Studies On The Molecular Mechanism Of Seed Coat Stay-Green Homologous Gene(G) Regulating Seed Dormancy In Cotton(Gossypium Spp)

Cotton is an annual herb or perennial woody plant of the genus Gossypium of the Malvaceae.Its seed fiber is an important raw material for the textile industry,has important economic value,and is one of the most important crops in the world.With the rapid rise of labor costs and the rapid development of agricultural science and technology,mechanized cultivation and harvesting is an inevitable trend in cotton production and development,which puts forward new requirements for cotton planting and breeding,such as neat emergence and consistent maturity.Cotton seeds have strong physical dormancy characteristics,which limits the uniformity of cotton seedlings to a certain extent.At present,the molecular regulation mechanism of cotton seed dormancy is not clear,but the G gene has been proved to regulate seed dormancy in many plants.In order to explore whether G homologous genes in cotton genome have similar regulatory effects on seed dormancy and its regulatory mechanism,this study first analyzes the genomic distribution,gene structure and evolutionary relationship of G homologous genes in different cotton species by bioinformatics.Subsequently,the G homologous genes of TM-1 and G.darwinii were obtained by homologous cloning technology,and recombinant expression vectors were constructed to analyze gene function and subcellular localization in Arabidopsis thaliana and tobacco.Finally,the yeast two-hybrid system was used to screen its interacting proteins,in order to provide a basis for further elucidating their regulatory mechanisms.The main results of this paper are as follows:1.The results of bioinformatics analysis showed that the G homologous genes of different cotton species could be classified into three groups,the G homologous genes of G.thurberi and G.davidsonii were located on chromosome 4,and the G homologous genes of the other eight cotton species were located on chromosome 8.The A subgroup of G.hirsutum has a close evolutionary relationship with G.herbaceum,and the evolutionary relationship between G.darwinii and G.barbadense is close.The G gene is relatively conservative in structure,and the homologous genes of G in most cotton species have 9 motifs and 9 exons,while Gmus＿A08G174400 is special,with only 3 motifs and3 exons.2.The results of protein subcellular localization showed that the expression products of G homologous gene(GhGA,GhGD)in the A and D subgenomes of G.hirsutum were localized in chloroplast.3.The results of transgenic experiments showed that the seeds of GhGA and GhGD Arabidopsis overexpression lines and Dar GA and Dar GD atg mutant complementation lines showed delayed germination.When the seed dormancy was broken by stratification treatment,the seeds of transgenic lines showed similar germination rate to WT and atg mutant seeds,indicating that G homologous genes in cotton affected seed dormancy,but did not affect germination rate.4.The expression levels of GhGA/GD,Gh NA13/ND13/NA8/ND8 and Gh PD7/PA9 in the dehulled seeds of TM-1 were decreased by 100μM ABA treatment,indicating that this concentration of ABA inhibited the expression of the above genes.The expression levels of Dar GA/GD,Dar NA13/ND13/NA8/ND8 and Dar PD7/PA9 in the dehulled seeds of G.darwinii were higher than those in the control group,indicating that this concentration of ABA promoted the expression of the above genes.The reason for the above differences may be that G.darwinii is a wild cotton with strong dormancy.In addition,by comparing the germination rate of dehulled and unhulled seeds of TM-1 and G.darwinii,it was found that the germination rate of dehulled seeds of the two cotton species was 100% at 32 h,the germination rate of unhulled seeds of TM-1 was was 83.3 %at 32 h and 100% at 48 h,while the germination rate of unhulled seeds of G.darwinii was0 within the statistical time,indicating that cotton seeds would be affected by a certain degree of physical dormancy.5.A total of 26 proteins that may interact with GhGA / GhGD were screened from the c DNA library of cotton ovules by yeast two-hybrid system.Further yeast point-topoint verification and luciferase complementation experiments showed that GhGA/GhGD could interact with 1-aminocyclopropane-1-carboxylic acid oxidase 1(ACO1)and dihydroflavonol reductase(DFR)at the protein level,indicating that GhGA/GhGD may regulate cotton seed dormancy by participating in the synthesis of ethylene or proanthocyanidins.In summary,this paper systematically analyzed the G homologous genes in different cotton species of Gossypium,and clarified the chromosome distribution,gene structure and phylogenetic relationship of G homologous genes in different cotton species.The role and regulatory mechanism of G homologous genes of TM-1 and G.darwinii in seed dormancy were discussed,which laid a foundation for further elucidating the biological functions of G homologous genes in cotton.