Asymmetric Protoplast Fusion And Expression Profile Analysis Of Genes Associated With Cell Wall Regeneration During Protoplast Culture In Cotton

Cotton is an important economic crop,is also important resources of oil and protein. Its production directly related to people's livelihood.But the unit yield increase has been limited by numerous diseases and pests that cause serious yield reduction.Now,the main problems are related to pests,diseases,and their control.The transfer of polygenic traits such as pathogen resistance from wild species to cultivars is of great importance in cotton. Although conventional breeding programs have made steady improvements in agronomic traits,it is becoming more and more difficult to develop new varieties.Protoplast culture emerging as a new cell engineering technology has take part in crop improvement.On this base,an efficient way to bypass sexual-crossing barriers and create new germplasm in cotton was developed by protoplast fusion through which we can transfer desirable agronomical relevant traits from wild cotton to cultivars.At the same time,protoplasts retaining the capacity to develop a normal cell wall under suitable culture conditions were regarded as particularly suitable for studying cell wall biosynthesis.Our studies involved protoplast culture,asymmetric protoplast fusion between cultivars and wild species and identification of genes associated with cell wall biosynthesis by suppression subtractive hybridization based on cotton protoplast.The main results of this research were as fellows:1.An effective protoplast culture system is necessary for obtaining somatic hybrids via cell fusion.In this study,Protoplast was isolated from wild cotton(Gossypium davidsonii) was cultured in KM₈P medium supplemented with different phytohormones. The most effective combination was 0.45μM 2,4-dichlorophenoxyacetic acid,2.68μMα-naphthalene acetic acid and 0.93μM kinetin and the division percentage at the 8^th day was 30.78±3.04%.The density of protoplast at 2 - 10×10⁵ cells/ml was suitable for protoplast division and calli formation,with a division percentage of 32.21±3.64%and a plating efficiency of 9.12±2.61%at the 40^th day.The optimal osmotic potential was achieved using 0.5 M glucose or 0.1 M glucose plus 0.5 M mannitol.Protoplasts were cultured in three ways,a double-layer culture system,with liquid over solid medium was proved to be the best way.2.For transferring partial genome or cytoplasm,the parent protoplasts were usually treated with chemical or physical methods prior to fusion.Ultraviolet(UV) irradiation was regarded as a substitute or alternative to ionizing irradiation in asymmetric protoplast fusion experiments.In this paper,we conducted four doses(0,38.7 J/cm²,77.4 J/cm², 116.1 J/cm²) of UV irradiation on G.klozschianum protoplasts.The results of agarose gel electrophoresis indicated that considerable fragmentations of the DNA happened when irradiated with the dose of 77.4 J/cm² and 116.1 J/cm²,but less fragment action with the dose of 38.7 J/cm² compared with control.The viability and first division percentage of the irradiated protoplasts decreased along with the increase of irradiation dose,and the viability of irradiated protoplasts decreased along with the increase of culture time.All the protoplasts treated with UV could not form mass callus although plantlets developed from untreated protoplasts.As a result,and the dose of 38.7 J/cm² UV treatment was chosen as the lethal dose for the following asymmetric protoplast fusion experiments.3.Asymmetric protoplast fusion was carried out between some combinations. Hybrid plants were obtained between two combinations Coker 201(as receptor) + G. klotzschianum(as donor) and EKang 8(as receptor) + G.stockii(as donor),and the hybrids were analyzed at morphological,cytological(chromosome counting and flow cytometric analysis) and molecular levels(RAPD,SSR,CAPS and cpSSR).Most regenerated plants derived from fused protoplasts displayed a recipient-like morphology, while some showed an intermediate phenotype.And some tissues showed heterosis. Chromosome numbers in these somatic hybrids displayed a large range.Cytology and cell flow cytometry showed that the nuclear material of these plants were between the receptor and the sum of the two parents.Absence or co-existence of parents' genome DNA fragments was