Transient expression of Zm CENH3-YFP in onion epidermal cellsThe vectors pCAMBLA3301-ZmCENH3-YFP and pCAMBLA3301-YFP was particle bombarded into the epidermal cells of onion by GeneGun.The result showed that,compared to ubiquitious distribution of YFP,the fusion ZmCENH3-YFP could be expressed and the ZmCENH3-YFP signals were only observed in the nucleus,indicating the correct nucleus localization of ZmCENH3 when transciently overexpressed in the epidermal cells of onion.2.The transformation of ZmCENH3 into wheat variety Yangmai 158 and characterization of the ZmCENH3 transgenic wheatThe expression vector pCAMBLA3301-ZmCENH3-YFP was transformed into immature embryos of wheat variety Yangmai158 by particle bombardment.A total of 426 independent T0 transgenic plants were obtained.PCR analysis using the primer pairs for the identification of both the ZmCENH3 and Bar genes showed that the specific bands for both genes could be amplified in five plants.pCAMBLA3301-ZmCENH3-YFP carries the GUS gene.Root tips of plants derived from the five lines were further GUS-stained and the successful blue staining of these lines further verified they were positive transgenic.RT-PCR was employed and it was found that,compared with that in the wheat receptor variety Yangmai 158,in the five positive transgenic lines there present an additional 405 bp specific amplicon,which was the same as the ZmCENH3 sequence by sequencing,indicating the transformed ZmCENH3 can be transcribed in the wheat background,however their expression levels were very low in all these five lines.qRT-PCR was further used to investigate the influence of alien ZmCENH3 introduction on the expression of endogenous CENH3 in these transgenic lines.The result showed that,contrasting to the low expression ZmCENH3 driven by constitutative CaMV35s promoter,the endogenous wheat CENH3 expression in these transgenic lines significantly up-regulated.We speculated that the transformation of alien ZmCENH3 into wheat could induce the competition expression of endogenous wheat CENH3 to supress the over-expression of alien Zm CENH3.3.Maize chromosome elimination was not supressed in the ZmCENH3 transgenic plants × maize hybrid embryosPrevious studies showed that most maize chromosomes were eliminated in most embryo cells one week after pollination of the maize pollen to wheat.In this study,408 maize SSRs distributed on different maize chromosomes were used for amplification using the hybrid embryos of two crosses:(untransformed Yangmai 158 x Maize)and(ZmCENH3 transgenic wheat x Maize).The numbers of specific amplicons from maize genome were compared to investigate whether the transformed ZmCENH3 can suppress the maize chromatin elimination.The result showed that there was no significant difference in 7-8 d hybrid embryos between the two crosses.GISH(Genomic in situ hybridization)using maize genomic DNA as probe found that,4-5 days after pollination,one or more micronucleis formed by maize chromatin were observed.This demonstrated the presence of maize chromatin elimination in(ZmCENH3 transgenic wheat × Maize)hybrid embryos.FISH(florescent in hybridization)using the 45 SrDNA as probe on the 9-10d hybrid embryos of(ZmCENH3 transgenic wheat x Maize)and(untransformed Yangmail58 x Maize)were conducted and the cell numbers with two(from wheat)or three(two from wheat and one from maize)45 SrDNA signals were compared.The results showed that there was no significant difference for the percentage of cells with two or three 45SrDNA signals between the two cross combinations.The results from the above three experiments all suggested that the transformation of ZmCENH3 into wheat could not suppress the maize chromatin elimination,at least in our study.4.ZmCENH3-YFP was not detected in the transgenic lines by Western blot and immunofluorescence assayEven though the transformed ZmCENH3-YFP could be transcribed in the transgenic wheat,we failed to observe the suppression of maize chromatin elimination in the(ZmCENH3transgenic wheat × Maize)hybrid embryos.The translation and allocation of ZmCENH3 protein were further investigated in the transgenic wheat.The YFP signals were used as the marker for the detection of the presence of the ZmCENH3-YFP fiusion pretein.The result showed that in the root tips of transgenic Yangmai 158 of ZmCENH3,no YFP signal could be observed.The transgenic wheat were further analyzed by Western blot using anti-YFP and ZmCENH3 antibody as probes and immunofluorescence using anti-YFP antibody.Both experiments failed to detect the presence of either ZmCENH3 or YFP in the transgenic Yangnail 58.These indicated the transformed ZmCEN3 either failed to translate into stable ZmCENH3 protein or the translated ZmCENH3 could not exist stably in the wheat background.The absence of stable ZmCENH3 in the transgenic wheat may be one of the most important reasons for the failure in suppression maize chromatin elimination of the(ZmCENH3 transgenic wheat × Maize)hybrid.5.The transformation of recombined wheat-maize CENH3 into wheat variety Yangmai 158The constitution of CENH3 has two parts,the histone fold domain(HFD)and the N-terminal domain.The N-terminal domain is quite diversed,even among species with close genetic relationship.However,the HFD is conserved,both for its sequence and function.To investigate whether the substitution of the maize HFD or the N-terminal domain by those from wheat CENH3 can affect the stabilization and function of the maize CENH3 in wheat background,two recombined wheat-maize CENH3 vectors were constructed and transformed into wheat variety Yangmai 158 by particle bombardment.A total of 196 independent T0 transgenic plants for the vector pZY101-C-ZmCENH3/N-wheat CENH3-GFP were obtained.Five plants were identified to be positive transgenic by PCR using primer pair CAMV35s-F/ZmCENH3-R and RT-PCR analysis using two primer pairs,WQMH-F1/ZmCENH3-R2 and WQMH-F1/ZmCENH3-R.They all amplified a specific amplicon for the recombined CENH3 gene,indicating C-ZmCENH3/N-wheat CENH3-GFP could be transcribed.A total of 52 independent T0 transgenic plants for the vector pCAMBLA3301-C-wheat CENH3/N-ZmCENH3-GFP were obtained.Nine plants were identified to be positive transgenic by PCR with primer pair Pinjie-F1/Pinjie-R2.The vector pCAMBLA3301-C-wheat CENH3/N-ZmCENH3-GFP carries the GUS gene.GUS staining showed that the root tips of five lines could be stained blue.RT-PCR showed that six of the nine transgenic plants amplified the specific amplicons,the sequences of which were identical with the C-wheat CENH3/N-ZmCENH3,indicating the transgene could be transcribed.Line CENH33-4 was identified to be positive,but could not be GUS stained,further sequencing of the amplicon showed that it was a negative transgenic line.Both recombined CENH3 were fused with GFP in the two vectors,GFP detection in the root tips of the transgenic lines can be used to trace their translation and loading.The result showed no GFP signal was observed in root tips of the C-ZmCENH3/N-wheat CENH3 transgenic plants.In the C-wheat CENH3/N-ZmCENH3 transgenic lines,GFP signals were only observed in some cells of line CENH33-8.This result indicated that the recombined CENH3 could not be translated or stabilized in wheat background.6.The transformation of wheat CENH3 into wheat variety Yangmai 158To study whether of the transformation of wheat CENH3 can be translated and stabilized in wheat,the expression vector pZY101-wheat CENH3-GFP was constructed and transformed into immature embryos of wheat variety Yangmai158.A total of 189 independent T0 transgenic plants were obtained.Western blot using anti-GFP antibody as probe showed that,compared with that in Yangmai 158,a specific band was observed in eight transgenic lines,indicating the transformed wheat CENH3 could be translated and was stably present in these transgenic lines.