| Papaya is a diploid dicotyledonous herb with 18 chromosomes and its draft genome has been sequenced.Papaya is one of the model plant species for sex chromosome reseach,because of its short life cycle,trioecy and the ability of producing large number of seeds.There are three sex forms in papaya,the female(XX),male(XY)and hermaphrodite(XYh).The preference sex type for papaya production is hermaphrodite.But the seeds of the hermaphrodite plants are difficult to obtain sustainably.Due to YhYh lethality,commercial papaya seeds segregate hermaphrodite and female,that are made from crossing hermaphrodite with female.Therefore,we need to adopt method of overplanting in the early stage and then thinning when flowered.In addition,the large number of seeds in papaya fruits makes it difficult to process papaya fruit.These two problems have a profound impact on cultivation and processing of papaya.This study analyzed papaya embryonic development,based on analysis of the function about the embryonic development candidate gene Cp49,and selection of papaya embryonic development mutants,in order to achieve true breeding hermaphrodite and create a new variety of seedless papaya.Based on previous studies,we have identified some important genes related to embryo development in the sex determination region.This project is to study the expression and regulation of these genes.The results are:1.Somatic embryogenesis and transcriptome analysis of papaya.Based on morphology and histology observation of papaya somatic embryo and the characteristics of different developmental stages,the papaya somatic embryogenesis is divided into four stages:globular embryo,heart embryo,torpedo embryo and cotyledon embryo.The differentially expressed genes in different developmental stages of papaya female somatic embryo and hermaphrodite somatic embryo were analyzed by transcriptome analysis.Two type of analysis methods are used:(1)To analyze the differentially expressed genes in somatic embryo of different sex forms at the same developmental period.The result shows that in the early stages of embryonic development,the female and the hermaphrodite somatic embryo apparent differences in gene expression.In the stage of globular embryo,the differentially expressed genes are mainly concentrated in the procession of cell wall formation.In the formation of heart embryo stage,the expression difference is related in matter and energy metabolism pathway.The main difference of the torpedo embryo stage is the genes involved in the assembly process of nucleosomes and chromatin.The differentiation genes of cotyledon embryo are mainly involved in the matter formation,metabolic processes and the formation of meristem.(2)Analyzing the gene expression trends in somatic embryo of the same sex forms.According to the results,the torpedo embryo and the heart embryo stage are the key stages in somatic embryogenesis.There are 89%and 83%genes expression changed in these two stages.2.TILLING was used to screen for mutants related to the embryonic development.Preliminary study shows that the genes in the sex determination region could affect the embryonic development.Papaya seeds were treated with a chemical mutagenesis agent ethyl methane sulfonate,M1 gene mutant population was created and high-throughput sequencing was screened for the mutants.The mutants were vertified by PCR.Three positive mutant plants were discovered,including cpxyh3,emb506 and pxcpxyh14,wiht missense mutation.3.The expression patterns of Cp49 gene.The gene expression result showed that in the development of somatic embryogenesis,the Cp49 gene was expressed in globular embryo,heart embryo,torpedo embryo and cotyledon embryo stages with dismishing gene expression.The globular embryo stage is the highest expression stage of Cp49 gene expression,the cotyledon embryo period exhibited the lowest expression compared with the other three.4.emb976 mutants,the Cp49 homologous gene in Arabidopsis.The gene evolutionary tree of Cp49 was constructed in order to find the closest homologous gene.We found the EMB976 is the homologous genes of the Cp49.The experimental result shows that the T-DNA insertion of emb976 in Arabidopsis showed partial embryo abortion during the cotyledon embryo stage in heterozygous mutants,and the abortion rate was about 25%.To figure out whether Cp49 and EMB976 have the conserved function,we build the expression construct with EMB976 promoter and Cp49 CDS,using floral dip method.It was transformaed into Arabidopsis mutants EMB976.We found that Cp49 gene expression can partly resuce the abortion phenotype of emb976,which shows conserved function in Cp49 and EMB976.5.Transgenic study of papaya.In order to futher explore the function of Cp49 gene,we build the papaya Cp49-X-CRISPR/Cas9,Cp49-XYh-CRISPR/Cas9 gene editing construct and Cp49-XYh-RNAi vector.They were transformed into the papaya calli by agrobacterium-mediated transformation.At present,we have obtained Cp49-XYh-CRISPR/Cas9 positive hygromycin resistant plants and Cp49-XYh-RNAi positive hygromycin resistance calli,these results laid a foundation for further study of the Cp49 gene function. |
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