identified through molecular analysis.The heredity of cytoplasm was investigated by cleaved amplified polymorphic sequence(CAPS) analysis and cpSSR. The results indicated that recombination and rearrangements might have occurred in some regions of mitochondria(mt) and chloroplast(cp) DNA.To our knowledge,this is the first report about asymmetric protoplast fusion in cotton.Protoplast fusion has provided a new way for cotton genetics and breeding,as well as the basis for creation cotton genetic resources,and the hybrids obtained would be useful for breeding programs.4.The plant cell wall is of super molecular architecture,and is composed of various types of heterogeneous polymers.The genes expression during cell wall biosynthesis attracts much attention for biologist.However,the molecular mechanisms underlying cell wall biosynthesis are currently poorly understood.Microscopic analysis,using Calcofluor White to stain cellulose,showed that the protoplasts generated a new cell wall in the first 48 h after transfer to a wall-regeneration medium.To identify genes related to cell wall biosynthesis in cotton,suppression subtractive hybridization was used to visualize differential gene expression at seven distinct time-points within these first 48 h.Around 412 differentially expressed ESTs(＞3 fold) were identified,and 210 unigenes were sequenced successfully.As confirmed by real-time RCR and quantitative RT-PCR analysis,the selected genes displayed complex expression pattern during cell wall regeneration in protoplast,including most previously published cell wall associated genes. Some ESTs similar to cell wall protein genes such as proline-rich protein(PRPL), glycine-rich protein(GRP),extension(EPR1) and fasciclin-like arabinogalactan protein (FLA2) which might participate in primary cell wall or secondary cell wall construction and modification were up-regulated during cell wall regeneration in cotton protoplast. Sucrose synthase,an important enzyme in sugar signal pathway,played important roles in cell wall regeneration for cellulose biosynthesis.This study highlighted the function of some transcription factors on cell wall biosynthesis,including SPL14,NAC,Gbiaa-re, MYB,WRKY,SMP1,RAD5 and zinc finger family protein.Enrichment of Ca2+-CaM signal molecules in cell wall biosynthesis was also displayed.On the basis of expression profiles of genes involved in cell wall regeneration during protoplast culture,we proposed a model of cell wall regeneration from protoplast derived of cotton suspension cultures.5.Based on the cell wall regeneration SSH library screen and Gene-RACE strategies, four genes encoding plant cell wall related expansin-like proteins have been isolated from upland cotton,named GhEXLA1,GhEXLA2,GhEXLB1 and GhEXLB2 respectively. GhEXLA1 was 971bp in length,and had an open reading frame(ORF) of 783bp which encoded a predicted polypeptide of 260 amino acids(aa).GhEXLA2 was 961bp in length, and contained an ORF of 780bp which encoded a predicted polypeptide of 259 aa. GhEXLB1 was 1028bp in length,and contained an ORF of 780bp which encoded a predicted polypeptide of 259 aa.GhEXLB2 was 1172bp in length,and contained an ORF of 771bp which encoded a predicted polypeptide of 255 aa.Gene structure analysis showed that GhEXLA1,GhEXLB1 and GhEXLB2 have 3 introns,but GhEXLA2 have 4 introns.Homologic searching against protein database using tBLASTx in GenBank,the deduced protein sequences of GhEXLA1 and GhEXLA2 were found to share the highest protein homology with robur expansin-like protein(CAE12163),GhEXLB1 with Populus trichocarpa hypothetical protein(PtrEXLB1),and GhEXLB2 with Vitis vinifera hypothetical protein(LOC100262694).The bioinformatics analysis of their proteins revealed they owned an N-terminal signal sequence and EG45-like domain,C-terminal cellulose-binding-like domain.Furthermore we verify the precise functions of the genes on cell wall regeneration by RNA interference and GFP-fusion expression approach.We have constructed the RNA interference(RNAi) and GFP-fusion expression vector.These vectors were transformed into upland cotton mediated by Agrobacterium,and we have obtained RNAi transgenic plants of GhEXLA1 and GhEXLB1.At the same time,the GFP-fusion vectors have transformed into Arabidopisis,and T2 transgenic plants were obtained.The analysis of regeneration plants are on going.