| Castanea henryi Rehd.Wils(Chinese chinquapin),is a unique woody grain tree species in southern China.It has been regarded as "hard-core crops" and "woody grains"since ancient times.It occupies a very important position in my country’s forestry production,economic construction and people’s daily life.However,the problem of excessive male flowers and too few female flowers(the ratio of male and female flowers is 1:500 to 1500)that are common in the production of Chinese chinquapin has not been well resolved,resulting in low and unstable yields,which has become a constraint The bottleneck of the healthy development of my country’s industry.The previous research of the research group showed that cytokinin is a hormone that plays an important role in the process of sex differentiation of Chinese chinquapin.6-BA spraying can significantly improve the differentiation rate of female flowers of Chinese chinquapin and increase the ratio of male and female flowers.In this study,the main cultivar ’Huali 2’ in Hunan Province was used as the material,and research methods such as tissue dissection microscopy technology HPLC-MSn analysis technology,transcriptomics,bioinformatics,fluorescence quantitative PCR technology and transgenic technology were used.From the cellular level of hormone metabolism and molecular level,with unsprayed exogenous 6-BA as a control,the morphological characteristics,dynamic changes of endogenous hormone content,hormone synthesis and signal pathway related gene expression during the formation process of the female flower of Chinese chinquapin.The correlation analysis between these characteristics and laws aims to clarify the mechanism of cytokinin in the formation of female chestnut flowers,and to lay a theoretical basis for further artificially regulating the sex differentiation of Chinese chinquapin flowers to improve yield.The main findings are as follows:1.It was found that the sex plasticity of Chinese chinquapin was found,and the male inflorescence adjacent to the lower end of the mixed inflorescence could be induced to become a female flower during the differentiation stage of the stamen primordium.The cytological process of female flower differentiation and development was studied by microsection technique with unsprayed 6-BA as control.The results showed that there were two types of inflorescences in the fruiting branches of the Chinese chinquapin,one was male inflorescence,and the other was mixed inflorescence,the ratio of male inflorescence to mixed inflorescence was 8.03:3.77,and the female flowers were 1～2 at the base of the mixed inflorescence.Spraying 6-BA in the male inflorescence elongation period(April 5)can induce 1～2 female flowers at the lower end of 3～5 male inflorescences adjacent to the mixed inflorescence;the induced female flowers consist of sepals,pistils(clusters)and degenerated stamens The composition is similar to the morphological characteristics of naturally formed female flowers on mixed inflorescences,but the number of stigmas is more than the latter;the induced differentiation time of female flowers can be divided into five stages:pistil primordium differentiation stage,pistil development stage,stigma elongation stage and flowering stage,which is consistent with the division of the female flowers naturally formed on the mixed inflorescence,and the differentiation time is about 4 days later than the latter.In the control group,the lower ends of the male inflorescences were all male flowers,which consisted of sepals and stamen groups,and only the stamen primordia were differentiated and developed in the male flowers.That is,cytokinin induces the differentiation and development of the pistil primordia of 1～2 flowers at the lower end of the male inflorescence adjacent to the mixed inflorescence to produce female flowers.2.The dynamic changes of endogenous hormones that influence the formation of female flowers of Chinese chinquapin by exogenous 6-BA were analyzed.HPLC-MSn analysis technique was used to study the dynamic changes of endogenous hormones during the differentiation of the pistil and stamen primordia of Chinese chinquapin.The results showed that after 6-BA treatment,the II-derived cytokinin in the differentiation stage of the pistil and stamen primordia showed a downward trend,especially the contents of CZR,iP,iP+ZT(with biological activity)were significantly lower than those of the control group;The contents of auxin(IAA)and jasmonic acid(JA)were significantly higher than those of the control;while endogenous gibberellin(GA1,GA3,GA7),abscisic acid(ABA),salicylic acid(SA)and methyl jasmonate(MEJA)was significantly lower than the control most of the time.6-BA alters the balance of endogenous hormones,high ratios IAA/(GA1+GA4),IAA/ABA,JA/(GA1+GA4),JA/ABA,JA/SA and low ratios(iP+ZT)/IAA is beneficial to the formation of the female flower of the chestnut,that is,6-BA promotes the formation of the female flower of the chestnut by affecting the changes in the content of endogenous hormones during the differentiation of the pistil primordium of the Chinese chinquapin.3.Key candidate genes associated with the formation of female flower formation in the Chinese chinquapin were identified.Transcriptome sequencing analysis of the meristems of A.chinensis at different time points after cytokinin treatment showed that there were 5 149 DEGs,2 491 up-regulated genes and 2 658 down-regulated genes between the five samples and the control.The differential genes related to the sex differentiation of Chinese chinquapin can be divided into four categories:phytohormone and signal transduction-related genes,floral organ homologous genes,meristem activity-related genes,cell cycle and nutrient transport-related genes.According to the expression patterns of hormone metabolism and flower developmentrelated genes,cytokinin metabolism genes(ChCKX1,ChRR5,ChRR9,AHK3.3,AHK3.3,etc.)and flower development-related genes(AP2,AP3,PI,SEP1,AGL2,AGL19,MYB108、UFO、CYP78A9C)were significantly differentially expressed.This indicates that exogenous 6-BA promotes the differentiation of the female flower of Chinese chinquapin by regulating the expression of cytokinin metabolism genes and genes related to flower development during the critical period of female flower formation.4.The biological functions of the key candidate genes ChCKX1,ChRR5,and ChRR9 in the formation of the female flower of the Chinese chinquapin were elucidated.Using different tissues(root,stem,leaf),different developmental stages of male and female flowers,and inflorescences 1,6,12,24 and 48 hours after exogenous 6-BA treatment as test materials,ChCKX1,ChRR5 were tested by qRT-PCR technology,ChRR9 gene expression analysis,the results showed that ChCKX1,ChRR5,ChRR9 were significantly differentially expressed relative to the control during the critical period of female flower formation.Through genetic transformation technology,it was found that 35S::ChRR9 developed slowly,bloomed later,and the stamen length was significantly lower than that of the wild type.35S::ChCKX1 corolla diameter is significantly smaller than that of wild type,35S::ChRR5 pedicel length is significantly lower than that of wild type This indicated that ChRR9 may be a key gene for the formation of female flowers. |
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