Study On Sex Differentiation Of 'Longyan Yeshi 1'

Most of the domesticated persimmon(Diospyros kaki Thunb.)cultivars are gynoecious;a few are either monoecious or androgynomonoecious.Androecious persimmons can be occasionally found in wild persimmon populations that show scattered distribution in remote,mountainous areas.In this study,male and hermaphrodite floral buds of'Longyan Yeshi l'were used as materials,and technologies including paraffin section,scanning electron microscope,high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry(HPLC-MSn)and real time-quantitative PCR(qRT-PCR)were used to systematically reveal the regulation mechanism of sex differentiation of `Longyan Yeshi l' from the aspects of morphological observation,hormone regulation and key gene expression.The main results are as follows:(1)The development process of male and hermaphrodite floral buds was observed by external morphology,scanning electron microscope and paraffin section.The results showed that male and hermaphrodite floral buds development basic synchronization.The floral buds differentiation of 'Longyan Yeshi l' from June to May of the next year,the microsporogenesis stage in the middle of April(2?3 days before and after April 17)is the critical morphological period.At this time,the stamen and pistil primordia of hermaphrodite floral buds were normal,but the pistil primordia of male floral buds aborted.(2)Mixed,male and hermaphrodite floral buds were collected on June 17(initiation of flower bud differentiation),April 6(early visible stage of three-flower cyme)and April 17(the critical morphological period).The contents of IAA,GA3,ZT,ABA and JA were determined by HPLC-MSn,and the regulation effect of endogenous hormones on sex differentiation was analyzed.The results showed that high content of IAA and JA on April 17 and the high content of ZT on April 6 and April 17 may be beneficial to the development of hermaphrodite floral buds of'Longyan Yeshi l'.On April 6 and April 17,the high content of GA3 and ABA may be beneficial to the development of male floral buds of'Longyan Yeshi 1'.(3)Eight genes highly related to sex differentiation were selected(OGI(the gene can promote male plant formation in Diospyros lotus L.),MeGI(high expression of transcription factors was beneficial to persimmon female flower formation),AMC9(genes associated with programmed cell death),AifA(genes associated with programmed cell death),IAA32(negative regulators of IAA signal transduction),ACO(key enzyme gene for ethylene biosynthesis),WRKY71(WRKY family transcription factors negatively regulate ABA signal transduction)and GA200X2(key genes for gibberellin biosynthesis))and qRT-PCR was used to analyze the expression patterns of these genes in the mixed,male and hermaphrodite floral buds of'Longyan Yeshi 1'at different development periods.To study the variation of the expression levels of these genes involved in sex differentiation,and to elucidate the molecular regulation mechanism of sex differentiation of persimmon.The results show that:the expression levels of these genes homologous to OGI,MeGI,AifA,IAA32,ACO,WRKY71 and GA20OX2 in male and hermaphrodite floral buds were the most different on April 17(the critical morphological period),while the expression levels of the gene homologous to AMC9 were the most different on Apri 20,indicating that the differential expression of these genes is crucial to the appearance of the critical morphological period of sex differentiation.In addition,in early and middle April(except April 14),the expression level of the gene homologous to OGI in male floral buds was significantly higher than that in hermaphrodite floral buds,suggesting that this period may be a key regulatory period for the gene to promote the formation of male floral buds.Except April 14,the expression level of the gene homologous to MeGI in hermaphrodite floral buds was significantly higher than that in male floral buds.At this time,the high expression of the gene homologous to MeGI may promote the formation of hermaphrodite floral buds of'Longyan Yeshi l'.April 6 to April 20(except April 14),the expression of the gene homologous to AMC9 in male floral buds was significantly higher than that in hermaphrodite floral buds,suggesting that the high expression of this gene may play an important role in promoting the abortion of pistils in male floral buds.On April 17,the gene homologous to AifA expression level in hermaphrodite floral buds was significantly higher than that in male floral buds,indicating that high expression of this gene may promote the formation of hermaphrodite floral buds of'Longyan Yeshi 1'.April 14 and April 17,the gene homologous to IAA32 expression level of male floral buds was significantly higher than that of hermaphrodite floral buds,indicating that the gene may promote the formation of male floral buds by inhibiting IAA signal transduction.April 14 to April 23,the gene homologous to ACO expression level in hermaphrodite floral buds was significantly higher than that in male floral buds,indicating that high the gene homologous to ACO expression may be conducive to the formation of hermaphrodite floral buds of 'Longyan Yeshi l' by increasing the release of ethylene.April 14 and April 17,the expression level of the gene homologous to WRKY71 in male floral buds was significantly higher than that in hermaphrodite floral buds.The transcription factor family has the effect of inhibiting ABA signal transduction,but endogenous hormone assay results showed that the ABA content in male floral buds on April 17 was higher than that in hermaphrodite floral buds,and the reason for this result still needs further analysis.Except April 14,the expression level of the gene homologous to GA20OX2 in hermaphrodite floral buds was significantly higher than that in male floral buds.The high expression of the gene may promote the development of hermaphrodite floral buds by promoting the gibberellin biosynthesis,but the endogenous hormone determination results showed that the content of GA3 in male floral buds on April 17 was higher than that in hermaphrodite:floral buds,and the specific reasons still need to be further studied.