Association Analysis Between Temporal And Spatial Expression Of Fiber Development Genes And Fiber Quality And The Transgenic Functional Confirmation Of β-1,4-Glucosidase (GhBG) Gene

Thousands of genes play role during fiber development,though the identification and function research of these genes was initiated recently,the progress was very quick.In this paper,we selected 10 preferentially expressed genes for fiber development which had reported previously and 14 cotton varieties(or strains) with significantly different fiber quality.Using Q-PCR technique to evaluate the relative expression value of 10 tested genes in different fiber developmental stages in 14 cotton varieties(or strains),in order to study the relationship between temporal and spatial expression of these genes and fiber quality traits.The analysis of expression profile of these genes in different cotton varieties(or strains) indicate:GhExp1,GhPL and GhCIPK1 gene preferentially express during fiber elongation;GhRacA and GhRacB gene have high expression level in earlier stage of fiber elongation and the thickening period of secondary cell wall;The predominant expression periods of GhACT1 are fiber elongation and the secondary cell wall thickening period;Two cellulose synthase genes(GhCelA1 and GhCelA3) preferentially express during late stage of fiber elongation and the thickening period of secondary cell wall;GhSus1 have high expression level during thickening period of secondary cell wall and fiber elongation stage, and GhSusA1 gene preferentially express during fiber initiation stage and the earlier stage of fiber elongation.For most genes,the expression value in low expression level period has significant correlation with fiber quality,while no significant correlation is detected when the expression of these genes at the peak except GhRacA gene.The expression level of GhExp1 in 20DPA fiber has a significant negative correlation with fiber strength and uniformity and it also has a significant positive correlation with fiber elongation;the expression level of GhPL gene in 23DPA has a significant negative correlation with fiber length;the expression level of GhRacA gene in 5DPA and 23DPA both have a high significant positive correlation with elongation;the expression level of GhRacB gene in 10DPA has a significant negative correlation with length;the expression level of GhCelA1 gene in 5DPA has a significant positive correlation with length and a significant negative correlation with Mic value;the expression level of GhCelA1 gene in 10DPA has a significant negative correlation with fiber length and a significant positive correlation with micronaire value and a high significant correlation with elongation;the expression level of GhCIPK1,GhACT1,GhSus1,GhSusA1and GhCelA3 gene have no significant correlation with fiber quality traits.To characterize the possible function ofβ-1,4-glucosidase(GhBG) in cotton fiber,in this study,the sense coding sequence of the GhBG gene was fused with fiber-specific E6 promoter to build the constructs of sense gene,and with which to transform into Simian3 via Agrobacterium-mediated transformation.PCR amplification of the regenerated plants with primers of marker gene and target gene indicated that 56 plants from 6 clones were positive,of which 48 were survived by grafting.Southern blot analysis of the transgenic cotton plants showed that the introduction of GhBG gene into cotton genome was successful.Genetic Analysis of 17 T₁ and T₂ regenerated plants from 6 different transgenic lines by PCR analysis and Kanamycin-resistance assay indicated that the segregation ratio of GhBG gene in 6 transgenic lines were inherited in a classical Mendelian manner(3:1) and one pure line(504-5) was screened.For analyzing the expression level of this pure line, 0DPA,5DPA,10DPA,15DPA,20DPA and 23DPA ovule or fiber RNA of pure line was extracted,with Simian3 as control.The analysis of Q-PCR results showed that the expression level of GhBG gene in 5DPA,10DPA and 20DPA ovule or fiber of pure line was higher than that of control and the differences were significant.The result of mature celullose content test of T₁ and T₂ indicted that there was no significant difference between transgenic lines and control.Analysis of fiber quality traits showed that the fiber length and strength in pure line decreased and fiber elongation percentage increased.These results demonstrate that the overexpression of GhBG gene have effect to fiber length and strength.