Mechanism Of ClBBM And ClPLT2 Regulating Gametophyte Development In Citrullus Lanatus L. And Parthenogenesis Induced By BBM

Watermelon,(Citrullus lanatus(Thunb.)Matsum.& Nakai),which is an important cash crop of Cucurbitaceae in the world.Cross-breeding is the main method for watermelon breeding,and the production of high-quality seeds plays a key role in promoting the development of watermelon industry.The production of seed is highly dependent on sexual reproduction.The formation of gamete,which is acts as a prerequisite for sexual reproduction,is precisely regulated by complex genetic networks.So,the exploration of gametophyte development was helpful to analyze the reproduction process of crops,and promote the production of high-quality seeds to improve economic benefits.Up to now,studies on watermelon gametophyte development are insufficient,and there is still much to learn about the underlying regulation mechanisms.Gene BBM has been reported to regulate reproductive development and induce parthenogenesis in many plants.At BBM and At PLT2 were functionally redundant in regulating early embryo and endosperm development in Arabidopsis.And the ectopic expression of BBM in egg cells of monocotyledon plants such as Pennisetum squamulatum,maize and rice were able to induce parthenogenesis,which were significant for double haploid breeding and hybrid vigor fixation.In this work,we verified that Cl BBM and Cl PLT2 regulated the development of male and female gametophyte by expression pattern analysis,phenotype analysis of mutants and RNA-seq.On the other hand,we found that the ectopic expression of Cl BBM and At BBM in watermelon and Arabidopsis egg cells were inhibited.To overcome the inhibition,we proposed a variety of strategies and finally realized the ectopic expression of BBM in egg cell to obtained parthenogenetic haploid offspring.This work laid a foundation for the study of watermelon gametophyte development and its regulation mechanism,and provided a new theoretical basis for the establishment of parthenogenesis induction system in watermelon and more dicotyledonous plants.The main results are as follows:1.Cl BBM and Cl PLT2 were expressed during the gametophyte development of watermelon.Watermelon Cl BBM and Cl PLT2 encoded nuclear localization proteins,which belonged to the AIL branch of the AP2/ERF transcription factor family.During male gametophyte development,Cl BBM and Cl PLT2 were expressed in microspore and tapetal cells at stage 9(microspores were released from tetrads).And in female gametophyte development,Cl BBM and Cl PLT2 were expressed in tetranuclear embryo sacs at stage FG4.2.Cl BBM and Cl PLT2 were functionally redundant in regulating male and female gametophyte development in watermelon.The single mutant of Cl BBM and Cl PLT2 had no defective phenotype,and the double mutant caused a large amount of pollen abortion and significantly reduced in seed setting rate.In Clbbm/Clplt2 double mutant,male gametophyte development began to be abnormal at stage S9,which showed that premature degradation of tapetum affected microspore development and eventually leading to pollen abortion.And the female gametophyte development of double mutant stagnated in the stage of tetranuclear embryo sac,which could not form functional female gametophyte,resulting in significant decrease in seed setting rate.3.Cl BBM and Cl PLT2 were regulated tapetum development and pollen wall formation.Cl BBM and Cl PLT2 were participated in the genetic pathway of DYT-TDF1-AMS-MS188-MS1,which affected the development of tapetum.Cl BBM and Cl PLT2 affected outer exine formation and the supply of nutrients for microspore development by participating in fatty acid synthesis,extension and degradation.Cl BBM and Cl PLT2 regulated the expression of genes related to cellulose anabolism,glucose metabolism and transmembrane transport and participated in the formation of inner intine.4.The expression of At BBM and Cl BBM were inhibited in egg cells.Genes Cl BBM and Cl PLT2 could not be ectopic expressed in watermelon egg cells.The expression of Cl BBM,Cl PLT2 and At BBM were also inhibited in Arabidopsis egg cells,and the inhibition did not affect the ectopic expression of rice Os BBM.Heat shock protein At HSF4 could be used as an activator for At BBM to improve the ectopic expression of At BBM in egg cells,but could not induce parthenogenesis.At EIN3 and At RKD5 were bound to the exon of At BBM,which inhibited the expression of At BBM.5.Ectopic expression of BBM in egg cell was able to induce parthenogenesis.Rewriting the coding sequence of At BBM into bacterial Ecoli BBML was able to reduce the effect of potential suppressors in egg cell without changing the amino acid sequence.Ecoli BBML could induced the production of parthenogenetic embryos,but failed to generate haploid.Partial substitution of rice Os BBM for At BBM were also able to achieve the ectopic expression of recombinant BBM in egg cells and induced parthenogenesis.Among them,the expression ratio in egg cells of At Ba Os Bbc was the highest and it was able to significantly increase the haploid induction rate of dmp8/dmp9.Besides,the inhibition of At BBM in egg cells could be relieved in Atrkd5 mutant,which was able to exert the parthenogenesis induction of At BBM and obtain haploid offspring.5.Transcriptomic study of pollens,unpollinated ovules and ovule 1 day after pollination in watermelon.The pollen of watermelon contained a large number of transcripts involved in regulating pollen development and function,which was of great significance for male gametes formation.Genes related to cell proliferation and metabolism and maintaining the quiescent state of mature embryo sac before fertilization were highly expressed in the unpollinated ovule,which acted as a protection for sexual reproduction and inhibited the induction of parthenogenesis.After 1 day of pollination,the ovule contained a large number of maternal transcripts,and the transcription levels of genes related to embryo development were increased.There were 46 and 21 genes encoded the parent transcript and embryo specific transcript that regulate embryo development in watermelon,respectively,which could be used to explore the key factors in embryogenesis.