Study On The Roles Of Blue Copper-binding Protein(BCP4)and Annexins(AnnGh3)in Fiber Elongation Of Cotton(Gossypium Hirsutum)

Cotton is one of the most important economic crops in the world,and as natural fibers,cotton fibers provide the main kind of raw material for for textile industry.Furthermore,cotton fiber,the single cell trichomes which elongate from the seed coat epidermal cells,provide an ideal model for studying plant cell polar elongation and secondary wall synthsis because of its unicellular and linear structures.Cotton fiber development can be divided into four distinct but overlapping phases:fiber initiation,fiber elongation,secondary wall biosynthesis and maturation.The vital traits of cotton fiber quality are fiber length and strength,which are mostly determined by their growth rate and mode during fiber elongation phase.As a result,it's very necessary to study the molecular regulation mechanism of related gene in cotton fiber cell elongation stage.Blue copper-binding proteins(BCPs)are small and generally soluble copper binding proteins,Annexins are a multigene family of calcium-dependent phospholipids binding proteins,which are both widespread in all kinds of plants and proved to play an important role in plant growth and development.We isolated from upland cotton and identified a blue copper protein gene GhBCP4 and an annexin gene AnnGh3,both the two genes expressed preferentially during elongation stage,suggested that they play an important role in the cotton fiber elongation development.As a consequence,we use the two genes as the research object by the technology of excessive expression,RNAi disturbances,yeast two-hybrid to study the function and the interacting proteins of the two genes.The main results were described as follows:1.Analysis of Subcellular localization of GhBCP4 proteinBy converting transformed into the fusion expression vector GhBCP4::eGFP into cotton,more than fifty lines callus cells of transgenic cotton were obtained.The callus cells with GFP fluorescence signal were chose and made temporary slide,then we observed and photographed it under a confocal microscopy,but we can't find the green fluorescence.After analyzing the structure of the GhBCP4 protein,we found that there is a signal peptide of transmembrane domain in the N-terminus and a GPI anchor site in the C-terminus,so we decided to cut off the SP from the ORF and rebuild the fusion expression vector to transform into cotton.In a result,we found that GhBCP4 protein is mainly distributed in the vacuole.2.Analysis of GhBCP4 Promoter ActivityAccording to the whole genome sequence of heterotetraploid upland cotton published by The Chinese Academy of Agricultural Sciences and other units,we isolated the GhBCP4 promoter that sequence with length of 2089bp.GhBCP4p::GUS fusion expression vector was constructed and transformed into Arabidopsis thaliana to obtain heterologous transgenic Arabidopsis plants.Histochemical staining of GUS was revealed in various organs,the results showed that there was a strong GUS signal in the root of the seedlings and in the vascular tissue of rosette leaves,but the GUS activity was weak in the stem,leaves,flowers and pods.While in the treatment of different hormones(such as auxin,jasmonic acid),the activity of the GhBCP4 promoter was affected.The results suggested that GhBCP4 may be involved in the early development of plant growth and may also be involved in some hormone signaling pathways.3.Identification and phenotypic analysis of GhBCP4 transgenic cottonFirstly,we identified the generation of 35Sp::GhBCP4 RNAi(T3)transgenic cotton,two generations of 35Sp::GhBCP4 overexpression(T2,T3)transgenic cotton and two generations of the RDLlp::GhBCP4 RNAi(T2,T3)transgenic cotton in gDNA level,and the expression of GhBCP4 gene in the transgenic cotton plants were analyzed by real-time quantitative RT-PCR.The results showed that the expression of GhBCP4 gene in 10DPA fiber of the overexpression transgenic cotton was significantly up-regulated compared with wild type and the mature fibers length was longer than wild type.On the contrary,the expression of GhBCP4 gene in RNAi transgenic cotton were detected in different degree of down-regulation compared with wild type,and the elongation of mature fibers were obviously supressed.These results indicated that the GhBCP4 gene is involved in regulating and promoting the elongation of cotton fibers.4.Screening and identification of GhBCP4 interacting proteinsIn order to further analyze how the GhBCP4 gene plays a role in regulating the elongation of cotton fibers,we can proceed from the proteins that interact with it.According to the structural analysis of GhBCP4 protein,the pGBKT7-GhBCP4 expression fusion vector was constructed,that the ORF of the GhBCP4 gene was cutted off the signal peptide of the transmembrane domain in N-terminus and the GPI anchor site in the C-terminus.The yeast two-hybrid system was used to screen the interaction protein in the 9DPA fiber cDNA library,we identified that five kinds of GhPGM1,GhPGM-LIKE,GhPMM,GhWD40R,GhATS3 showed strong interaction with GhBCP4.The expression pattern of the five genes in different tissues of the cotton was analyzed,the results indicated that GhPGMl is preferentially expressed both in fibers,especially in 9DPA fibers,suggested that GhPGM1 may play an important role in the elongation of cotton fibers.The expression of GhPGM1 in the 10DPA fibers of the GhBCP4 transgenic cotton lines was detected,found that the expression of GhPGM1 was up-regulated in the overexpression lines and down-regulated in the RNAi lines compared with the wild type,the expression pattern is consistent with GhBCP4.The results indicated that that GhPGM1 may be synergistic with GhBCP4 involved in the regulation of cotton fiber elongation development.5.Identification analysis of AnnGh3 transgenic cottonIn order to explore the function of the annexin gene AnnGh3 in fiber development,firstly transgenic cotton should be obtained for analysis.The overexpression vector TUA9::AnnGh3 OE and the two RNA interference vectors TUA9::AnnGh3RNAi GS,TUA9::AnnGh3RNAi C were respectively transformed into wild type cotton.We get a lot of transgenic cotton,but unfortunately,mostly transgenic cotton grow unnormally and pollen sterility,only a few normal and flowering transgenic cotton lines.However,further experiments also need to obtain more transgenic cotton lines and their progeny plants to explore the specific function of the AnnGh3 gene in cotton fiber elongation development.