| Chrysanthemum(Chrysanthemum morifolium)originates from China,with rich varieties,varied colors,and various forms,which have high ornamental and economic value.In production,distant hybridization is often used to improve the quality of cultivated chrysanthemum by transferring excellent genes from wild species.However,embryonic abortion often occurs during this process.In recent years,researchers have studied the phenomenon of aborted embryos by cytology and morphology and high-throughput sequencing.Based on the previous studies,in this paper,we analyzes the regulation of miRNA in embryo abortion.CmERF12 that related to embryonic development were cloned and plant expression vectors were constructed.Expression characteristics,subcellular localization,and transcriptional activation activity have been studied.The system of efficient regeneration and genetic transformation of ’Yuhuanying’ was established,and the transgenic chrysanthemum was obtained,which provided the basis for the subsequent functional identification of the gene.Through the transformation of Arabidopsis thaliana,the molecular mechanism of embryo abortion was initially revealed.The main results are as follows:1.In order to understand the roles of unigenes,miRNAs and their target genes play in chrysanthemum embryonic abortion,distant hybridization between C.morifolium’Yuhualuoying’ and diploid C.nankingenese was carried out,the normal and abnormal embroyos at 12d and 18d after pollination were obtained and three libraries were performed by RNA-Seq.As a result,among the 12 genes that were verified quantitatively,9 genes had the highest expression levels in NE18,such as Dof,WRI1,2S,OLE,etc,suggesting that they may play a role in the maturation stage of embryonic development.Transcription factor NAP had the lowest expression in NE18 and the highest expression in AE18;The expression of ERF 12 in aborted embryos(AE18)was significantly higher than that of normal embryos,which is similar to the result by RNA-Seq,suggesting that the overexpression of ERF 12 may lead to embryonic abortion.Additionally,a total of 170 miRNAs were identified.miR169b,miR440 and miR528-5p.miR164c and miR159a were highly expressed in normal embryo at 18 days after pollination,suggesting the regulatory role at the late stage of embryonic development.miR172c was only detected in normal embryo at 18 days after pollination,which means that miR172c mainly mediate genes expression in postembryonic development and these genes may promote embryo maturation.Other miRNAs,including miR414,miR2661 and miR5021,may regulate the genes participated in pathways of auxin response and energy metabolism,then they regulate the complex embryonic development together.2.To screen the key genes in embryonic abortion,the transcriptome data was analyzed.As a result,CmERF12 was screened from the libraries.Specific primers were designed and the gene was cloned by use of the ordinary PCR method.As a result,the ORF of CmERF12 has a full length of 336 bp and encodes 112 amino acids,and the encoded CmERF12 protein has the highest similarity to ERF 12 protein in Arabidopsis thaliana.The results of subcellular localization showed that CmERF12 were localized to the nucleus in vivo.Recombinant vector pDEST-GBKT7-CmERF12 was constructed and transformed into the yeast strains Y2H.The results showed that CmERF12 have no obvious transcriptional activation ability.3.In early studies,researchers discovered that chrysanthemums were susceptible to infection by A.tumefaciens.Therefore,Agrobacterium-medidted transformation has become one of the most common method for transforming chrysanthemums.In this study,leaf disk method was used to get the transformants.The results showed that different ages of tissue culture seedlings and hormone ratios had significant effects on the regeneration of;Yuhuluoying’.The regeneration rate of leaf disks from 30-day-age seelings was significantly higher of than that of 25 days and 40 days.When the plant material was too old,the regeneration frequency was less than 65%;and the leaves with a seedling age of 25 days were cultured in MS+6-BA 1.0 mg/L+NAA 1.0 mg/L,although the regeneration rate reached 80%,which was higher than that of the 30-day tissue culture seedlings,there were vitrification which is not suitable to be used as a regeneration condition.When it comes to hormone ratios,it was found that when the concentration of 6-BA was consistent and the concentration of NAA increased,the regeneration rate of buds increased.When the concentration of 6-BA was increased to 