Analysis Of Apomixis Characteristics And Functional Verification Of Key Genes In Zanthoxylum Bungeanum

Zanthoxylum bungeanum is an important economic species in the Rutaceae family.Its peel has a unique hemp aroma and is one of the eight major condiments in China.In addition,it is also a traditional Chinese medicine that has the effects of treating roundworms,diarrhea,dysentery,and inflammatory diseases.Studies have shown that Zanthoxylum bungeanum has the characteristics of apomixis,apomixis is a kind of asexual reproduction without fusion of male and female gametes.This reproductive method can directly produce offspring from the female parent and the genotype of the offspring is consistent with that of the female parent.Therefore,the offspring can inherit the excellent traits of the female parent and have great application potential in agricultural hybrid breeding and seed production.However,the regulation mechanism of apomixis is still unclear.This thesis uses Zanthoxylum bungeanum fruits at different developmental stages as materials,combined with Zanthoxylum bungeanum transcriptome and embryo cytology analysis,to screen the key regulatory genes of Zanthoxylum bungeanum apomixis and verify the functions of the key genes.The main findings are as follows:1.Zanthoxylum bungeanum has sexual reproductive obstacles,which are mainly manifested as low pollen germination rate and difficulty in extending the pollen tube into the embryo sac to combine with egg cells.The in vitro germination results of Zanthoxylum bungeanum pollen showed that the germination rate at 12 h was only 11%.In addition,pollen can germinate on the stigma,but only a few pollen tubes extend normally,and most of the pollen tubes are coiled in the stigma and do not extend to the ovary.Eventually,the pollen tubes decompose and cannot enter the ovary to complete fertilization.2.The differentially expressed genes of the pollinated and unpollinated groups are mainly enriched in the plant hormone signal transduction pathway.In addition,pollination cannot complete the fertilization effect but can significantly increase the abscisic acid(ABA)content and fruit setting rate.The transcriptome sequencing analysis results of Zanthoxylum bungeanum pollinated group and non-pollinated group showed that transcriptome sequencing produced a total of 69,131 sequences.Among them,there are13593 differentially expressed genes,including 7102 up-regulated genes and 6491down-regulated genes.KEGG enrichment analysis of differentially expressed genes showed that 144 genes were enriched in the hormone signal transduction pathway(ko04075).Pollination cannot make Zanthoxylum bungeanum complete the sexual reproduction process,but it significantly increases the concentration of abscisic acid(ABA)and fruit setting rate.In addition,spraying 100 ?g/L ABA can also improve the fruit set rate.Therefore,it is inferred that pollination cannot promote the completion of fertilization but can significantly increase the concentration of abscisic acid(ABA)and promote fruit setting.It is recommended to plant the Zanthoxylum bungeanum male plants in the plantation or spray an appropriate concentration of ABA during the flowering period to increase the fruit setting rate.3.The type of Zanthoxylum bungeanum apomixis is sporophyte apomixis.The embryo sac development of Zanthoxylum bungeanum was observed by paraffin section and safranine fast green staining,which proved that Zanthoxylum bungeanum had the characteristics of apomixis,and the reproductive type was sporophyte apomixis.In order to further determine the origin of embryos,we carried out cell ploidy tests on different tissues of the progeny and parents of Zanthoxylum bungeanum.The results of flow cytometry showed that the ploidy of endosperm and somatic cells were both 2n,indicating that Zanthoxylum bungeanum endosperm(2n)could form spontaneously independently of fertilization,and no 3n endosperm produced by double fertilization was detected.Using 12 pairs of polymorphic SSR(Simple Sequence Repeat)markers,3 female parents and 45 offspring were detected by capillary electrophoresis.The results showed that the fluorescent signals of the polymorphic SSR primer amplified products of the female parent material and the offspring material were completely consistent,indicating that no trait separation occurred during the reproduction of Zanthoxylum bungeanum Maxim.This result can be used as a molecular evidence supporting the characteristics of apomixis in Zanthoxylum bungeanum.4.ZbAGL11 is a key gene for apomixis in Zanthoxylum bungeanum,which can lead to the apomixis phenotype of transgenic Arabidopsis.Through bioinformatics analysis and gene-specific expression analysis,we screened a candidate gene that regulates apomixis,a member of the MADS-Box transcription factor family,ZbAGL11.ZbAGL11 is almost not expressed in roots and stems,and the relative expression level in leaves is very low,but it has a high expression level in the critical period of apomictic nucellar embryogenesis.In order to further study the relationship between ZbAGL11 and apomixis,the researchers verified the transgenic function of Zanthoxylum bungeanum and Arabidopsis thaliana.The results showed that ZbAGL11 can cause abnormal development of floral organs in Arabidopsis thaliana,and the unpollinated ZbAGL11-OE Arabidopsis produces fertile offspring with apomictic phenotype.In addition,the study also found that the overexpression of ZbAGL11 increased the induction rate of callus cultured in vitro of Zanthoxylum bungeanum.5.ZbAGL11 may participate in the regulation of apomixis by interacting with the peptide-prolyl cis-trans isomerase CYP65(Peptidyl-prolyl cis-trans isomerase CYP65,Zb CYP65)and the accelerated cell death 11 gene(Zb ACD11).The results of yeast two-hybrid test showed that ZbAGL11 can interact with Zb CYP65 and Zb CAD11 proteins.Through the STRING database and Cytoscape software,the visualization of the MADS-box transcription factor interaction network was realized.The results show that MADS-box transcription factors have a close interaction with each other,and they may regulate the development of floral organs in the form of complexes.In addition,the members of MADS-box transcription factor are also closely related to FT,CO,GI,PHYB,etc.In summary,the apomixis of Zanthoxylum bungeanum is sporophyte apomixis,and ZbAGL11 plays an important role in the apomixis of Zanthoxylum bungeanum.ZbAGL11 is a key gene that regulates the formation of apomictic nacelle embryos.Overexpression of ZbAGL11 in Arabidopsis can cause floral organ development disorders and produce apomictic phenotypes.