Preliminary Study On Molecular Mechanism Of GhUGT3 And GhrbcMT Genes Regulating Pollen Development In G. Hirsutum L.

Cotton is one of the important fiber crops and oil crops in the world with significant hybrid advantages.The advantages of cotton hybridization are manifested in many aspects such as growing stronger than parents,salt and alkali resistance,drought resistance and pest resistance,as well as the improved fiber quality and uniformity of the hybrid offspring.The cotton significant hybridization is essential for increasing cotton yield and improving cotton germplasm resources.Male sterile lines,restorer line selection and related gene cloning become the effective ways to develop dominant cotton hybrid.At present,many germplasm resources of cotton male sterility and nuclear-cytoplasmic male sterility have been discovered.Moreover,molecular markers and cloning of fertility-related genes have also been studied However,due to the complex cotton genome and long growth cycle,cotton is still one of the crops which difficultly genetically modified.The long period of cotton genetic transformation,the recalcitrant genotype and high frequency of variation of somatic embryognesis and regenerated plants,resulted in laborious and time-consuming transformation of cotton,seriously hindered the analysis and utilization of cotton fertility-related functional genes,and also hindered the research and utilization of the genetic mechanism of heterosis.In recent years,virus-induced gene silencing technology has been widely used in genetic transformation and functional gene analysis in Arabidopsis thaliana,rice,cotton and other crops because of the high efficiency,simplicity and time saving.It also provides the research platform and technical support for the preliminary study of cotton gene function.In this paper,we used the CLCrV-based vector system to construct the viral silence vectors pCLCrVA-GhUGT3 and pCLCrVA-GhrbcMT.The genes:GhUGT3(UDP-glucosyltransferase)and GhrbcMT(ribulose 1,5 bisphosphate carboxylase/oxygenase large subunit ε-N-Methyltransferase)are related to pollen development Agrobacterium lumefaciens transformation was used to transform C3 12 to silence the expression of endogenous GhUGT3 and GhrbcMT in Gossypium hirsutum.The phenotypic analysis and gene expression combined with pollen activity detection,anther development observation,chlorophyll content test was conducted to verify the effect of GhUGT3 gene on the accumulation of callose in cotton microspores and to explore the relationship between the subunits of callose synthase.On the other hand,the biological function of GhrbcMT gene was explored in the development of G.hirsutum.The results are as follows:(1)GhrbcMT belongs to the non-genetic substance methyltransferase,which is high homology and rich in incomplete-LRX-leucine motif in amino acid N-terminal.There is no signal peptide sequence and transmembrane domain in GhrbcMT.(2)During the vegetative growth period,the GhrbcMTi plants were dwarfed and grew slowly.The pitches of GhrbcMTi plants were shortened.In the flowering,GhrbcMTi plants had few stamens and the stigma was shortened.GhrbcMTi plants are self-fertile.Pollen staining showed that the pollen activity of GhrbcMTi plants were poor,indicating that GhrbcMT had the significant effect on cotton fertility.(3)Tissue-specific expression analysis indicated that the GhrbcMT was predominantly expressed in leaves.The chlorophyll content was further determined.The results showed that the chlorophyll content of leaves from GhrbcMTi plants were not significantly different from WT,which indicates the expression level of GhrbcMT had no significant effect on the process of chlorophyll synthesis,but maybe regulate plant vegetative growth and reproductive growth.(4)GhUGT3 is a major member of glycosyltransferase family I and contains a conserved PSPG sequence at the carboxy terminus of the amino acid sequence.It is presumed that the PSPG sequence is a substrate binding site.Furthermore,GhUGT3 is one of the subunits that constitute callose synthase which is involved in the synthesis of callose.(5)GhUGT3i plants were infertile during flowering with fewer stamens.Anthers of GhUGT3i plants could not release pollen.The results of pollen activity detection showed the pollens of GhUGT3i plants were inactive and fragile.The callose wall of the GhUGT3i plants seemed thin by tetrad staining.The tetraploid microspores of GhUGT3i plants was abnormal and the boundaries of the microspores were not clear which indicates that the GhUGT3 may be involved in the process of callose synthesis.(6)Expression level of GhROP1 and GhSusC were significantly decreased in GhUGT3i plants.However,the expression level of GhANN3 was different.Tissue-specific analysis of GhSusC showed that GhSusC was predominantly expressed in cotton stamens and stigmas,suggesting that GhROP1,GhSusC,GhANN3 and GhUGT3 may be coordinated and affected in the process of callose synthesis.The results indicated that the UDP-glycosyltransferase encoded by GhUGT3,ANN3,SusC and ROP1 in G.hirsutum may be involved in the synthesis of cotton callose and UGT3 may have regulatory effects on SusC and ROP1 during the process of synthesis of callose.On the other hand,the GhrbcMT gene may affect the vegetative growth of the plant,thereby affect the flowering and development of the plant and ultimately resulted in fertility change.