| Vitis amurensis Rupr.is an important material for wine production as well as for cold hardiness grapevine breeding in northeast China.However,the wild varieties are mostly unisexual and the male plants cannot bear fruit in the field.Studying the sex differentiation mechanism of Vitis amurensis Rupr.and establishing stable and reliable sex reversal technology of male plants has positive significance for the comprehensive utilization of germplasm resources and breeding of varieties of Vitis amurensis and further enrichment of the mechanism study of sex differentiation in higher plants.In this study,the sex differentiation and reversal mechanism of Vitis amurensis including male and bisexual flower plants was studied in term of morphology,physiology and molecular biology.The main findings are as follows:1.The sex differentiation was studied from the perspective of morphology and cytology.CPPU treatment significantly promoted the expansion of male flower buds and ovary.In the process of pistil development,the integument morphology,integument cell morphology and organelle development of ovules were different between treatment and control;the differences between treatments in the integument morphology,integument cell morphology and organelle development began earlier than the abnormal division of nucleus of pistil abortion,and the abnormal development of integument of male plant may directly affect the development of embryo sac,resulting in abnormal embryo sac caryomitosis development and the subsequent abortion of pistils.Histochemical results showed that CPPU treatment could regulate sexual differentiation of Vitis amurensis by promoting starch and protein production in ovules.2.The sex differentiation of Vitis amurensis was studied from the perspective of hormone content and the ratio between the different hormones.The high content of IAA,and low content of ZT,i.e.the low GA3 / IAA,and the high IAA / ZT in flower buds during the critical gender differentiation period was in favor of the development of bisexual flowers;hormone content and the ratio in leaves did not show correlation with plant gender;high IAA content in roots had effects on promoting male development;the content of ABA in the stem tips and the high content of IAA and the high ratio of GA3 / IAA in the tendrils could promote male development.In the process of gender transformation of male plants in CPPU treatment,low content of ZT and high content of IAA were advantageous to the development of4 amphoteric flowers in early sex determination,and low content of GA3 and high content of IAA and ABA in middle stage were advantageous to the mitosis of binuclear embryo sac.3.The sex differentiation of Vitis amurensis was studied from the perspective of polyamine content and their ratios.During the period of critical gender differentiation,high Put content in flower buds promoted male growth,while high Spd / Put and（Spd + Spm）/Put promoted female growth;high Spd and Put content in leaves promoted male growth,but high Spd / Put,Spm / Put and（Spd + Spm）/ Put promoted female growth;the content and ratio of three polyamines in roots did not show correlations with flower type.The high Put content of stem tip in male plant promoted male development,while the high Spm / Put and（Spd+Spm）/Put promoted the female development.In the process of gender transformation of male plants in CPPU treatment,the high level of Spd and Spm in the early phase may be related to the early gender determination,whereas the low level of Spd and Spm in the late phase may be related to the further development of pistils.The change of Put content in the early phase may be related to the early gender determination,and the increase of Put content in the late phase may be related to the further development of pistils.4.Transcriptome analysis of sex reversal in Vitis amurensis was studied by high-throughput sequencing.A total of 19.32 Gb Clean Data was obtained,therein,a total of 90,028 Unigenes were assembled,and the N50 of Unigene was 1204 showing the high assembly integrity.Through the database annotation such as COG,38935 Unigene with annotated information were finally obtained.There were 501 differentially expressed genes between the treatments.The 335 differentially expressed genes annotated by GO database referred to the level of biological processes,cellular components and molecular functions.Comparing the COG databases,functional predicted Unigene was the largest,and accounted for 55% of the 168 differentially expressed genes,which might be attributed to the unique population of Vitis amurensis,and the different flower pattern from the common hermaphroditic grape population,and the relatively less research,and the gene of Vitis amurensis not reported in the database;the second largest was signal transduction genes,accounting for 38%.According to the KEGG classification results of the differentially expressed genes,161 differentially expressed Unigene genes were annotated in the KEGG metabolic map,which involved 50 metabolic pathways,including metabolism,organic system,genetic information processing,cell process and environmental information processing.Among them,14 differentially expressed Unigene genes were involved in plant hormone signal transduction,and 13 differentially expressed Unigene genes were involved in pentose-glucuronide conversion pathways and starch-sucrose metabolism pathways respectively.Therefore,overall,plantsignal transduction and sugar metabolism were closely related to the formation of sex in Vitis amurensis.5.Screening and expression analysis of differentially expressed genes in Vitis amurensis was studied by quantitative fluorescence method based on results of the transcriptome analysis.Firstly,the selected 24 candidate genes were done the analysis of semiquantitative expression,then the fluorescence quantitative analysis of 11 differentially expressed genes was carried out in eight different stages after semi-quantitative screening.Combined with sequence functional analysis,we speculated that c16214（auxin-responsive protein）may be a key gene for early sex determination,and also an important factor affecting the development of floral organs in the late sex-specific stage,which runs through the whole process of sexual differentiation of Vitis amurensis;expression changes of c47987（cytokinin dehydrogenase 3[Vitis vinifera]）may be also an important factor affecting the process of gender differentiation.After the flower buds of male plants were treated with exogenous cytokinin,the level of extracellular cytokinin were decreased,therefore the information was transmitted between cells and led to the effects on the gender differentiation.The regular change of c25650（indole-3-pyruvate-monooxygenase YUCCA6 [Vitis vinifer] A]）during different stages may affect the early sex determination and the late pistil development. |
