The Study On Molecular Mechanism Of GhGLP4 Regulating Stamen Development In G. Hirsutum L

Cotton is one of the most important economic crops,an important source of natural textile fibers,and also an important source of vegetable oil and protein.About 95%of the world’s cotton yield comes from upland cotton(Gossypium hirsutum L.),the utilization of cotton heterosis has become an important means to improve cotton yield and fiber quality,it is of great practical significance to selece and popularize hybrid cotton with strong heterosis by using cotton sterile lines for cotton production in China.However,it is difficult to breed hybrid parents such as sterile lines in the utilization of cotton heterosis.The source of male sterile lines is relatively single,and the restoring ability of restorer lines is limited.At present,it is still difficult to select hybrids of strong heterosis "three lines"(male sterile line,maintainer line and restorer line)for largescale application,and the excavation and function research of fertility-related genes are relatively slow,the molecular mechanism related to male sterility is still unclear.Based on the transcriptome data of anther development of upland cotton C312(G.hirsutum)and its male sterile mutant ms1,we screened out that GhGLP genes encoding Germin like protein(GLP)in G.hirsutum and GhGLP genes had significant differences at different anther development stages,and were speculated to be related to the fertility of G.hirsutum.The GhGLP genes were analyzed and identified by bioinformatics methods,and the GhGLP4 gene with significant differential expression level in anther development was screened,and its property,structure and function of the gene were predicted.Virus induced gene silencing(VIGS)technology and Agrobacteriummediated genetic transformation technology were used to reduce the expression of GhGLP4 gene in upland cotton C312,GhGLP4i lines and resistant embryonic callus were obtained respectively.The phenotype observation,SOD and H2O2 dynamic change and gene expression level were analyzed,the regulatory mechanism of GhGLP4 involved in anther development was preliminarily revealed.The main results are as follows:(1)66 GhGLPs genes were identified in G.hirsutum genome database.Among them,the GhGLP4 gene has significant differences in the early,middle and late stages of the anther development of C312 and ms1.The gene may be related to the development of stamens,the tissue-specific expression analysis showed that it was predominantly expressed in the stamens.(2)More than 20 GhGLP4 interference lines GhGLP4i were obtained by VIGS technology.The gene expression analysis of the interference lines showed that the gene expression was extremely significantly down-regulated.The GhGLP4i plants showed male sterility,that is,the stigma exsertion,the number of anthers is less,the pollen sac is closed without cracking,the number of pollen is small and non-viable,the ovule browning and boll falling.After pollination with wild-type C312 pollen,the boll setting was normal.We preliminarily judged that the decrease of GhGLP4 gene expression affected the development of cotton stamens,which made it unable to produce normal and active pollen,but did not affect the development of stigma and normal hybridization.(3)We measured the SOD activity and H2O2 content in the anthers of the interference lines,and analyzed the expression levels of related genes.We found that the reason of GhGLP4 induced abortion was that the ROS scavenging ability of the interference lines was impaired,the rapid release of ROS and high content of H2O2 affected the normal development of stamens of GhGLP4 lines,leading to pollen abortion.(4)The constructed recombinant pK7GWIWG2-GhGLP4 was used to infect the hypocotyl of upland cotton YZ1 by Agrobacterium-mediated genetic transformation,the regeneration system of transgenic plants was established,the transgenic embryonic callus and plants have been obtained.Molecular biological identification and agronomic traits were carried out.The function of GhGLPs gene family was analyzed by bioinformatics methods,and the fertility-related genes were screened,then the function of GhGLPs gene family was analyzed by VIGS and RNAi.The results showed that reducing the expression of GhGLP4 gene in C312,the plants showed male sterility phenotype.Through the determination of its biochemical indicators and the expression analysis of related molecules,we speculated that the mechanism of pollen abortion may be caused by ROS in anthers,the elimination ability is impaired,and the high content of H2O2 disrupts the endogenous balance of ROS,leading to plant abortion.