3.0 mg/L,the leaf disc regeneration rate began to decrease,the regeneration buds showed vitrification in different degrees as well.Comprehensively,the best regeneration system contains the medium MS+6-BA 1.5 mg/L+NAA 0.8 mg/L,with the seedling age was 30 days.the highest leaf regeneration rate was up to 91.11%,while the average number of buds is as high as 5.90.4.On the basis of the best regeneration system,we screened the concentration of hygromycin and carbenicillin,explored the influence of seveal conditions such as preculture and delayed culture on transformation efficiency.What’s more,the vitrifications were restored to normal seedlings by adjusting the culture regulation.As a rusult,the Agrobacterium-medidted genetic transformation system of ’Yuhualuoying’ was initially established and genetic chrysanthemum lines were obtained.The results showed that the leaf disk was sensitive to hygromycin and the optimal selection pressure was 8-10 mg/L,and the optimal concentration for rooting screening was 11 mg/L;Carbenicillin is a broad-spectrum antibiotic which has a strong inhibitory effect on Agrobacterium.In this experiment,the optimum concentration of carbenicillin in the delayed culture medium was 400 mg/L,because in this condition,the callus was healthy and the Agrobacterium was completely inhibited;The amount of both hygromycin and carbenicillin was gradually decreased in order to reduce the negative impact on leaf disks.The optimal genetic transformation for ’Yuhuanying’ was supposed to be:pre-cultured for 2 days,infection for 7 minutes,co-cultured for 3 days,postponed antibiotics selecting for 5 days.What’s more,studies have shown that 6-KT can effectively increase the regeneration rate of resistant shoots.Finally,we obtained transgenic resistant lines successfully.Among the 15 resistant lines,there were 10 positive seedlings.The relative expression level of CmERF12 was identified,and found that the use of this system can successfully obtain the CmERF12 transformation.The construction of an efficient regeneration and genetic transformation system can make a good foundation for the verification of gene function and revealing the mechanism of embryonic abortion.5.In order to verify the function of CmERF12 in embryonic development,we constructed the plant expression vector pMDC32-CmERF12,and obtained the transgenic positive Arabidopsis thaliana through seveal screening.The process of embryo abortion was obaerved and the abortion rate was analyzed.What’s more,we used qRT-PCR to verify the expression level of genes related to embryonic development.The results showed that three overexpressed lines were obtained by Agrobacterium-mediated inflorescence transformation,named as ox-1,ox-2 and ox-4 respectively.There were no significant changes in the floral structure of the three transgenic lines,while the pistil developed normally,and the longer four stamens were the same tall as the pistil so that the pollen could adhere to the stigma regularly.The early embryonic development of Arabidopsis is normal,eliminating the possibility of pre-fertilization disorders.On the other hand,at different growth stages,the transgenic lines showed obvious abortion.In the early stage of embryo development,the ovules of wild-type and transgenic lines were normal and full,with no obvious abortion phenomenon.The abortion phenomenon mainly occurred in the third period(about 10 d after pollination).The development of embryos in the transgenic plants was obviously abnormal and the surface of ovules was shrinking,deep browning.In the fourth period,which was the seed maturation period,the wild-type seeds were substantial and healthy,however,seeds from transgenic lines were shriveled and died abnormally.Among the three transgenic lines,the abortion phenomenon of ox-1 and ox-2 was obvious.The atrophic pods were about 46.7%and 40.0%,while the ox-4 was about 16.7%.The results showed that overexpression of CmERF12 in Arabidopsis affected the normal development of Arabidopsis embryos.These results provide the basic regulation functions of the of CmERF12,which plays a role in embryo late development and maturation through the expression inhibition of transcription factors in mature stages and synthesis inhibition of protein,oil and other storage substances.In addition,it may also promote the expression of embryonic abortion related genes.These results may lay a good foundation for the study of the molecular mechanism of chrysanthemum embryonic abortion and development. |
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